Masatoshi Esaki

Tim50 Is a Subunit of the TIM23 Complex that Links Protein Translocation across the Outer and Inner Mitochondrial Membranes

Based on the results of site-specific photocrosslinking of translocation intermediates, we have identified Tim50, a component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex

Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles

Nearly all mitochondrial proteins are synthesized in the cytosol and subsequently imported into mitochondria with the aid of translocators: the TOM complex in the outer membrane, and the TIM23 and TIM22 complexes in the inner membrane. The TOM complex and the TIM complexes cooperate to achieve efficient transport of proteins to the matrix or into the inner membrane and several components, including Tom22, Tim23, Tim50 and small Tim proteins, mediate functional coupling of the two translocator systems. The TOM complex can be disconnected from the TIM systems and their energy sources (ATP andΔΨ ), however, using alternative mechanisms to achieve vectorial protein translocation across the outer membrane

Sec16 is a Determinant of Transitional ER Organization

BACKGROUND Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER sites and is, therefore, an ideal system for studying tER organization. RESULTS We show that the integrity of tER sites in P. pastoris requires the peripheral membrane protein Sec16. P. pastoris Sec16 is an order of magnitude less abundant than a COPII-coat protein at tER sites and seems to show a saturable association with these sites. A temperature-sensitive mutation in Sec16 causes tER fragmentation at elevated temperature. This effect is specific because when COPII assembly is inhibited with a dominant-negative form of the Sar1 GTPase, tER sites remain intact. The tER fragmentation in the sec16 mutant is accompanied by disruption of Golgi stacks. CONCLUSIONS Our data suggest that Sec16 helps to organize patches of COPII-coat proteins into clusters that represent tER sites. The Golgi disruption that occurs in the sec16 mutant provides evidence that Golgi structure in budding yeasts depends on tER organization.

Tom40 protein import channel binds to non-native proteins and prevents their aggregation.

Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel. Here we report the role of Tom40 in protein translocation across the membrane. The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40. Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate. A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria. These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation.

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.

Sec16 influences transitional ER sites by regulating rather than organizing COPII

It has been proposed that during the budding of COPII vesicles from transitional ER (tER) sites, Sec16 plays two distinct roles: negatively regulating COPII turnover and organizing COPII assembly. New data suggest that Sec16 does not in fact organize COPII and that regulation of COPII turnover can explain the influence of Sec16 on tER sites.

The budding yeast Pichia pastoris has a novel Sec23p homolog

In Pichia pastoris, coat protein complex II (COPII) vesicles form at discrete transitional ER (tER) sites. Analyzing COPII coat proteins in this yeast will help to reveal the mechanisms of tER organization. Here, we show that like Saccharomyces cerevisiae, P. pastoris contains essential SEC23 and SEC24 genes, as well as the non‐essential SEC24 homolog LST1. In addition, P. pastoris contains a novel non‐essential SEC23 homolog that we have designated SHL23. The products of all four genes are concentrated at tER sites. Deletion of SHL23 does not disrupt tER morphology. As judged by two‐hybrid analysis, Sec23p associates with both Sec24p and Lst1p, whereas Shl23p associates selectively with Lst1p. These results suggest that P. pastoris COPII vesicles contain an Shl23p/Lst1p complex that is absent in S. cerevisiae.

The phosphate carrier has an ability to be sorted to either the TIM22 pathway or the TIM23 pathway for its import into yeast mitochondria

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 (translocase of the mitochondrial outer membrane 40) complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments. Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1–TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane. Here we analyzed an in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase. The fusion protein without TM1 and TM2 was translocated across the outer membrane but was not inserted into the inner membrane. The fusion proteins without TM1–TM4 were not inserted into the inner membrane but instead translocated across the inner membrane. Functional defects of Tim50 of the TIM23 complex caused either by depletion of the protein or the addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1–TM4 across the inner membrane, suggesting that lack of TM1–TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway. PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence.

The Tom40 assembly process probed using the attachment of different intramitochondrial sorting signals

The β-barrel protein Tom40 functions as a protein-conducting channel in the mitochondrial outer membrane. By attaching mitochondrial presequences for various mitochondrial destinations to Tom40, it is possible to follow its sorting process. The results provide insight into the mechanism for the precise delivery of β-barrel proteins to the outer membrane.