KR20250022285A - SCOTIN Proline-Rich Domain Biomolecular Condensates via Liquid-liquid Phase Separation and Applications Thereof - Google Patents

Abstract

The present invention relates to a biomolecular condensate of a SCOTIN proline-rich domain (PRD) formed through liquid-liquid phase separation, and materials, methods, and uses related thereto. Specifically, the present invention provides a recombinant protein, nucleic acid, or vector for targeting a biomolecular condensate to a membrane of a cell organelle; a method for forming a biomolecular condensate of a PRD or a protein comprising the PRD, a method for regulating the function of a cell organelle or endoplasmic reticulum using the same, and a method for selecting a condensate-regulating substance.

Description

SCOTIN Proline-Rich Domain Biomolecular Condensates via Liquid-Liquid Phase Separation and Applications Thereof

The present invention relates to biomolecular condensates of SCOTIN proline-rich domains formed through liquid-liquid phase separation and materials, methods, and uses related thereto.

Eukaryotic cells compartmentalize cellular space through organelles and perform specialized biological functions within them. Among the organelles, membrane-less compartments or biomolecular condensates are regions that create a physically and chemically separated internal environment without being bordered by an external lipid membrane and can condense biomolecules within it to perform unique biological functions. Representative condensate structures include the nucleolus, paraspeckle, Cajal body, P granules, and stress granules (reference [Banani, Salman F., et al. (2017)]).

When a biomolecular condensate has liquid-liquid phase separation characteristics, the condensate is compartmentalized in a liquid or gel-like state, exhibits the material properties of a liquid droplet, and biomolecules can be freely exchanged inside and outside the condensate. As the liquid-liquid phase separation characteristics of biomolecular condensates are known to be involved in the regulation of a wide range of cellular functions, such as cell signaling, neural synapses, gene expression, and protein degradation, as well as being closely related to the pathogenesis of cancer and degenerative diseases, the field of biomolecular condensates has attracted extensive attention from academia and industry (e.g., Hyman, Anthony A., Christoph A. Weber, and Frank Julicher. (2014)). Research on medical or biotechnological applications of biomolecular condensates is known, including methods for characterizing condensates (US Pat. No. 10,533,167 B2 and US Pat. No. 10,538,756 B2 and literature [Bracha, Dan, Mackenzie T. Walls, and Clifford P. Brangwynne (2019)]) and methods for screening condensate-modulating substances (US Pat. No. 11,340,231 B2 and US Pat. No. 11,493,519 B2; literature [Boija, Ann, Isaac A. Klein, and Richard A. Young. (2021)]).

Meanwhile, SCOTIN protein is a protein mainly located in the cell nucleus or ER (Endoplasmic Reticulum) membrane, and its expression is known to be induced in stressful environments such as gamma-ray irradiation or interferon stimulation, and to induce cellular responses such as apoptosis induction by p53 (literature [Bourdon, J-C., et al. (2002)]). Through previous studies, the present inventors have reported that the proline-rich domain (PRD), a cytoplasmically exposed region of SCOTIN protein, has the function of inducing autolysosomal degradation of HCV (hepatitis C virus) protein (nonstuructural 5A; NS5A) and inhibiting autophagy induction by inhibiting the contact between ERES (ER exit site) and phagophore (literature [Kim, Nari, et al. (2016)], literature [Lee, Jee-Eun, et al. (2021)], etc.). However, it is not known whether the SCOTIN protein or its subdomain PRD can form biomolecular condensates through liquid-liquid phase separation.

United States Patent US10,533,167 B2 United States Patent US10,538,756 B2 United States Patent US11,340,231 B2 United States Patent US11,493,519 B2 International Patent Publication WO2021/094746 A1 International Patent Publication WO2021/150937 A1

Hyman, Anthony A., Christoph A. Weber, and Frank Julicher. “Liquid-liquid phase separation in biology.” Annual review of cell and developmental biology 30 (2014): 39-58. Bracha, Dan, Mackenzie T. Walls, and Clifford P. Brangwynne. “Probing and engineering liquid-phase organelles.” Nature biotechnology 37.12 (2019): 1435-1445. Banani, Salman F., et al. “Biomolecular condensates: organizers of cellular biochemistry.” Nature reviews Molecular cell biology 18.5 (2017): 285-298. Boija, Ann, Isaac A. Klein, and Richard A. Young. “Biomolecular condensates and cancer.” Cancer cells 39.2 (2021): 174-192. Bourdon, J-C., et al. “Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane.” The Journal of cell biology 158.2 (2002): 235-246. Kim, Nari, et al. “Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A.” Nature communications 7.1 (2016): 1-12. Lee, Jee-Eun, et al. “SHISA5/SCOTIN restrains spontaneous autophagy induction by blocking contact between the ERES and phagophores.” Autophagy (2021): 1-16.

An object of the present invention is to provide materials, uses and methods relating to biomolecular condensates of the proline-rich domain (PRD) of the SCOTIN protein formed through liquid-liquid phase separation.

More specifically, the purpose of the present invention is to provide a recombinant protein, nucleic acid or vector for targeting a biomolecular condensate to a membrane of a cell organelle; a method for forming a biomolecular condensate of a PRD or a protein comprising the same, a method for regulating the function of a cell organelle or endoplasmic reticulum using the same, and a method for selecting a condensate-regulating substance.

All terms described in the present invention are used with the same meaning as widely known in the life sciences field, unless otherwise defined, and the cited documents are incorporated herein by reference in their entirety and are treated as if they were directly described in the specification of the present invention according to the descriptive context in which the cited documents are mentioned.

SCOTIN protein and proline-rich domain (PRD)

In the present invention, the SCOTIN protein is a protein expressed by the SCOTIN/SHISA5 gene. The human SCOTIN gene is located on chromosome 3p21.3 and is composed of, for example, an amino acid sequence of sequence number 1 (see NCBI Accession No. NP_057563.3, UniProt Accession Number Q8N114, etc.).

In the present invention, the SCOTIN protein includes a protein having homology with an amino acid sequence represented by the amino acid sequence of SEQ ID NO. 1, taking into consideration interspecies sequence homology, intracellular expression context, possibility of conservative amino acid substitution, etc., and for convenience, the sequence information of SEQ ID NO. 1 among human SCOTIN proteins is described below.

The SCOTIN protein is composed of a total of 240 amino acids, and amino acid sequences 1-24 from the amine group terminus are defined as a signal peptide (SS), amino acid sequences 25-103 as a cysteine-rich domain (CRD), amino acid sequences 104-128 as a transmembrane domain (TM), and amino acid sequences 129-240 as a proline-rich domain (PRD). In the present invention, unless otherwise specified, the proline-rich domain (PRD) refers to the PRD of the SCOTIN protein, and is represented by the amino acid sequence of sequence number 2 in the present invention.

Liquid-liquid phase separation characteristics of PRD

In the present invention, the PRD can form a biomolecular condensate by inducing liquid-liquid phase separation spontaneously in vitro or in cells. A biomolecular condensate refers to a membrane-free compartment formed by biomolecules such as proteins in a eukaryotic cell, and can form a compartment that is not bounded by a membrane and whose molecular composition and concentration are distinguished from the external environment by the phase separation characteristics of key molecules forming the condensate. When liquid-liquid phase separation occurs in vitro, the condensate can be observed in the form of a spherical liquid droplet.

Biomolecular condensates can have various phases depending on their phase separation characteristics, and condensates formed by liquid-liquid phase separation exhibit the material properties of liquid droplets, maintain a biochemical environment separated from the outside in a liquid or gel-like state, and have the characteristics of dynamic material exchange with the outside environment of the condensate. Therefore, biomolecular condensates with liquid-liquid phase separation are physically and biochemically distinct from crystals, aggregates, or stoichiometric protein complexes in the solid phase.

Biomolecular condensates formed by liquid-liquid phase separation can be observed and measured by various observation means in vitro or in cells. In one embodiment, biomolecular condensates formed in vitro can be measured by observing the spontaneous formation of spherical liquid droplets, and biomolecular condensates formed in cells can be measured by observing the condensate structures on the nanometer to micrometer scale. The liquid-liquid phase separation characteristics of biomolecular condensates can be identified by observing the coalescence and fission behavior of the condensates through fluorescence live imaging of the condensates, and can be observed by fluorescence techniques such as fluorescence bleaching, e.g., fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), or fluorescence correlation spectroscopy (FCS), wherein the molecular mobility or fluidity of the resulting condensates can be extended to seconds to minutes compared to stoichiometric protein complexes (e.g., Banani, Salman F., et al. (2017)). Additionally, established optogenetic tools can be used to characterize the liquid-liquid phase separation properties of condensates (e.g., Bracha, Dan, Mackenzie T. Walls, and Clifford P. Brangwynne (2019)).

In an embodiment of the present invention, it was confirmed that the fusion and fission of the condensate dynamically occurred through fluorescence live imaging of the organelle-targeting PRD recombinant protein or SCOTIN protein, and that the molecular rearrangement of the condensate actively occurred through FRAP observation (Example 2; FIGS. 2c to 2f). This shows that the PRD condensate of the present invention has liquid or gel-like phase properties with liquid-liquid phase separation properties.

In the present invention, an intrinsically disordered region (IDR) refers to an amorphous region in a protein region that fails to fold into a tertiary structure and spontaneously switches between various unfolded forms with similar energy. The weak and transient multivalent interactions of the IDR can form a biomolecular condensate in a liquid or gel-like phase by inducing liquid-liquid phase separation. In an embodiment of the present invention, the PRD includes an IDR consisting of 28 amino acid sequences therein (amino acid sequences 150 to 177 of SCOTIN protein; SEQ ID NO: 3), and it was confirmed that a PRD or SCOTIN protein lacking the IDR represented by SEQ ID NO: 3 lost the functions of forming liquid droplets in vitro through liquid-liquid phase separation and forming biomolecular condensates with liquid-liquid phase separation in cells (Example 2; FIGS. 2a and 2e). This shows that the PRD of the present invention or a protein comprising it (e.g., a recombinant PRD protein targeting an organelle membrane or a SCOTIN protein, etc.) can form a biomolecule condensate or liquid droplet by inducing liquid-liquid phase separation mediated by IDR. Through this, the intracellular function of the condensate formed through liquid-liquid phase separation can be confirmed by observing the cellular function regulation phenomenon of the PRD dependent on IDR.

Proteins containing PRD as a liquid-liquid phase separation domain

In an embodiment of the present invention, it was confirmed that various forms of proteins having PRD as a basic domain can commonly form condensates (specifically, liquid droplets in vitro and/or biomolecule condensates within cells) through the liquid-liquid phase separation function of PRD (Example 1; FIGS. 1A to 1C and FIG. 1F). Therefore, an appropriate form of protein having PRD as a basic domain can be utilized to exhibit the biomolecule condensate properties of PRD in vitro or within cells.

In one embodiment, the protein comprising the PRD is in the form of a membrane protein, such as an integral or peripheral membrane protein. In an embodiment of the present invention, when the PRD is implemented in the form of a membrane protein that binds to a lipid membrane, it was confirmed that PRD condensates were formed on the lipid membrane (Examples 1, 3 and 6; FIGS. 1A to 1C, FIG. 1F, FIGS. 3A to 3C, FIGS. 6A to 6D). In the case of PRD expressed in the form of membrane proteins, unlike PRD condensates that can move freely in the three-dimensional space of the cytoplasm or in vitro, their movement is limited to the two-dimensional space where the lipid bilayer is formed (reference [Su, Xiaolei, et al. "Phase separation of signaling molecules promotes T cell receptor signal transduction." Science 352.6285 (2016): 595-599.] and reference [Beutel, O., and R. MARASPINI. POMBO-GARCIA K., MARTIN-LEMAITRE C., HONIGMANN A. 2019." Phase separation of zonula occludens proteins drives formation of tight junctions" Cell (2019) 179: 923-936.], etc.)

In another embodiment, the protein comprising the PRD may be in the form of a non-membrane protein that is freely mobile in the cytoplasm or in vitro . In an embodiment of the present invention, it was confirmed that a condensate was formed through liquid-liquid phase separation within a cell or in vitro even with only a PRD without a membrane protein domain connected (Examples 1 and 2; FIGS. 1c and 2a).

In one embodiment, the PRD can be conjugated with one or more foreign substances (e.g., small molecule compounds, lipids, carbohydrates, nucleic acids, polypeptides, nanoparticles, vesicles, viruses, cells, etc.) to achieve the purpose of characterizing or regulating the function of the condensate. More specifically, the foreign substance can be a foreign protein or polypeptide, and in this case, the foreign protein or polypeptide can be fused to the end of the PRD using a genetic expression system.

In an embodiment of the present invention, by expressing PRD fused with an epitope tag (Myc) or fluorescent protein (mDsRed, GFP, YFP, mEmerald, etc.) in cells, analysis of the characteristics of condensate formation location, size, shape, etc. was performed (Examples 1 to 3; FIGS. 1b, 1c, 1e to 1f, 2c to 2f, 3a to 3c), and by treating PRD fused with an affinity tag (12-His) in vitro , condensate function control was performed to induce membrane contact by attaching the condensate to the endoplasmic reticulum membrane (Example 6; FIGS. 6a to 6d). Since the fusion of a foreign protein (e.g., a protein of several to several tens of kDa in size) to the PRD does not affect the essential condensate-forming function of the PRD, an appropriate foreign protein (e.g., an enzyme, a tag protein, a fluorescent protein, a structural protein, a signaling protein, etc.) for medical or biotechnological purposes can be fused to the PRD to exhibit the biomolecular condensate function.

The foreign protein fused to the PRD can be linked via an appropriate linker peptide depending on the intended use, and can be subjected to enzymatic or chemical modification, thereby allowing one or more foreign substances to be additionally bound. In addition, after expression or purification of the entire protein, the foreign protein can be removed by a protease, etc. under specific conditions.

Below, proteins with PRD as a liquid-liquid phase separation domain are broadly classified into three types and described.

1. Use of organelle-targeting PRD recombinant protein and its condensates

PRD recombinant protein

In one aspect, the present invention provides a recombinant protein for targeting PRD biomolecule condensates on the membrane of an organelle, comprising (i) a PRD (proline-rich domain) represented by the amino acid sequence of SEQ ID NO: 2; and (ii) an organelle-targeting membrane protein fused to the PRD, wherein the membrane protein is not derived from a SCOTIN protein.

In the above recombinant protein, the organelle-targeting membrane protein is a protein that is expressed in a eukaryotic cell and then transported to the membrane of a specific organelle by a protein sorting system, and functions to enable the entire recombinant protein to be targeted on the membrane of the organelle. The organelle targeted by the above recombinant protein may be a membrane organelle belonging to the endomembrane system of a eukaryotic cell, and includes not only membrane organelles located inside a cell (e.g., the nucleus, ER, Golgi apparatus, endosomes, autophagosomes, lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, peroxisomes, secretory granules, etc.), but also the cell plasma membrane and exosomes.

In one embodiment, the organelle targeted by the recombinant protein is ER, endosome, or mitochondria. In an embodiment of the present invention, after producing a recombinant protein targeted to ER, endosome, and mitochondria and overexpressing it in cells, it was observed that PRD condensates were formed on the membranes of the desired organelles (Examples 1 and 3; FIGS. 1f, 3b, and 3c).

More specifically, the organelle-targeting membrane protein may be (a) a transmembrane domain fused with an organelle marker protein or (b) a signal peptide.

In one embodiment, the (a) organelle marker protein is a membrane marker protein known to be located in the membrane of a specific organelle, and an appropriate type can be selected and used depending on the organelle to be targeted. More specifically, the membrane marker protein is characterized by being a protein that (1) is specifically located in the membrane of the organelle to be targeted, (2) has relatively little or no specific enzymatic activity or post-translational modification, so as not to substantially affect the function of the targeted protein, and (3) does not have its own condensation ability simply by overexpression of the membrane marker protein, so as not to affect the condensation of the recombinant protein. According to the above criteria, for example, marker proteins of ER (Sec61β, VAPA, RTN4, CLIMP63), Golgi apparatus (Giantin, GM130), early endosome (EEA1, Rab5), late endosome (CD63, Rab7), recycling endosome (transferrin receptor; TfR), autophagosome (LC3A, LC3B, LC3C, GABARAP, GABARAP L1, GABARAP L2), lysosome (LAMP1, LAMP2), cell membrane (E-cadherin, GPI, VSVG), nuclear membrane (Lamin B1, NUP98, Merin), or exosome (CD9) can be used as the organelle marker proteins of the present invention by referring to known sequence information, etc.

In an embodiment of the present invention, when recombinant proteins (mEmerald-SCOTIN PRD-Sec61β and CD63-SCOTIN PRD-Myc) in which PRD is fused with Sec61β, an ER membrane marker protein, or CD63, a late endosome membrane marker protein, were expressed in cells, it was confirmed that the recombinant proteins were targeted to the membrane of the corresponding organelle to form PRD condensates (Examples 1 and 3; FIGS. 1f and 3b).

In one embodiment, the transmembrane domain to which the signal peptide is fused (b) is a membrane protein to which a signal peptide consisting of several tens of amino acid sequences is fused to the end of a transmembrane domain region that does not have a protein targeting function by itself. Here, the signal peptide performs the function of directing the membrane protein to a specific organelle by the protein sorting system of a eukaryotic cell. A typical signal peptide acts as a protein targeting signal consisting of about 13 to 36 amino acids, and may be composed of, for example, a positively charged hydrophilic peptide near the N'-terminus of the membrane protein, a hydrophobic amino acid present in the signal peptide, and a somewhat polar region near the C-terminus. However, a signal peptide for a specific organelle such as a peroxisome may also be fused to the C'-terminus of the membrane protein. The signal peptide can be cleaved by a signal peptidase once it is directed to the target organelle. The above signal peptide can be used by selecting an appropriate type depending on the cell organelle to be targeted, and for example, ER (KDEL, MHRRRSRSCREDQKPV), Golgi apparatus (SXYQRL), mitochondrial targeting sequence (MTS), peroxisome targeting sequence (peroxisome targeting signal; PTS), and known sequence information can be appropriately utilized (literature [Nielsen, Henrik, et al. "A brief history of protein sorting prediction." The protein journal 38 (2019): 200-216.], etc.).

In one embodiment, a membrane protein fused with the N'-terminal signal peptide of TOM20 can be utilized to target the mitochondrial outer membrane (Literature [Kanaji, Sachiko, et al. "Characterization of the signal that directs Tom20 to the mitochondrial outer membrane." The Journal of cell biology 151.2 (2000): 277-288.]). In an embodiment of the present invention, when a recombinant protein (TOM20(N33)-PRD-YFP) fused with the N'-terminal 33-signal peptide of TOM20 capable of targeting the outer membrane of mitochondria with the transmembrane domain fused with the signal peptide was expressed in cells, it was confirmed that the recombinant protein was targeted to the membrane of the corresponding organelle to form a PRD condensate (Example 3; FIG. 3c).

In the above recombinant protein, the membrane protein is not derived from the SCOTIN protein, and is specifically composed of an amino acid sequence that is different from the TM of the SCOTIN protein. Preferably, the recombinant protein is characterized in that any region except the PRD is different from a functional domain constituting the SCOTIN protein (e.g., SS, CRD, TM of the SCOTIN protein).

In one embodiment, when the recombinant protein according to the present invention targets an organelle belonging to the endocytic membrane system, the PRD can be fused to the N'-terminus (mEmerald-SCOTIN PRD-Sec61β) or C'-terminus (CD63-SCOTIN PRD-Myc; MTS-PRD-YFP) of the membrane protein and exposed to the cytoplasmic region.

In one embodiment, one or more foreign proteins may be fused to the PRD end of the recombinant protein for characterization of condensates or regulation of condensate function. In an embodiment of the present invention, as an example of a foreign protein fused to the PRD, a short epitope tag (Myc) or a fluorescent protein (mEmerald, YFP) having a molecular weight of about 27 kDa was used to observe the location, size, and shape of condensates targeted to the membrane of cell organelles (Examples 1 and 3; FIGS. 1f and 3c).

In one aspect, the present invention provides a nucleic acid encoding the recombinant protein described above. Specifically, it is DNA or RNA comprising a nucleic acid sequence encoding the recombinant protein, which can be physically or chemically linked to a suitable carrier capable of being introduced into a eukaryotic cell. In one embodiment, the carrier is a recombinant vector (e.g., a plasmid or a viral vector) comprising the nucleic acid, and can be commercialized in the form of a reagent or kit.

In one aspect, the present invention provides a cell into which the recombinant vector described above has been introduced. In one embodiment, the cell may be a eukaryotic cell, and depending on the cellular organelle to be targeted, the eukaryotic cell may be an animal cell (e.g., a mammalian cell including a human), a plant cell, or a fungal cell (e.g., yeast, etc.).

In one aspect, the present invention provides a method for targeting biomolecule condensates on the membrane of an organelle, comprising the steps of: expressing the recombinant protein described above in an isolated eukaryotic cell; allowing the protein sorting system of the eukaryotic cell to transport the recombinant protein to an organelle; and inducing liquid-liquid phase separation of the PRD on the membrane of the organelle.

In the present invention, the isolated cell includes all cells, tissues, and organs that are physically separated from a living organism and can be cultured in vitro, as well as cells or cell lines derived from cells once separated. That is, if it can be cultured in a medium, it is included in the isolated cell of the present invention regardless of whether it is physically separated from the living organism.

Expression of a recombinant protein in a eukaryotic cell can be accomplished by genetic means, such as introducing a nucleic acid sequence encoding the protein into the cell via a recombinant vector. In one embodiment, the recombinant protein can be introduced into the cell together with a gene transfer vehicle by inserting the nucleic acid encoding the protein into a recombinant vector (e.g., a plasmid, viral vector, cosmid, artificial chromosome, etc.) having a promoter and an origin of replication operably linked thereto.

In an embodiment of the present invention, when recombinant proteins for targeting PRD condensates on the membranes of ER (mEmerald-SCOTIN PRD-Sec61β), late endosomes (CD63-SCOTIN PRD-Myc), and mitochondria (TOM20(N33)-PRD-YFP) were expressed in human cell lines (HeLa or HEK293), it was confirmed that the recombinant proteins moved onto the membranes of the corresponding cellular organelles and formed biomolecule condensates on the membranes by liquid-liquid phase separation of PRD (Examples 1 to 3; FIGS. 1f, 3b, and 3c).

Regulation of membrane contacts between organelles

In one aspect, the present invention provides a method for inducing membrane contact of first and second organelles in a cell, comprising the steps of: expressing the first and second recombinant proteins described above in an isolated eukaryotic cell; allowing the first and second recombinant proteins to be transported to first and second organelles, respectively, by a protein sorting system of the eukaryotic cell; and inducing liquid-liquid phase separation of the PRD on the membranes of the first and second organelles, wherein the first and second recombinant proteins comprise first and second organelle-targeting membrane proteins, respectively, and both PRDs are exposed to a cytoplasmic region.

In the present invention, membrane contact of organelles means that the membranes of two different organelles are physically placed in close proximity. Organelles form contact with each other through membrane contact sites (MCS), and through this, they can perform biological functions such as signal transmission and biomolecule movement. For example, ER of eukaryotic cells can control the spatiotemporal arrangement of intracellular membrane systems such as endolysosomes through membrane contact. Such membrane contact can be mediated by interaction or tethering between membrane proteins of both organelles.

The level of membrane contact between organelles can be observed by observing the physical arrangement between two organelles using fluorescence or electron microscopy, or by techniques that measure the distance between marker proteins of two organelles, such as the proximity ligation assay (PLA).

In one embodiment, among the organelles that are subject to membrane contact by the recombinant protein described above, the first organelle may be the ER, and the second organelle may be the membrane of an endosome, a lysosome, a peroxisome, a mitochondrion, or a plasma membrane.

In an embodiment of the present invention, it was confirmed that the PRD of SCOTIN protein is located on the membranes of two different organelles (ER and late endosomes) and can induce membrane contact between the two organelles through homotypic interaction or tethering, and that this process is mediated through the formation of PRD condensates (Example 5; FIGS. 5a, 5c, and 5d). Furthermore, it was confirmed that the induction of membrane contact between the ER and late endosomes can regulate the spatial arrangement of endosomes within a cell (Example 7; FIG. 7).

Regulation of cell organelle shape

Biomolecular condensates can recruit structural proteins onto membranes or induce changes in organelle shape through their material properties that exert surface tension and adhesion (reviewed in [Agudo-Canalejo, Jaime, et al. "Wetting regulates autophagy of phase-separated compartments and the cytosol." Nature 591.7848 (2021): 142-146.]).

In one embodiment, the recombinant protein according to the present invention can modulate the structure or morphology of an organelle by targeting PRD condensates on the membrane of the desired organelle. In an embodiment of the present invention, it was confirmed that PRD condensates formed on the ER membrane induced condensation of lower structures of the ER, PRD condensates formed on the late endosome membrane induced clustered structures of endosomes, and PRD condensates formed on the mitochondrial outer membrane induced fragmentation morphology of some mitochondria (Example 3; FIG. 1f, FIG. 3b, and FIG. 3c).

2. Utilization of SCOTIN protein and its condensates

In one aspect, the present invention provides a method for forming a biomolecular condensate of a SCOTIN protein on a membrane of an organelle, comprising the steps of: inducing expression of a SCOTIN protein in an isolated cell; and inducing liquid-liquid phase separation of a PRD represented by the amino acid sequence of SEQ ID NO: 2 among the SCOTIN proteins.

After intracellular expression, SCOTIN protein is located in various intracellular endoplasmic reticulum compartments such as endosomes as well as the ER and nuclear membrane. In an example of the present invention, when SCOTIN protein was overexpressed in cells, SCOTIN protein condensates were spontaneously formed on the membranes of cellular organelles, and it was confirmed that these condensates had liquid or gel-like properties induced by liquid-liquid phase separation mediated by IDR in the PRD of SCOTIN protein (Examples 1 and 2; FIGS. 1e and 2e).

In one embodiment, the induction of expression of the SCOTIN protein can be performed by a method of introducing a nucleic acid encoding the SCOTIN protein into a cell, such as a method of transfecting a cell with a recombinant vector having inserted a nucleic acid encoding the SCOTIN protein represented by SEQ ID NO: 1. In another embodiment, the induction of expression of the SCOTIN protein can be performed by creating an environment for inducing SCOTIN expression, which is at least one of interferon treatment and a DNA damage signal. The DNA damage signal refers to a physical or chemical environment known to directly or indirectly damage DNA within a cell, and includes, but is not limited to, irradiation with radiation (e.g., gamma rays, X-rays, ultraviolet rays, and other ionizing radiation) or treatment with a DNA-damaging agent (e.g., a genotoxic chemical such as an alkylating agent or a topoisomerase inhibitor).

Cellular stress environments that can induce SCOTIN protein expression are partly known in the art (e.g., Bourdon, J-C., et al. (2002) and Kim, Nari, et al. (2016)).

In one embodiment, the organelle where the SCOTIN protein forms a condensate is the ER. In this case, the biomolecular condensate of the SCOTIN protein can perform the function of condensing the ER membrane structure and/or inhibiting ER transport from the ER to the Golgi apparatus. In an embodiment of the present invention, in a SCOTIN protein-induced environment, the SCOTIN protein condensate is formed on the ER membrane to form a structure that entangles ER tubules in one place (Example 3; FIG. 3a), and it was confirmed that the ER transport function inhibition mediated by the SCOTIN protein condensate occurs (Example 8; FIGS. 8a and 8b).

In one embodiment, the organelles in which the SCOTIN protein forms condensates are the ER and endosomes (e.g., late endosomes).

In this case, the biomolecular condensate of SCOTIN protein can regulate the spatial arrangement of endosomes within the cell by inducing contact between ER and endosomes. In an example of the present invention, when SCOTIN protein was overexpressed in SCOTIN KO cells, it was confirmed that the SCOTIN protein condensate induced ER and endosomes to close locations (Example 5; FIGS. 5a to 5d) and induced the spatial arrangement of endosomes to a region near the cell nucleus (Example 7; FIG. 7).

Inhibition of endoplasmic reticulum transport from the ER to the Golgi apparatus

In one embodiment, biomolecular condensates of SCOTIN protein formed on the ER membrane can temporarily inhibit vesicle transport from the ER to the Golgi apparatus by creating an environment that induces SCOTIN protein expression (e.g., a cellular stress environment such as DNA damage or an inflammatory environment).

In eukaryotic cells, proteins that follow the secretory pathway are newly synthesized, concentrated in the ER (endoplasmic reticulum), sorted by the COPII (coat protein complex type II) complex at the ER exit site (ERES), and transported to their destination via the Golgi apparatus. The process by which the COPII complex transports transport proteins consists of a series of processes in which the GTPase Sar1 is recruited to Sec16 at the ERES, the inner coat protein Sec23-Sec24 dimer and the outer coat Sec13-31 tetramer bind, the Sec23-Sec24 dimer binds to membrane transport proteins, and the Sec31-Sec13 tetramer is recruited to the ERES to form a cage-shaped outer coat, thereby forming an endoplasmic reticulum surrounded by a stable COPII complex. The process by which proteins are transported from the ER to the Golgi apparatus by the COPII endoplasmic reticulum in eukaryotic cells is exquisitely regulated, and the endoplasmic reticulum transport function is appropriately controlled in response to external stimuli such as stressful environments.

In an embodiment of the present invention, through a protein transport tracing experiment using the RUSH system, it was confirmed that when SCOTIN protein was overexpressed or an environment inducing SCOTIN expression (e.g., interferon-gamma treatment) was created, COPII-mediated protein transport from the ER to the Golgi apparatus was inhibited through biomolecular condensation mediated by the IDR of SCOTIN PRD (Example 8; FIGS. 8a and 8b).

Accordingly, the present invention provides a method for inhibiting ER-to-Golgi transport, comprising the steps of inducing expression of a SCOTIN protein in an isolated eukaryotic cell; and the step of forming a biomolecular condensate of the SCOTIN protein by inducing liquid-liquid phase separation of a PRD represented by the amino acid sequence of SEQ ID NO: 2 among the SCOTIN proteins. The inhibition of ER-to-Golgi transport includes inhibition of transport of a protein along the secretory pathway.

In one embodiment, SCOTIN protein condensates can form on the ER membrane and sequester COPII complex components, specifically Sec31 or Sec13, within the SCOTIN protein condensate compartment.

Selective isolation of proteins in the condensate compartment

In one aspect, the present invention provides a method for screening a protein sequestered in a SCOTIN protein condensate, comprising the steps of: inducing expression of a SCOTIN protein in an isolated eukaryotic cell; inducing liquid-liquid phase separation of a PRD represented by the amino acid sequence of SEQ ID NO: 2 among the SCOTIN proteins, thereby forming a biomolecular condensate of the SCOTIN protein; and identifying a protein sequestered in the condensate compartment.

Biomolecular condensates can mediate specific biochemical reactions by concentrating the protein within the condensate compartment through selective sequestration of the protein, or by inhibiting the protein from acting outside the condensate compartment, thereby mediating specific cellular responses. Therefore, the method for forming a biomolecular condensate of a SCOTIN protein on the membrane of an organelle according to the present invention can be utilized for the identification of proteins that are selectively sequestered in a biomolecular condensate compartment.

In one embodiment, identification of a protein sequestered by a biomolecular condensate can be performed using a proximity labeling technique such as BioID. For example, after tagging the PRD end of a SCOTIN protein with a biotin ligase enzyme, a biotinylation reaction is performed on a protein that can be sequestered in the condensate compartment of the SCOTIN protein by treating it with biotin, which is an enzyme substrate, and then the biotinylated protein is pulled down using streptavidin beads, and the sequestered protein can be identified through mass spectrometry.

Examples of intracellular proteins that may be sequestered in SCOTIN protein condensate compartments include, but are not limited to, proteins involved in cell-cell adhesion, endoplasmic reticulum-mediated protein transport (e.g., formation of COPII endoplasmic reticulum, ER-to-Golgi protein transport, or post-Golgi endoplasmic reticulum transport), membrane fusion of cells or organelles, actin filament polymerization, viral entry into host cells, translational regulation to combat ER stress, etc.

In one embodiment, the protein selectively sequestered into the SCOTIN protein condensate compartment is Sec31 and/or Sec13, components of the COPII complex, and it was confirmed that protein transport from the ER to the Golgi apparatus can be inhibited through selective sequestration of the protein into the condensate compartment (Example 8; Figure 8c).

SCOTIN condensate mutation

The SCOTIN protein is known to be induced in cellular stress environments such as DNA damage or inflammatory environments, and mediates cellular functions to counter stress, such as apoptosis or sequestration and degradation of viral proteins.

In an embodiment of the present invention, point mutations (S151P, A157D, 161Stop) in the IDR of PRD were selected among SCOTIN protein mutations in cancer patients, and it was confirmed that formation of SCOTIN protein condensates and abnormalities in cell function occurred in cells overexpressing the mutant SCOTIN protein (Example 9; FIGS. 9a and 9b).

Accordingly, the present invention provides a composition for detecting abnormality in SCOTIN protein condensate formation or abnormality in transport inhibition from ER to Golgi apparatus in a SCOTIN expression-inducing environment, the composition comprising a probe or primer that can complementarily bind to a nucleic acid sequence encoding any one or more mutation sites among (i) a substitution of serine at position 151 with proline (S151P); (ii) alanine at position 157 with aspartic acid (A157D); and (iii) a substitution of amino acid at position 161 with a translation termination signal (161Stop), based on a SCOTIN protein represented by the amino acid sequence of SEQ ID NO: 1.

Regulation of contact between ER and endosomes

As described in the above-mentioned organelle membrane-targeting recombinant protein paragraph, organelles form contacts with each other through membrane contact sites, and induction of expression of SCOTIN protein can induce membrane contacts through formation of condensates of SCOTIN protein located on the membranes of each organelle by causing SCOTIN protein to be located on the membranes of the ER and endosomes (e.g., late endosomes), respectively.

In an embodiment of the present invention, it was confirmed that the SCOTIN protein is located on the membranes of two organelles (ER and endosomes), and can induce membrane contact between organelles through condensate formation mediated by IDR in the PRD of the SCOTIN protein, thereby inducing the spatial arrangement of intracellular endosomes near the cell nucleus, thereby regulating the intracellular dynamic behavior of the organelles (Examples 5 and 7; FIGS. 5A to 5D and FIG. 7).

3. Utilization of proline-rich domain (PRD) and its condensates

In one aspect, the present invention provides a method for forming a PRD condensate, comprising the steps of treating a PRD represented by the amino acid sequence of SEQ ID NO: 2 in vitro or in a cell; and inducing liquid-liquid phase separation of the PRD.

In the present invention, PRD acts as a basic domain that induces liquid-liquid phase separation, so that liquid droplets can be formed in vitro or biomolecule condensates can be formed within cells without the assistance of other domains.

The liquid-liquid phase separation of PRD in vitro is dependent on the concentration of the PRD protein, the foreign protein fused to the PRD, and the environment of the reaction system (e.g., pH and buffer composition, addition of a crowding agent, etc.) and forms a unique phase diagram (Example 2; Fig. 2b). In an embodiment of the present invention, the purified PRD was treated in vitro and spontaneously coalesced to form liquid droplets without external energy supply (Example 2; Fig. 2a).

Within the cell, PRD can form biomolecular condensate structures dispersed in the cytoplasmic space without being confined to the biomembrane. In an embodiment of the present invention, it was observed that spherical PRD biomolecular condensates were formed in the cytoplasmic space by transfecting the PRD expression vector into the cell (Example 1; Figure 1c). In an alternative embodiment, the purified PRD can be loaded onto an appropriate intracellular delivery vehicle, processed outside the cell, and introduced into the cell. For example, the PRD processed outside the cell can be introduced inside the cell by inserting the PRD into a lipid-based carrier such as a liposome, emulsion, or lipid nanoparticle, or by fusing a cell membrane receptor binding protein (e.g., an antibody or a fragment thereof) or a cell-penetrating peptide (CPP) to the PRD (see, e.g., Cerrato, Carmine Pasquale, and Ulo Langel. "An update on cell-penetrating peptides with intracellular organelle targeting." Expert Opinion on Drug Delivery 19.2 (2022): 133-146.).

PRD condensates formed in vitro or within cells can freely move and diffuse within the three-dimensional reaction space, and can be utilized to modulate the biochemical activity of biomolecules by sequestering specific biomolecules within the condensate compartments. For example, the overall reaction rate can be increased by concentrating enzyme and substrate concentrations within the PRD condensate compartments, and the activity of enzymes trapped in the condensate compartments can be modulated through macromolecular crowding effects. In addition, PRD condensates can follow embodiments that can be applied medically or biotechnologically through biomolecule condensates, such as sensing specific intracellular environments, buffering protein concentrations, activating or deactivating specific biological reactions, protein localization, and providing stresses for regulating the shape of organelles (e.g., Banani, Salman F., et al. (2017)).

Foreign protein fused to PRD

In the present invention, one or more foreign substances described above may be conjugated to the PRD. More specifically, the foreign substance is a foreign protein or polypeptide fused to the terminal of the PRD, and a nucleic acid sequence encoding the foreign substance may be expressed in the form of a recombinant vector linked to a nucleic acid sequence encoding the PRD. In an embodiment of the present invention, by fusing an epitope tag (Myc) to the PRD, the formation location, structure, and size of PRD condensates within a cell were observed, and by fusing an affinity tag (12-His) to the PRD, the PRD condensate was attached to the endoplasmic reticulum membrane in vitro to induce endoplasmic reticulum membrane contact (Example 6; FIG. 6a and FIG. 6b).

In one embodiment, one or more IDR sequences can be fused to the end of the PRD to modulate the liquid-liquid phase separation properties of the PRD. This allows for controlling the saturation concentration (C _sat ) at which liquid-liquid phase separation is induced, the size of the condensate, and the phase properties by increasing the multi-atom interactions by the IDRs within the PRD (Literature [Alberti, Simon, Amy Gladfelter, and Tanja Mittag. "Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates." Cell 176.3 (2019): 419-434.]).

In one embodiment, the PRD can be localized to a specific compartment and biomolecules that interact with the PRD can be concentrated by fusing a signal sequence, such as a nuclear localization signal (NLS), to the PRD to target it to a specific compartment within the cell.

In one embodiment, by fusing an enzyme protein to the PRD, the biological or biochemical function of the enzyme protein can be improved. For example, by fusing an enzyme utilized as a molecular biological tool, such as a nucleic acid polymerase (e.g., DNA polymerase, RNA polymerase, reverse transcriptase, or RNA replicase), a restriction enzyme (e.g., Type I to V enzyme), or a ligase, to the PRD, the enzyme activity can be enhanced by creating a macromolecular crowding effect on the enzyme (International Patent WO2021/094746, etc.).

In one embodiment, a PRD condensate compartment can be formed at an antigenic region of the antibody by fusing an antibody or fragment thereof to the PRD. For example, an antibody or fragment thereof that binds to a cell membrane receptor can be fused to the PRD to form a PRD condensate at the receptor. The PRD condensate can be used to introduce the PRD condensate into cells by binding to a receptor that undergoes a cell internalization process such as endocytosis.

In one embodiment, two or more foreign substances can be delivered to the same PRD condensate compartment by conjugating them to different PRDs, respectively. In an embodiment of the present invention, two SCOTIN proteins fused with two foreign tag proteins (Myc and GST) to the PRD were overexpressed in cells, and it was confirmed through GST pull-down that the two tag proteins were delivered to the same PRD condensate compartment and interacted (Example 4; FIG. 4). The method of conjugating two different foreign substances to the PRD and delivering them to the same condensate compartment can be usefully utilized as a medical or biotechnological tool. For example, by treating a first PRD in which an antibody or a fragment thereof for a cell receptor is fused to the PRD and a second PRD in which a drug molecule is conjugated together outside the cell, the first PRD can induce antibody-mediated endocytosis, and the second PRD can form a condensate compartment together with the first PRD, thereby delivering the drug molecule fused thereto together with the first PRD into the cell (see, e.g., Bai, Xun, et al. "Phase separation enhanced drug delivery system for anti-cancer therapy." (2022).).

Induction of inter-endoplasmic reticulum membrane contact by PRD membrane attachment

In one embodiment, the membrane contact function of an intrinsic membrane protein, such as the organelle-targeting PRD or SCOTIN protein, can be implemented by fusing a membrane attachment peptide having membrane attachment function to the PRD.

The above membrane attachment peptide is a protein or polypeptide having binding activity to the membrane of an endoplasmic reticulum or organelle, and the PRD to which the membrane attachment peptide is fused can be attached to the membrane of an endoplasmic reticulum or organelle in the form of a peripheral membrane protein to form a PRD condensate on the membrane. Through this, a function similar to that of an integral membrane protein containing a PRD can be implemented.

In one embodiment, an affinity tag protein, such as polyhistidine (poly-His), GST, MBP, CBP, Strep-tag, FLAG-tag, etc., is used as a membrane attachment peptide, and a ligand for the affinity tag protein is added to the biomembrane to which the PRD is to be attached, thereby enabling the PRD to be specifically attached to the biomembrane. For example, in the case of polyhistidine, which is a Ni ⁺ affinity tag protein, a system for attaching the PRD to the target endoplasmic reticulum surface in vitro can be created by including the chelator lipid DOGS-Ni-NTA as an endoplasmic reticulum component (Example 6; FIGS. 6A and 6B). By forming a PRD condensate on the surface of the endoplasmic reticulum, endoplasmic reticulum membrane contact can be induced similarly to an intrinsic membrane protein (organelle-targeting recombinant protein or SCOTIN protein).

In one aspect, the present invention provides a method for inducing membrane contact between vesicles, comprising: treating vesicles containing a lipid conjugated with a ligand in a test tube; treating a PRD represented by the amino acid sequence of SEQ ID NO: 2 before and after the vesicle treatment, wherein an affinity peptide for the ligand is fused to the PRD; attaching the PRD to a membrane of the vesicle; and inducing liquid-liquid phase separation of the PRD. Specifically, by using a polyhistidine tag protein as an affinity tag protein and adding a lipid conjugated with a Ni ⁺ chelating group (NI-NTA), which is a ligand therefor, to the vesicles, the PRD can be attached to the vesicle membrane, and membrane contact between vesicles can be induced through the liquid-liquid phase separation of the PRD (Example 6; FIGS. 6c and 6d).

The above vesicle may be a single type of vesicle, or may include two or more types of vesicles having different sizes or shapes.

Method for screening condensate control substances using PRD

In one aspect, the present invention provides a method for screening a liquid-liquid phase separation modulating substance of a PRD, comprising the steps of: treating a test substance; treating a PRD represented by the amino acid sequence of SEQ ID NO: 2 before and after the treatment of the test substance; inducing liquid-liquid phase separation of the PRD; and screening the test substance when the level of condensate formation of the PRD changes compared to a control environment in which the test substance is not treated.

The test substance may be a small molecule compound, peptide, antibody or fragment thereof, aptamer, oligonucleic acid, high molecular weight compound, multifunctional compound, natural product or a combination thereof that is processed outside the cell, or may be a gene construct that can be expressed inside the cell.

The level of the PRD condensate formation can be observed and measured through whether liquid droplets are formed; the location and/or level of condensate formation; the shape of the biofilm on which the condensate is formed; the level of contact between the biofilms on which the condensate is formed; the level of inhibition of vesicle transport by the condensate; and the level of sequestration of biomolecules by the condensate, or a combination thereof. For example, when the test substance is treated in vitro and there is no formation of liquid droplets after the test substance is treated, the test substance can be selected as a liquid-liquid phase separation inhibitor of the PRD, and 1,6-hexanediol can be selected through this (Example 2; FIG. 2a). The phase separation regulating substance can be a biomolecule condensate regulating substance, and specifically, can be a condensate formation inducing or inhibiting substance.

For the selection of the above liquid-liquid phase separation control material, one or more foreign proteins or polypeptides for condensate property analysis can be fused to the PRD and used.

The biomolecular condensate-targeting recombinant protein according to the present invention can regulate the structure and function of organelles by targeting condensates on the membranes of target organelles, and can regulate condensate-mediated cellular functions such as inducing contact between organelles and inhibiting the ER endoplasmic reticulum transport function through the formation of biomolecular condensates of PRD or SCOTIN proteins, and can be utilized for the selection of condensate-regulating substances. Therefore, the PRD biomolecular condensate according to the present invention can exhibit medical or biotechnological utility in various forms.

Figure 1a is a schematic diagram showing the domain-specific constructs of the SCOTIN protein;
Figures 1b and 1c show the results of immunofluorescence observation of cells overexpressing SCOTIN protein domain-specific constructs;
Figure 1d shows the results of predicting IDR characteristics of the SCOTIN protein region;
Figure 1e shows a fluorescence image of cells overexpressing IDR-deleted SCOTIN protein;
Figure 1f shows a fluorescence image of cells overexpressing chimeric PRD fused to an ER membrane protein;
Figure 2a shows the results of liquid droplet formation of purified PRD in vitro ;
Figure 2b shows a phase diagram for PRD liquid droplet formation conditions;
Figure 2c shows the live image results for intracellular SCOTIN protein condensations;
Figures 2d and 2e show FRAP results for intracellular SCOTIN protein;
Figure 2f shows FRAP results for ER-targeting PRD condensates;
Figure 3a shows the confocal microscopy results of SCOTIN condensates formed on the ER membrane;
Figure 3b shows fluorescence imaging results for SCOTIN condensates targeted to the late endosome membrane;
Figure 3c shows fluorescence imaging results for SCOTIN condensates targeted to the mitochondrial outer membrane;
Figure 4 shows the GST pull-down results showing the interaction of SCOTIN proteins tagged with GST and Myc, respectively;
Figures 5a and 5b show the results of PLA measurements between ER and endosomes in cells overexpressing various SCOTIN domains;
Figures 5c and 5d show the results of PLA measurements in cells co-expressing ER and endosome-targeting PRDs;
Figure 6a shows a schematic diagram of an in vitro liposome tethering experiment;
Figure 6b shows the results of attachment of purified PRD to the endoplasmic reticulum membrane;
Figure 6c shows the results of membrane contact between PRD-attached endoplasmic reticulum (SUV);
Figure 6d shows the results of membrane contact between PRD-attached vesicles (SUV and GUV);
Figure 7 shows the results of intracellular endosome placement by regulation of membrane contact between ER and endosome;
Figure 8a shows the results of inhibition of membrane transport from ER to Golgi apparatus in SCOTIN overexpressing cells via the RUSH system;
Figure 8b shows the results of inhibition of protein transport from the ER to the Golgi apparatus in interferon gamma-treated cells via the RUSH system;
Figure 8c shows fluorescence imaging results showing sequestration of Sec31, a COPII component, in the SCOTIN condensate compartment;
Figure 9a shows the results of observing condensate structures in cells overexpressing the IDR mutant of SCOTIN protein;
Figure 9b shows the results of measuring the protein transport function from the ER to the Golgi apparatus via the RUSH system in cells overexpressing the IDR mutant of SCOTIN protein.

<Materials and Experimental Methods>

Plasmid

SCOTIN-Myc and SCOTIN-mDsRed constructs were constructed by cloning the SCOTIN protein coding sequence (UniProt accession number Q8N114) into the pcDNA3.1-Myc (Invitrogen, V80020) vector or cloning it together with the mDsRed monomer (pDsRed-monomer-N1; Clontech, 632465) at the C-terminus into the pEF-DEST51 vector (Invitrogen, 12285011).

SCOTIN PRD-Myc, SCOTIN(TMPRD)-Myc, and SCOTIN(Δ150-177)-mDsRed constructs were produced by replacing the SCOTIN sequence with SCOTIN PRD, SCOTIN(TMPRD), or SCOTIN(Δ150-177) using the QuickChange site-directed mutagenesis kit (Stratagene, 200519).

Str-Ii-SCOTIN-SBP-EGFP or Str-Ii-SCOTIN(Δ150-177)-SBP-EGFP constructs were constructed by replacing VSVG with SCOTIN or SCOTIN(Δ150-177) in Str-Ii-VSVG-SBP-EGFP (Addgene, 65300). Str-Ii-SCOTIN-SBP-mDsRed and Str-Ii-SCOTIN(Δ150-177)-SBP-mDsRed were constructed by replacing EGFP with a DsRed monomer.

All constructs were verified by DNA sequence analysis.

Organelle membrane-targeting PRD chimeras

Plasmid vectors (gene constructs) containing nucleic acids encoding PRD recombinant proteins mEmerald-SCOTIN PRD-Sec61β (SEQ ID NO: 5), CD63-SCOTIN PRD-Myc (SEQ ID NO: 6), MTS-SCOTIN PRD-YFP (SEQ ID NO: 7), and PTS-SCOTIN PRD-Myc (SEQ ID NO: 8), which are targeted to the ER membrane, late endosome membrane, and mitochondrial outer membrane, respectively, were constructed as follows (MTS: mitochondria targeting sequence, PTS: peroxisome targeting sequence).

The mEmerald-SCOTIN PRD-Sec61β construct was generated by adding human SCOTIN PRD to the BglII site of mEmerald-Sec61β-C1 (Addgene, 90992) using Gibson assembly.

The CD63-SCOTIN PRD-Myc construct was constructed by cloning human SCOTIN PRD and human CD63 coding sequences into the pcDNA3.1-Myc vector, and CD63-SCOTIN PRD-YFP was constructed by replacing Myc with YFP in the pcDNA3.1-CD63-SCOTIN PRD-Myc recombinant vector.

The MTS-SCOTIN PRD-YFP construct was constructed by replacing CD63 with the human TOM20 (N-terminal 33 amino acids) coding sequence in pcDNA3.1-CD63-SCOTIN PRD-YFP (reviewed in Kanaji, Sachiko, et al. "Characterization of the signal that directs Tom20 to the mitochondrial outer membrane." The Journal of cell biology 151.2 (2000): 277-288.).

The PTS-SCOTIN PRD-Myc construct was constructed by replacing CD63 in pcDNA3.1-CD63 SCOTIN PRD-Myc with the peroxisome targeting sequence (amino acids 135 to 354) of human PEX13 (reference [Fransen, Marc, et al. "Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences." Molecular and cellular biology 21.13 (2001): 4413-4424.]).

Cell culture and transfection

HeLa and HEK293 cells were cultured in DMEM (Lonza, BE12-604F) supplemented with 10% FBS (Welgene, S001-01) and 1% penicillin-streptomycin (Gibco, 15140-122). Plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen, 1668019) according to the manufacturer's instructions. The medium was changed after 4 h, and cells were harvested or fixed after 48 h and used for subsequent experiments.

Immunofluorescence and fluorescence microscopy

Cells were fixed with 4% paraformaldehyde (Thermo Fisher Scientific, A11313) for 10 min at room temperature and then permeabilized with 0.2% Triton X-100 (Thermo Fisher Scientific, BP-151-500) in phosphate-buffered saline (PBS) (Thermo Fisher Scientific, 21600010) for 10 min. However, 0.2% saponin (Sigma, 47036-50 g-f) in PBS was used for RTN4 and Sec61β antibody staining. For blocking, 5% BSA in PBST or 5% BSA in 0.2% saponin-PBS was added for 1 h at room temperature, and then cells were incubated with primary antibodies overnight at 4 °C. Primary antibodies used for immunohistochemistry were rabbit anti-Sec61β (Atlas Antibodies, HPA049407), rabbit anti-RTN4 (Bio-Rad, AHP1799), mouse anti-giantin (Abcam, ab37266), rabbit anti-EEA1 (Cell Signaling, C45B10), mouse anti-CD63 (BD Pharmingen, 556019), mouse anti-LAMP1 (Abcam, ab25630), rabbit anti-TfR (Abcam, ab84036), and FITC anti-Myc (Abcam, ab1263). Secondary antibodies conjugated to Alexa Fluor 405 (Abcam, ab175651) or Alexa Fluor 488, 568, 647 (Invitrogen, A21206, A21202, A10042, A10037, A31573, A31571) in PBST were then incubated with the cells for 30 min at room temperature. Nuclei were then visualized with NucBlue Fixed Cell ReadyProbes reagent (Invitrogen, R37606). Mitochondria were stained using MitoTracker Deep Red FM (Invitrogen, M22426) according to the manufacturer's instructions. Coverslips were mounted with fluorescent mounting medium (Dako, s3023) and images were acquired with a 63X objective using an LSM800 confocal microscope.

SCOTIN PRD protein purification

To produce 12-His-tagged SCOTIN PRD protein (SEQ ID NO: 4), the plasmids for expression of SCOTIN PRD and PRD (Δ150-177) used for protein purification were constructed by inserting the corresponding DNA fragment amplified from SCOTIN cDNA into the pET28a vector together with a TEV cleavage site using Gibson assembly.

12His-SCOTIN PRD or 12His-SCOTIN(PRDΔ150-177) inserted into the pET28a vector was overexpressed in E. coli BL21(DE3). Bacteria were cultured in TB (Terrific Broth) medium with shaking at 37°C for 8 h, followed by subsequent culture at 17°C for 36 h to induce protein expression. The bacterial pellet was lysed by resuspension in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, and protease inhibitor cocktail; pH 7.9) and sonication. Afterwards, the lysate pellet was obtained through centrifugation (12,000×g, 4°C) and dissolved in binding buffer (8 M urea, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, and 25 mM imidazole; pH 7.9) by stirring at room temperature for 2 h. The cell debris in the pellet form in the binding buffer was removed through centrifugation (12,000×g, 4°C), and the supernatant was purified using a HisTrap™ HP column (Cytiva). The eluted protein was further purified by gel filtration (HiLoad® 16/600 Superdex® 200 pg) using FPLC (AKTA go, Cytiva). The protein was dialyzed against storage buffer (20 mM sodium acetate, 150 mM NaCl; pH 5) using a desalting column (PD-10, Cytiva). Next, the proteins were concentrated to the concentration required for the experiment using Vivaspin® concentrators (Sartorius) and stored at -80 °C.

In vitro Phase separation assay

For liquid-liquid phase separation (LLPS) analysis, purified proteins (10 μM) were mixed in LLPS buffer (50 mM Tris-HCl, 150 mM NaCl, and 10% PEG8000 at pH 7.5). 2 μL of protein was mixed with LLPS buffer and loaded onto 3% BSA-coated glass slides, covered with a coverslip, and LLPS droplets were observed. Droplet images were taken immediately after mixing the protein and LLPS buffer using an Axiovert 200 M microscope (Zeiss) or a FV3000 confocal microscope (Olympus).

Live imaging and FRAP (Fluorescence recovery after photobleaching) assays

For live imaging, SCOTIN-mDsRed expressing HeLa cells cultured with the medium in 35-mm dishes were imaged with an LSM800 confocal microscope. For FRAP analysis, a spherical area covering the entire condensate was bleached with the highest laser power. Fluorescence intensities were measured at minimum intervals. FRAP results were normalized to an unbleached control area of the same area in the same cell. Relative intensities were calculated by dividing the measured intensity by the initial intensity before bleaching. Plateau and t _1/2 values were calculated by nonlinear regression analysis with the GraphPad Prism 8 program.

GST pulldown

HEK-293 cells were lysed with NP-40 lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, and 1% Nonidet P-40 at pH 7.4). Cell lysate (2.5 mg) was incubated with 40 μL of glutathione-Sepharose beads (GE Healthcare) at 4 °C. The beads were then washed five times with NP-40 lysis buffer, and proteins were eluted from the beads by incubation for 10 min with Laemmli sample buffer supplemented with 5% β-mercaptoethanol at 95 °C. The tagged proteins were then detected by Western blotting.

Western blotting

For Western blotting analysis, cells were lysed in lysis buffer (25 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholine, 0.1% SDS, 1 mM DTT, 2 μg/ml pepstatin, 0.1 mg/ml PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM benzamidine) on ice for 30 min. After centrifugation at 13,000 rpm for 30 min, proteins in the supernatant were quantified by the Bradford method (Bio-Rad, BR 5000006). Cell lysates were mixed with 5X protein loading buffer (15% SDS, 0.825 M sucrose, 0.325 M Tris-Cl (pH 6.8), 0.002% bromophenol blue, 5% β-mercaptoethanol) and treated at 95 °C for 15 min. Proteins were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad, BR 162-0115), and treated with antibodies. The nitrocellulose membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, 31430 and 31460), and immunoreactive signals were visualized using a LAS4000 luminescent image analyzer (Fujifilm Life Science). Signals were acquired using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096).

PLA(proximity ligation assay)

The experiment was performed according to the manufacturer's instructions (Sigma-Aldrich, DUO92101), specifically, samples were incubated overnight at 4 °C with mouse anti-VAPA (Santa Cruz, sc-293278), anti-RTN4 (Santa Cruz, sc-271878), anti-Rab7 (Cell Signaling Technology, 9367S), or anti-MLN64 (Abcam, ab3478) antibodies, while negative control samples were incubated with single antibodies only. After washing, cells were incubated with anti-rabbit PLA PLUS and anti-mouse PLA MINUS probes (Duolink; Sigma-Aldrich, DUO92002 and DUO92004) at 37 °C for 1 h in a wet chamber. After ligation of the hybridized complementary probes (30 min, 37 °C), rolling circle amplification was performed with red or green fluorescently labeled nucleotides at 37 °C for 100 min. Fluorescent spots were then collected and quantified by confocal microscopy.

Preparation of artificial liposomes

To prepare small unilamellar vesicles (SUVs), 73% (mol/mol) POPC (Avanti Polar Lipids, 850457P), 20% DOPS (Avanti Polar Lipids, 840035C), 5% DOGS-NTA (Avanti Polar Lipids, 790404P), and 2% Rh-PE (Avanti Polar Lipids, 810146C) in chloroform were first mixed. After evaporation of chloroform, the thin lipid film was completely resuspended by vigorous vortexing and sonication for 30 min in 1 mL of resuspension buffer (20 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, and 10% [v/v] glycerol). Sonication was performed until the mixture became clear from a turbid state for SUV preparation. Then, the obtained liposome suspension was extruded 25 times through a mini-extruder equipped with a polycarbonate membrane (Avanti Polar Lipids, 610003, pore diameter 50 nm) at 40 °C. The liposome suspension with a lipid concentration of 4 mM was stored at 4 °C until use. The size distribution of liposomes was measured by dynamic light scattering (DLS; Malvern Instruments, Nano S90).

To prepare giant unilamellar vesicles (GUVs), first, a polyvinyl alcohol (PVA; Alfa Aesar, 41240, average MW 88,000-97,000) solution was prepared in deionized water (DW) at a concentration of 100 mg/mL, and 20-30 μL of the PVA solution was evenly spread on a slide glass, and then dried at 50 °C for 30 min to form a thin PVA film. A lipid mixture containing 93% (mol/mol) POPC (Avanti Polar Lipids, 850457P), 5% DOGS-NTA (Avanti Polar Lipids, 790404P), and 2% Cy5-PE (Avanti Polar Lipids, 810335C) was prepared in chloroform at a concentration of 1 mg/mL. A total of 20-30 μL of the lipid mixture was dropped onto a PVA-coated glass slide and spread gently. Then, chloroform was completely removed under vacuum for at least 1 h. To prepare a chamber for GUV formation, an O-ring was attached to the lipid-coated slide glass, and 200 μL of GUV formation buffer containing 200 mM sucrose, 50 mM NaCl, and 20 mM Tris (pH 8.2) was filled into the chamber. After incubation in the dark for 1 h, the GUV suspension in the chamber was recovered. The obtained GUV suspension was stored at 4 °C until use. The size was monitored using a confocal microscope.

Liposome tethering assay

A membrane-attached peptide, poly(His) tag (12-His), was fused to SCOTIN PRD to enable binding of SCOTIN PRD to DOGS-NTA, a membrane lipid component of SUVs and GUVs. Purified 12His-SCOTIN PRD was used in the experiment at a concentration of 10 μM, SUV at 2 mM, and GUV at 1 mg/mL, and attachment of SCOTIN PRD to the membrane of the endoplasmic reticulum was confirmed through a liposome co-sedimentation assay (reference [Segawa, Kazuya, Naoki Tamura, and Joji Mima. "Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering." Journal of Biological Chemistry 294.19 (2019): 7722-7739.] and reference [Wu, Xiandeng, et al. "Vesicle tethering on the surface of phase-separated active zone condensates." Molecular Cell 81.1 (2021): 13-24.] etc.).

Production of SCOTIN-KO cells

SCOTIN knockout (KO) cell lines were generated using SCOTIN-KO and HDR plasmids (Santa Cruz Biotechnology, sc-408226, sc-408226-HDR). The gRNA/Cas9 expression plasmid and donor plasmid were transfected into HeLa cells in medium containing puromycin. Deletion of SCOTIN protein expression was confirmed for each colony by Western blotting.

Statistical Analysis

All statistical analyses and graph generation were performed using GraphPad Prism. Statistical differences were assessed by two-tailed unpaired t-test for comparison between two groups. One-way ANOVA with Turkey's multiple comparison was used to compare the means of three or more unmatched groups. All data were expressed as mean ± standard error (or deviation), and statistical significance was expressed as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. (not significant).

<Example>

Example 1. Condensate-forming function of proline-rich domain (PRD)

After transfecting cells with various domain-specific constructs of the SCOTIN protein, SCOTIN, SCOTIN(ΔPRD), SCOTIN(TMPRD), SCOTIN PRD, and SCOTIN(Δ150-177), the formation of condensates was examined by fluorescence microscopy (Fig. 1a).

As a result, when SCOTIN-Myc or SCOTIN(TMPRD)-Myc protein was overexpressed in HeLa cells, condensates were observed when immunofluorescence was observed, but no condensates were observed when SCOTIN(ΔPRD)-Myc protein was overexpressed (Fig. 1b; scale bar 10 μm). In contrast, when PRD-Myc without a transmembrane domain was overexpressed, condensates were observed when confocal microscopy was performed, similar to in SCOTIN(TMPRD)-Myc overexpressing cells (Fig. 1c; scale bar 10 μm).

AlphaFold program analysis predicted the PRD domain to be an intrinsically disordered region with a weak structural signature, and in particular, the IDR prediction program analysis predicted that the 28 amino acid region (amino acid sequences 150-177 of SCOTIN protein) in the PRD had strong IDR (intrinsically disordered region) characteristics (Fig. 1d). When the SCOTIN (Δ150-177) construct, in which the IDR among SCOTIN proteins was deleted, was transfected into HeLa cells and observed under a confocal microscope, the condensate structure observed in SCOTIN overexpressing cells was no longer observed (Fig. 1e; scale bar, 10 μm).

A chimeric PRD (Sec61β-PRD-mEmerald) construct, in which the SCOTIN membrane protein region excluding the PRD is replaced with the integral ER membrane protein Sec61β, was generated and transfected into HeLa cells, followed by microscopic observation. As a result, distinct spherical condensate structures were observed in chimeric PRD overexpressing cells, whereas no condensate structures were observed in Sec61β-mEmerald overexpressing cells without PRD fusion or in Sec61β-PRD(Δ150-177)-mEmerald overexpressing cells with IDR deletion (Fig. 1f; scale bar, 10 μm).

These experimental results demonstrate that PRD can form IDR-mediated condensates within cells, and that PRD alone can form condensates within the cytoplasm or on organelle membranes via its extracellular membrane protein domain.

Example 2. Liquid-liquid phase separation function of PRD condensate

To investigate the phase separation characteristics of PRD condensates mediated by IDR, purified PRD was treated in vitro and then observed under a microscope. As a result, PRD (10 μm) treated at a physiological salt concentration (150 mM) and neutral pH (7.0) condensed to form liquid droplets (Fig. 2a; scale bar 10 μm). The phase diagram of protein concentrations and pH that can form liquid droplets is shown in Fig. 2b. In contrast, when 1,6-hexanediol (5% w/v), a liquid-liquid phase separation inhibitor, was further treated in an environment where liquid droplets were formed, or when PRD (Δ150-177) was treated instead of PRD, liquid droplets were not formed (Fig. 2a).

These results demonstrate that PRD can form IDR-mediated condensates in vitro , and that these condensates are liquid- or gel-based condensates formed via liquid-liquid phase separation.

To observe the liquid-liquid phase separation characteristics of PRD condensates within cells, confocal live imaging was performed in SCOTIN-mDsRed expressing HeLa cells. As a result, it was confirmed that fusion and fission in the condensates were dynamically developed during the observation period of several to tens of seconds (Fig. 2c; scale bar 10 μm).

Next, to investigate whether molecular rearrangement occurs at the condensate boundary, FRAP experiments were performed to measure the recovery of fluorescence signals after fluorescence bleaching of the condensate region in SCOTIN-mDsRed expressing cells. As a result, fluorescence signal recovery occurred slowly after bleaching of the condensate region in SCOTIN-mDsRed expressing cells (Fig. 2d; t _1/2 = 122.6 s; Plateau = 62.53), whereas fluorescence signal recovery occurred very rapidly in SCOTIN(Δ150-177)-mDsRed expressing cells (Fig. 2e; t _1/2 = 10.72 s; Plateau = 83.16), similar to that of ER membrane proteins that do not undergo liquid-liquid phase separation.

Furthermore, FRAP measurements of the molecular rearrangement rate in the ER-targeting chimeric PRD condensate of Example 1 showed that the ER marker protein (Emerald-Sec61β) exhibited rapid fluorescence signal recovery (Fig. 2f; t _1/2 = 6.737 s; Plateau = 93.61), whereas the fluorescence recovery rate of the chimeric PRD (mEmerald-PRD-Sec61β) was much slower (Fig. 2f; t _1/2 = 162.1 s; Plateau = 66.21).

The above results show that PRD condensates formed on the membranes of cytoplasmic organelles within cells are liquid or gel-based condensates formed through liquid-liquid phase separation.

Example 3. Observation of PRD condensates targeted on the membrane of cell organelles

After co-staining SCOTIN-Myc expressing cells with RTN4, the ER region was observed by confocal microscopy. SCOTIN was co-localized with the ER tubule marker (RTN4) (Image I) and formed small spherical condensate structures (Image II) or large, irregular condensate compartments that entangled ER tubules (Image III) (Fig. 3a). When PRD was targeted to the ER membrane through overexpression of the chimeric PRD (mEmerald-SCOTIN PRD-Sec61β), PRD condensates formed distinct spherical PRD condensates on the ER membrane (Fig. 1e).

When CD63-SCOTIN PRD-Myc, which links CD63 to a membrane protein that targets late endosomes, was overexpressed in cells, it was observed that PRD condensates were targeted to the endosomal membrane to form condensates, and endosomes formed clustered compartments (Fig. 3b).

When TOM20(N33)-PRD-YFP, which links the N-terminal 33 amino acid sequence of the mitochondrial signal sequence (MTS) TOM20 that targets the mitochondrial outer membrane to a membrane protein, was overexpressed in cells, PRD condensates were targeted to the mitochondrial outer membrane to form condensates, and condensation and fragmentation of mitochondria were observed (Fig. 3c).

The above experimental results demonstrate that PRD condensates can be targeted to the membrane of an organelle using a membrane protein that targets the membrane of the organelle, thereby inducing morphological changes such as condensation of the organelle.

Example 4. Delivery of foreign proteins into the PRD condensate compartment

HEK293 cells were transfected with SCOTIN-GST plasmid, SCOTIN-Myc or SCOTIN(Δ150-177)-Myc plasmid for 48 h. Pulldown of SCOTIN-GST was performed on whole cell lysates using glutathione-Sepharose beads, and proteins interacting with SCOTIN-GST were analyzed by immunoblotting.

As a result, Myc-tagged proteins were detected in the pulled-down precipitates when SCOTIN-Myc was transfected, whereas Myc-tagged proteins were not detected when SCOTIN (Δ150-177)-Myc was transfected (Fig. 4).

The above experimental results demonstrate that foreign proteins can be delivered to the PRD condensate compartment by fusing the foreign proteins to the PRD.

Example 5. Regulation of organelle membrane contact by PRD condensates

PLA (proximity ligation assay) can detect an amplified signal when two proteins are placed in close proximity (e.g., less than about 40 nm), and by performing PLA on membrane proteins of organelles, the level of membrane contact between two organelles can be measured.

First, PLA was performed on ER-endosome tethering proteins VAPA (ER protein) and Rab7 (late endosome protein) in SCOTIN-KO HeLa cells overexpressing SCOTIN-mDsRed to measure the level of membrane contact between ER and late endosomes. As a result, the PLA signal in SCOTIN-KO HeLa cells was significantly reduced compared to wild-type HeLa cells, but when full-length SCOTIN was overexpressed, the membrane contact level was restored to the wild-type level. However, the membrane contact level was not restored when SCOTIN (ΔPRD) or SCOTIN (Δ150-177) was overexpressed (Fig. 5a). When SCOTIN, TMPRD, or PRD were individually expressed in SCOTIN-KO cells, SCOTIN and TMPRD restored the membrane contact level of ER and endosomes, whereas PRD did not (Fig. 5b).

After co-expressing chimeric PRD constructs (Sec61β-PRD-mEmerald and CD63-PRD-Myc) targeting the ER and late endosomes, respectively, in cells, we examined whether membrane contact could be induced by PRD condensates targeted to the membranes of the two organelles by immunofluorescence observation. As a result, the two chimeric PRDs targeted to the membranes of the ER and late endosomes, respectively, were observed at the same locations (Fig. 5c). In addition, PLA performed on VAPA and Rab7 showed that the proximity between the ER and endosomes increased when the two chimeric PRDs were co-expressed (Fig. 5d).

The above experimental results demonstrate that PRD is located on the membranes of two organelles (ER and endosomes) and can induce membrane contact between organelles by forming PRD condensates.

Example 6. Control of membrane contact by PRD condensates attached to the endoplasmic reticulum membrane

To investigate whether attachment of PRD onto the ER membrane can induce membrane contact between ERs, an in vitro liposome tethering assay was performed (Fig. 6a). For the experiment, two types of liposomes (GUV, SUV) with different sizes were prepared. GUV is a giant liposome with a diameter of approximately 10 μm and has a negligible membrane curvature, making it easy to monitor membrane alignment, and SUV is a liposome with a diameter of approximately 30 nm and has a shape similar to intracellular spherical ER. For liposome observation, SUV and GUV were labeled with rhodamine B and Cy5-conjugated lipids, respectively. To attach 12-His-tagged PRD onto the ER membrane, liposomes were doped with 5% DOGS-Ni ²⁺ -NTA. Liposome attachment of 12His-PRD was confirmed through a liposome coprecipitation assay (Fig. 6b).

Confocal microscopy observations of fluorescently stained liposomes showed that, unlike the environment where only SUVs were treated, when PRDs were attached to SUVs, liposome clusters were formed among SUVs, whereas when PRD(Δ150-177) was attached to SUVs instead of PRDs, liposome clusters were not formed (Fig. 6c; scale bar 10 μm).

When SUV and GUV were treated together, aligned PRD-SUV condensates were observed on the surface of PRD-GUV with attached PRD (Fig. 6d, Type I) or clustered PRD-SUV condensates on the surface of PRD-GUV (Fig. 6d, Type II), but when PRDΔ150-177-SUV with attached IDR PRD was treated, membrane contact with PRD-GUV was not formed (Fig. 6d).

The above experimental results demonstrate that inter-endoplasmic reticulum membrane contact can be induced by PRD condensates by attaching PRD to the endoplasmic reticulum membrane surface.

Example 7. Regulation of intracellular organelle arrangement by PRD condensates

Late endosomes are known to be mainly arranged in the perinuclear (PN) region within cells and form PN clouds. We investigated the effect of regulation of contact between two organelles (ER and endosomes) by PRD condensates within cells on the spatial arrangement of endosomes. SCOTIN KO cells were transfected with Rab7-EGFP and the intracellular distribution of late endosomes was observed by fluorescence microscopy. In wild-type cells, late endosomes were mainly arranged in the PN cloud, but in SCOTIN KO cells, late endosomes were often observed in a more dispersed form at the cell periphery. When SCOTIN-mDsRed was overexpressed in SCOTIN KO cells, late endosomes were re-arrested in the PN cloud, but this recovery of endosome arrangement was not observed when the condensate mutant SCOTINΔ150-177 was expressed (Fig. 7).

The above experimental results demonstrate that the spatial arrangement of organelles can be controlled by regulating organelle contact by PRD condensates.

Example 8. Regulation of transport from the ER to the Golgi apparatus by PRD condensates

The RUSH (retention using selective hook) system consists of a bicistronic expression plasmid capable of co-expressing an ER hook protein and a reporter protein, wherein the hook protein is Str (streptavidin) tagged with an ER retention sequence (e.g., MHRRRSRSCREDQKPV, KDEL, etc.), and the reporter protein is fused with SBP (streptavidin-binding protein) that binds to Str and a fluorescent protein for tracking (e.g., EGFP, etc.). When the hook and reporter proteins are co-expressed in a cell, the reporter protein is artificially retained in the ER by the binding between Str and SBP, but when biotin is treated externally, the binding between Str and SBP is disrupted and the reporter protein is released from the hook protein. At this time, by observing the fluorescent protein signal fused to SBP under a fluorescence microscope, the intracellular transport pathway of the reporter protein can be monitored (see literature [Boncompain et al.], etc.).

When the RUSH system, which uses E-cadherin as a reporter protein that moves from the ER to the cell membrane via the Golgi apparatus along the secretory pathway, was expressed together with an ER hook protein and fluorescence observation was performed, SBP-EGFP-E-cadherin, which was detained in the ER before biotin treatment, moved to the Golgi apparatus (G130: Golgi apparatus marker protein) 15 min after biotin treatment in wild-type cells. On the other hand, in SCOTIN-overexpressing cells, the level of E-cadherin Golgi movement was significantly reduced compared to wild-type cells after biotin treatment, and in SCOTIN (Δ150-177) expressing cells, it was restored to the level of the control Sec61β-overexpressing cells (Fig. 8a; scale bar, 10 μm).

Meanwhile, when interferon γ (IFN-γ), a SCOTIN expression inducer, was externally treated in wild-type cells introduced with the RUSH system, the Golgi movement of E-cadherin, which appeared 15 minutes after biotin treatment, was inhibited. However, when interferon γ was treated in SCOTIN KO cells instead of wild-type cells, the inhibition of E-cadherin Golgi movement was not observed (Fig. 8b).

ER-to-Golgi transport of endoplasmic reticulum is accomplished through the binding of COPII components, including Sec16, Sec23/24, and Sec31/13. In cells overexpressing SCOTIN, Sec31 was observed in the SCOTIN condensate region instead of Sec16, but in cells overexpressing SCOTIN (Δ150-177) instead of SCOTIN, sequestration of Sec31 in SCOTIN condensates was not observed (Fig. 8c).

These results demonstrate that SCOTIN condensates formed on the ER membrane can inhibit ER-to-Golgi transport in the presence of SCOTIN protein expression by sequestering COPII components (Sec31).

Example 9. Loss of PRD condensate formation function due to IDR mutations

We analyzed cancer mutation data reported in OncoMX (Dingerdissen et al., 2020) to investigate point mutation regions likely to affect SCOTIN protein function. Among them, point mutations that occurred in the 150th to 177th regions, which are IDR regions of SCOTIN protein, were introduced into the SCOTIN-mDsRed construct and transfected into HeLa cells to investigate whether condensate formation occurred. As a result, it was confirmed that condensate formation function was lost in S151P, A157, and 161stop mutations (Fig. 9a).

To investigate the effect of IDR point mutations on the ER-to-Golgi transport inhibitory function of SCOTIN protein, ER transport function was traced with E-cadherin RUSH reporter in cells expressing mutant SCOTIN proteins. As a result, S151P, A157D, and 161stop mutations did not inhibit RUSH reporter (E-cadherin) transport from ER to Golgi (Fig. 9b).

The above experimental results show that mutations in the IDR of PRD can result in the loss of condensate formation function and the endoplasmic reticulum transport regulation function mediated by it.

Attach electronic file of sequence list

Claims (22)

(i) a PRD (proline-rich domain) represented by the amino acid sequence of sequence number 2; and
(ii) comprising a membrane protein targeting an organelle fused to the PRD (wherein the membrane protein is not derived from a SCOTIN protein);
Recombinant proteins for targeting biomolecule condensation onto the membranes of organelles. In the first paragraph, the cell organelle targeting membrane protein is
(a) a cell organelle marker protein; or
(b) a transmembrane domain fused with a signal peptide,
Recombinant protein. A recombinant protein in which a foreign protein is fused to the PRD in claim 1. In the first paragraph, a recombinant protein wherein the cell organelle is ER, endosome or mitochondria. A nucleic acid encoding a protein according to any one of claims 1 to 4. A recombinant vector comprising the nucleic acid of claim 5. A cell into which the recombinant vector of clause 6 has been introduced. A step of expressing a recombinant protein according to any one of claims 1 to 4 in an isolated eukaryotic cell;
a step of allowing the recombinant protein to be targeted to a cellular organelle by the protein sorting system of the eukaryotic cell; and
Comprising a step of inducing liquid-liquid phase separation of PRD on the membrane of the above cell organelle.
A method for targeting biomolecule condensation on the membrane of a cell organelle. A step of expressing the first and second recombinant proteins according to any one of claims 1 to 4 in an isolated eukaryotic cell;
A step of allowing the first and second recombinant proteins to be targeted to the first and second organelles, respectively, by the protein sorting system of the eukaryotic cell; and
Comprising a step of inducing liquid-liquid phase separation of PRD on the membrane of the first and second cell organelles,
wherein the first and second recombinant proteins comprise first and second organelle-targeting membrane proteins, respectively, and the PRDs are both exposed to the cytoplasmic region.
A method for inducing membrane contact between first and second organelles within a cell. A method according to claim 9, wherein the first organelle is ER and the second organelle is endosome. A step of inducing expression of SCOTIN protein in isolated eukaryotic cells; and
A step of inducing liquid-liquid phase separation of PRD represented by the amino acid sequence of sequence number 2 among the above SCOTIN proteins
A method for forming a biomolecular condensate of SCOTIN protein on the membrane of a cell organelle, comprising: In the 11th paragraph, the induction of expression of SCOTIN protein
(i) introducing a nucleic acid encoding a SCOTIN protein into a cell; or
(ii) A method performed by creating an environment in which one or more of interferon treatment and DNA damage signals induce SCOTIN expression. A method according to claim 12, wherein the cell organelle comprises ER. A method according to claim 13, wherein the biomolecular condensate of SCOTIN protein formed on the ER membrane induces one or more of the following:
(i) condensation of ER membrane structures;
(ii) inhibition of endoplasmic reticulum transport from the ER to the Golgi apparatus; and
(iii) Contact between ER and endosomes. A step of processing a PRD represented by the amino acid sequence of sequence number 2; and
Steps for inducing liquid-liquid phase separation of PRD
A method for forming a biomolecular condensate comprising: In claim 15, a method wherein the PRD condensate forms liquid droplets in vitro . A method according to claim 16, wherein a foreign peptide is tagged to the PRD. A step of treating an endoplasmic reticulum containing a lipid conjugated with a ligand in a test tube;
A step of processing a PRD represented by the amino acid sequence of SEQ ID NO: 2 before or after the above vesicle processing, wherein an affinity peptide for the ligand is fused to the PRD;
a step of attaching the PRD to the membrane of the endoplasmic reticulum; and
Step for inducing liquid-liquid phase separation of the above PRD
A method for inducing membrane contact between endoplasmic reticulum, comprising: A method according to claim 18, wherein the ligand is Ni-NTA (Nitrilotriacetic acid) and the affinity peptide is polyhistidine. A method according to claim 18, wherein the endoplasmic reticulum comprises two or more types of endoplasmic reticulum. Step of processing the test material;
A step of processing a PRD represented by the amino acid sequence of sequence number 2 before and after the above test substance treatment;
A step of inducing liquid-liquid phase separation of PRD; and
A step for selecting a test substance when the level of condensate formation of the above PRD changes compared to a control environment in which the test substance is not treated.
A method for selecting a liquid-liquid phase separation control substance of PRD, comprising: In the 21st paragraph, the liquid-liquid phase separation control substance of the PRD is a screening method in which at least one of the following control substances is used:
(i) a substance that regulates contact between the ER and endosomes; or
(ii) Substances that regulate endoplasmic reticulum transport from the ER to the Golgi apparatus.