ABSTRACTMutations in theTransportandGolgiOrganization 2 (TANGO2) gene are associated with intellectual deficit, neurodevelopmental delay and regression. Individuals can also present with an acute metabolic crisis that includes... more
ABSTRACTMutations in theTransportandGolgiOrganization 2 (TANGO2) gene are associated with intellectual deficit, neurodevelopmental delay and regression. Individuals can also present with an acute metabolic crisis that includes rhabdomyolysis, cardiomyopathy and cardiac arrhythmias, the latter of which are potentially lethal. While preventing metabolic crises has the potential to reduce mortality, no treatments currently exist for this condition. The function of TANGO2 remains unknown but is suspected to be involved in some aspect of lipid metabolism. Here, we describe a model ofTANGO2-related disease in the fruit flyDrosophila melanogasterthat recapitulates crucial disease traits. Pairing a new fly model with human cells, we examined the effects of vitamin B5, a Coenzyme A (CoA) precursor, on alleviating the cellular and organismal defects associated withTANGO2deficiency. We demonstrate that vitamin B5 specifically improves multiple defects associated with TANGO2 loss-of-function in...
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Research Interests: Biology, Medicine, Multidisciplinary, Macromolecular X-Ray Crystallography, Crystal structure, and 15 moreCell Differentiation, Humans, Mutation, Animals, P-glycoprotein, Glycoproteins, Cell Proliferation, Amino Acid Sequence, Protein Quaternary Structure, Protein Binding, X Ray Crystallography, Insulin Like Growth Factor, Dimerization, Cell Growth, and Embryonal carcinoma
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The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential... more
The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G...
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The highly evolutionarily conserved transport protein particle (TRAPP) complexes (TRAPP II and III) perform fundamental roles in subcellular trafficking pathways. Here we identified biallelic variants in TRAPPC10, a component of the TRAPP... more
The highly evolutionarily conserved transport protein particle (TRAPP) complexes (TRAPP II and III) perform fundamental roles in subcellular trafficking pathways. Here we identified biallelic variants in TRAPPC10, a component of the TRAPP II complex, in individuals with a severe microcephalic neurodevelopmental disorder. Molecular studies revealed a weakened interaction between mutant TRAPPC10 and its putative adaptor protein TRAPPC2L. Studies of patient lymphoblastoid cells revealed an absence of TRAPPC10 alongside a concomitant absence of TRAPPC9, another key TRAPP II complex component associated with a clinically overlapping neurodevelopmental disorder. The TRAPPC9/10 reduction phenotype was recapitulated in TRAPPC10-/- knockout cells, which also displayed a membrane trafficking defect. Notably, both the reduction in TRAPPC9 levels and the trafficking defect in these cells could be rescued by wild type but not mutant TRAPPC10 gene constructs. Moreover, studies of Trappc10-/- knoc...
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BackgroundThe combination of febrile illness-induced encephalopathy and rhabdomyolysis has thus far only been described in disorders that affect cellular energy status. In the absence of specific metabolic abnormalities, diagnosis can be... more
BackgroundThe combination of febrile illness-induced encephalopathy and rhabdomyolysis has thus far only been described in disorders that affect cellular energy status. In the absence of specific metabolic abnormalities, diagnosis can be challenging.ObjectiveThe objective of this study was to identify and characterise pathogenic variants in two individuals from unrelated families, both of whom presented clinically with a similar phenotype that included neurodevelopmental delay, febrile illness-induced encephalopathy and episodes of rhabdomyolysis, followed by developmental arrest, epilepsy and tetraplegia.MethodsWhole exome sequencing was used to identify pathogenic variants in the two individuals. Biochemical and cell biological analyses were performed on fibroblasts from these individuals and a yeast two-hybrid analysis was used to assess protein-protein interactions.ResultsProbands shared a homozygous TRAPPC2L variant (c.109G>T) resulting in a p.Asp37Tyr missense variant. TRAP...
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Research Interests: Biology, Limb Girdle Muscular Dystrophy, Adolescent, Intellectual Disability, Medicine, and 15 moreMovement disorders, Biological Sciences, Humans, Muscular Dystrophy, Female, Male, Endoplasmic Reticulum, Creatine Kinase, Adult, Consanguinity, Ataxia, Lysosomes, Golgi Apparatus, Exome, and Medical and Health Sciences
TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic... more
TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.
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The cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that inhibits proliferation and enhances differentiation of human embryonal carcinoma cells. CREG binds to the cation-independent mannose 6-phosphate... more
The cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that inhibits proliferation and enhances differentiation of human embryonal carcinoma cells. CREG binds to the cation-independent mannose 6-phosphate (M6P)/insulin-like growth factor II (IGF2) receptor (IGF2R) (M6P/IGF2R), and this receptor has been shown to be required for CREG-induced growth suppression. To better understand CREG function in cellular growth and differentiation, we solved the 3D crystal structure of this protein to 1.9-Å resolution. CREG forms a tight homodimeric complex, and CREG monomers display a β-barrel fold. The three potential glycosylation sites on CREG map to a confined patch opposite the dimer interface. Thus, dimerization of glycosylated CREG likely presents a bivalent ligand for the M6P/IGF2R. Closely related structural homologs of CREG are FMN-binding split-barrel fold proteins that bind flavin mononucleotide. Our structure shows that the putative flavin mononucleotide-bin...
Research Interests: Biology, Medicine, Multidisciplinary, Crystal structure, Cell Differentiation, and 15 moreHumans, Mutation, Animals, P-glycoprotein, X-ray crystallography, Glycoproteins, Mannose-6-Phosphate, Cell Proliferation, Amino Acid Sequence, Protein Quaternary Structure, Protein Binding, Insulin Like Growth Factor, Dimerization, Cell Growth, and Embryonal carcinoma
TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of... more
TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present ...
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Research Interests: Biology, Membrane Proteins, Biological Chemistry, Medicine, Biological Sciences, and 15 moreHumans, Mice, Animals, Biological, Proteins, CHEMICAL SCIENCES, Molecular cloning, Protein Conformation, Amino Acid Sequence, MAP Kinase, Protein Binding, Endosomes, Molecular Sequence Data, conserved sequence , and Medical and Health Sciences
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Research Interests: Mixed Methods, Flow Cytometry, Fluorescence Microscopy, Biology, Health, and 15 moreAutophagy, Apoptosis, Medicine, Cell Culture, Biological Sciences, Humans, Animals, Cell Death, ER stress, Knowledge base, Eukaryotic Cells, Human Disease, Biological Assay, Biochemistry and cell biology, and Immunoblotting
Abstract: It is clear that plasma fibrinogen levels are strongly influenced by genetic factors. To date 14 polymorphic sites have been identified within the fibrinogen gene cluster, mainly by restriction fragment length polymorphism... more
Abstract: It is clear that plasma fibrinogen levels are strongly influenced by genetic factors. To date 14 polymorphic sites have been identified within the fibrinogen gene cluster, mainly by restriction fragment length polymorphism (RFLP) and single‐stranded conformation polymorphism (SSCP) analyses. Since elevated plasma fibrinogen is an independent risk factor for cardiovascular disease, these and other polymorphisms are of practical interest in defining haplotypes that correlate with fibrinogen levels. Here, DNA sequencing of fibrinogen genes from four patients led to the identification of 17 variations from the published sequence. Nine of these occurred in all chromosomes sequenced and were considered to be errors in the published data. Of the remaining eight, five represented novel variations, three having been previously described. The population frequency of the five novel variations, together with six known polymorphisms, was estimated by genotyping 50 normal individuals at each locus. The five new variations were all found at polymorphic frequencies in this group. Two of these new polymorphisms, Bβ intron 2 and Bβ codon 159, belong to the Bβ linkage group defined by Behague et al., 1 since their rare alleles occurred in complete concordance with the rare alleles of BβMnl I and BβBcl I. Calculation of pairwise linkage disequilibrium coefficients showed that the three remaining novel polymorphisms, AαDde I, BβHinf I, and γ intron 9 exhibited linkage equilibrium with respect to all other loci examined, including nearby polymorphisms that are themselves in strong linkage disequilibrium. This data indicates that these polymorphisms occur randomly with respect to background haplotype, and suggests that they are mutational hot spots.
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A number of the factors mentioned above, as well as their mammalian orthologs, are believed to act in several stages of membrane traffic. Ypt1p, the yeast ortholog
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Research Interests: Magnetic Resonance Imaging, Biology, Transcription Factors, Medicine, Biological Sciences, and 15 moreBrain, Humans, Female, Male, Endoplasmic Reticulum, Infant, Membrane transport proteins, Atrophy, Base Sequence, Golgi Apparatus, Protein Transport, Exome, Brain Diseases, Child preschool, and Medical and Health Sciences
TRAPPC11 was identified as a component of the TRAPP III complex that functions in membrane trafficking and autophagy. Variants in TRAPPC11 have been reported to be associated with a broad spectrum of phenotypes but all affected... more
TRAPPC11 was identified as a component of the TRAPP III complex that functions in membrane trafficking and autophagy. Variants in TRAPPC11 have been reported to be associated with a broad spectrum of phenotypes but all affected individuals display muscular pathology. Identifying additional variants will further our understanding of the clinical spectrum of phenotypes and will reveal regions of the protein critical for its functions. Here we report three individuals from unrelated families that have bi-allellic TRAPPC11 variants. Subject 1 harbors a compound heterozygous variant (c.1287 + 5G > A and c.3379_3380insT). The former variant results in a partial deletion of the foie gras domain (p.Ala372_Ser429del), while the latter variant results in a frame-shift and extension at the carboxy terminus (p.Asp1127Valfs*47). Subjects 2 and 3 both harbour a homozygous missense variant (c.2938G > A; p.Gly980Arg). Fibroblasts from all three subjects displayed membrane trafficking defects ...
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Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound... more
Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation. Subject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and...
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Triple A syndrome (MIM #231550) is associated with mutations in the AAAS gene. However, about 30% of patients with triple A syndrome symptoms but an unresolved diagnosis do not harbour mutations in AAAS. Search for novel genetic defects... more
Triple A syndrome (MIM #231550) is associated with mutations in the AAAS gene. However, about 30% of patients with triple A syndrome symptoms but an unresolved diagnosis do not harbour mutations in AAAS. Search for novel genetic defects in families with a triple A-like phenotype in whom AAAS mutations are not detected. Genome-wide linkage analysis, whole-exome sequencing and functional analyses were used to discover and verify a novel genetic defect in two families with achalasia, alacrima, myopathy and further symptoms. Effect and pathogenicity of the mutation were verified by cell biological studies. We identified a homozygous splice mutation in TRAPPC11 (c.1893+3A>G, [NM_021942.5], g.4:184,607,904A>G [hg19]) in four patients from two unrelated families leading to incomplete exon skipping and reduction in full-length mRNA levels. TRAPPC11 encodes for trafficking protein particle complex subunit 11 (TRAPPC11), a protein of the transport protein particle (TRAPP) complex. Western blot analysis revealed a dramatic decrease in full-length TRAPPC11 protein levels and hypoglycosylation of LAMP1. Trafficking experiments in patient fibroblasts revealed a delayed arrival of marker proteins in the Golgi and a delay in their release from the Golgi to the plasma membrane. Mutations in TRAPPC11 have previously been described to cause limb-girdle muscular dystrophy type 2S (MIM #615356). Indeed, muscle histology of our patients also revealed mild dystrophic changes. Immunohistochemically, β-sarcoglycan was absent from focal patches. The identified novel TRAPPC11 mutation represents an expansion of the myopathy phenotype described before and is characterised particularly by achalasia, alacrima, neurological and muscular phenotypes.
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Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We termed the pathological UPR that causes fatty liver disease a "stressed UPR". Here, we investigated the mechanism of stressed UPR... more
Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We termed the pathological UPR that causes fatty liver disease a "stressed UPR". Here, we investigated the mechanism of stressed UPR activation in zebrafish bearing mutation in the trappc11 gene that encodes a component of the transport protein particle (TRAPP) complex. trappc11 mutants are characterized by secretory pathway defects, reflecting disruption of the TRAPP complex. Additionally, we uncovered a defect in protein glycosylation in trappc11 mutants which was associated with reduced levels of lipid linked oligosaccharides (LLO) and compensatory upregulation of genes in the terpenoid biosynthetic pathway which produces the LLO anchor, dolichol. Treating wild-type larvae with terpenoid or LLO synthesis inhibitors phenocopied the stressed UPR seen in trappc11 mutants and was synthetically lethal with trappc11 mutation. We propose reduced LLO levels causing hypoglycosylation is a m...
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Transport protein particle (TRAPP) is a multiprotein complex involved in endoplasmic reticulum-to-Golgi trafficking. Zebrafish with a mutation in the TRAPPC11 orthologue showed hepatomegaly with steatosis and defects in visual system... more
Transport protein particle (TRAPP) is a multiprotein complex involved in endoplasmic reticulum-to-Golgi trafficking. Zebrafish with a mutation in the TRAPPC11 orthologue showed hepatomegaly with steatosis and defects in visual system development. In humans, TRAPPC11 mutations have been reported in only three families showing limb-girdle muscular dystrophy (LGMD) or myopathy with movement disorders and intellectual disability. We screened muscular dystrophy genes using next-generation sequencing and performed associated molecular and biochemical analyses in a patient with fatty liver and cataract in addition to infantile-onset muscle weakness. We identified the first Asian patient with TRAPPC11 mutations. Muscle pathology demonstrated typical dystrophic changes and liver biopsy revealed steatosis. The patient carried compound heterozygous mutations of a previously reported missense and a novel splice-site mutation. The splice-site change produced two aberrantly-spliced transcripts th...
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Secretion of proteins from eucaryotic cells involves the vesicular transport of proteins between a series of compartments. These compartments differ in their lipid and membrane protein composition and thus have distinct identities.... more
Secretion of proteins from eucaryotic cells involves the vesicular transport of proteins between a series of compartments. These compartments differ in their lipid and membrane protein composition and thus have distinct identities. signals, which determine the ...
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The TRAPP subunit TrappC12/TTC15, here renamed TRAMM, plays a role in the regulation of kinetochore stability and CENP-E recruitment during mitosis.
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Since the late 1990s, a number of multisubunit tethering complexes (MTCs) have been described that function in membrane trafficking events: TRAPP I, TRAPP II, TRAPP III, COG, HOPS, CORVET, Dsl1, GARP and exocyst. On the basis of... more
Since the late 1990s, a number of multisubunit tethering complexes (MTCs) have been described that function in membrane trafficking events: TRAPP I, TRAPP II, TRAPP III, COG, HOPS, CORVET, Dsl1, GARP and exocyst. On the basis of structural and sequence similarities, they have been categorized as complexes associated with tethering containing helical rods (CATCHR) (Dsl1, COG, GARP and exocyst) or non-CATCHR (TRAPP I, II and III, HOPS and CORVET) complexes (Yu IM, Hughson FM. Tethering factors as organizers of intracellular vesicular traffic. Annu Rev Cell Dev Biol 2010;26:137-156). Both acronyms (CATCHR and MTC) imply these complexes tether opposing membranes to facilitate fusion. The main question we will address is: have these complexes been formally demonstrated to function as tethers? If the answer is no, then is it premature or even correct to refer to them as tethers? In this commentary, we will argue that the vast majority of MTCs have not been demonstrated to act as a tether....
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Transport protein particle (TRAPP) represents a series of related protein complexes that function in specific stages of inter-organelle traffic. They share a core of subunits that can activate the GTPase Rab1 through a guanine nucleotide... more
Transport protein particle (TRAPP) represents a series of related protein complexes that function in specific stages of inter-organelle traffic. They share a core of subunits that can activate the GTPase Rab1 through a guanine nucleotide exchange factor (GEF) activity and are distinguished by 'accessory' subunits giving each complex its distinct function. The subunits are ubiquitously expressed and, thus, mutations in TRAPP subunits would be expected to be embryonic lethal. However, since its discovery, a number of subunits have been found to be mutated in several diverse human disorders suggesting that some of these subunits may have cell- or tissue-specific functions. Here we review the current state of knowledge with respect to TRAPP subunit mutations in human disease. We suggest ideas to explain their tissue-specific phenotypes and present avenues for future investigation.
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Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two... more
Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane...
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Treatment of rat dorsal root ganglion cultures with 1 microM okadaic acid leads to a fragmentation of neurofilaments and a reduction in the electrophoretic mobilities of the three subunits on SDS-polyacrylamide gels (Sacher, M. G.,... more
Treatment of rat dorsal root ganglion cultures with 1 microM okadaic acid leads to a fragmentation of neurofilaments and a reduction in the electrophoretic mobilities of the three subunits on SDS-polyacrylamide gels (Sacher, M. G., Athlan, E. S., and Mushynski, W. E. (1992) Biochem. Biophys. Res. Commun. 186, 524-530). Based on the observed response to varying concentrations of okadaic acid, fragmentation was inferred to be due to inhibition of protein phosphatase-2A activity and reduction in electrophoretic mobility to inhibition of protein phosphatase-1. Okadaic acid treatment led to an increase in amino-terminal, relative to carboxyl-terminal, domain phosphorylation in the low molecular weight (NF-L) subunit in the Triton X-100-soluble and -insoluble fractions. The purified catalytic subunit of protein phosphatase-2A dephosphorylated 32P-labeled NF-L and the middle molecular weight subunit from okadaic acid-treated cultures, whereas the catalytic subunit of protein phosphatase-1 ...
Research Interests: Biological Chemistry, Cytoskeleton, Western blotting, Biological Sciences, Animals, and 13 morePhosphorylation, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Neurons, Immunoprecipitation, Protein Phosphatase 2A, CHEMICAL SCIENCES, Rats, Rat, Gel electrophoresis, Subunit, Okadaic acid, Neurofilament proteins, and Medical and Health Sciences
ABSTRACT This chapter discusses the purification of TRAPP from Saccharomyces cerevisiae and identification of its mammalian counterpart. Primarily construction of Bet3p-protein A fusion used to purify TRAPP is discussed. TRAPP is... more
ABSTRACT This chapter discusses the purification of TRAPP from Saccharomyces cerevisiae and identification of its mammalian counterpart. Primarily construction of Bet3p-protein A fusion used to purify TRAPP is discussed. TRAPP is initially purified using a strain in which the sole copy of Bet3p is tagged at its carboxy terminus with three c-myc epitopes. The Bet3p-associated proteins are then purified from this strain by precipitating the complex from a lysate. This is done by incubating a clarified detergent extract with affinity purified anti-c-myc antibody bound to Affi-Gel. The bound protein is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This affinity purification led to the identity of several TRAPP subunits. In addition, large-scale purification of TRAPP and identification of TRAPP subunits, small-scale precipitation of TRAPP, and identification of mammalian TRAPP from HeLa lysates are described.