Toshiya Endo

The TOM40 complex is a protein translocator in the mitochondrial outer membrane and consists of several different subunits. Among them, Tom40 is a central subunit that constitutes a protein-conducting channel by forming a β-barrel structure. To probe the nature of the assembly process of Tom40 in the outer membrane, we attached various mitochondrial presequences to Tom40 that possess sorting information for the intermembrane space (IMS), inner membrane, and matrix and would compete with the inherent Tom40 assembly process. We analyzed the mitochondrial import of those fusion proteins in vitro. Tom40 crossed the outer membrane and/or inner membrane even in the presence of various sorting signals. N-terminal anchorage of the attached presequence to the inner membrane did not prevent Tom40 from associating with the TOB/SAM complex, although it impaired its efficient release from the TOB complex in vitro but not in vivo. The IMS or matrix-targeting presequence attached to Tom40 was effe...

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. ThepH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidicpH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for He-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.

Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway. Amino acids with benzophenone side chains of various lengths [DL-2-amino-3-(p-benzoylphenyl)propanoic acid (1) and DL-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)] were incorporated at specific sites throughout the cytochrome b2-dihydrofolate reductase fusion proteins, pb2(220)-DHFR and pb2 delta 19(220)-DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively. In vitro import of pb2(220)-DHFR and pb2 delta 19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted pb2 delta 19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels. On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb2(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of pb2(220)-DHFR, but not pb2 delta 19(220)-DHFR, was photocrosslinked to an as-yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.

Mitochondria are two-membrane bounded organelles consisting of 1000-2000 different proteins, most of which are synthesized in the cytosol and subsequently imported into mitochondria. The imported proteins are further sorted to one of the four compartments, the outer membrane, intermembrane space, inner membrane, and matrix, mostly following one of the five major pathways. Mitochondrial protein import and sorting are mediated by the translocator complexes in the membranes and chaperones in the aqueous compartments operating along the import pathways. Here, we summarize the expanding knowledge on the roles of translocators, chaperones, and related components in the multiple pathways for mitochondrial protein trafficking.