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

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.

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 and ∆Ψ), however, using alternative mechanisms to achieve vectorial protein translocation across the outer membrane

Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import.

Many newly synthesized proteins have to become unfolded during translocation across biological membranes. We have analyzed the effects of various stabilization͞destabilization mutations in the Ig-like module of the muscle protein titin upon its import from the N terminus or C terminus into mitochondria. The effects of mutations on the import of the titin module from the C terminus correlate well with those on forced mechanical unfolding in atomicforce microscopy (AFM) measurements. On the other hand, as long as turnover of the mitochondrial Hsp70 system is not rate-limiting for the import, import of the titin module from the N terminus is sensitive to mutations in the N-terminal region but not the ones in the C-terminal region that affect resistance to global unfolding in AFM experiments. We propose that the mitochondrial-import system can catalyze precursor-unfolding by reducing the stability of unfolding intermediates. mitochondria ͉ titin ͉ Hsp70 ͉ mechanical stability A protein's function relies on its native folded conformation. However, normal cell functions also require transient unfolding of proteins in such processes as translocation across biological membranes and selective degradation by ATPdependent proteases. In both cases, polypeptide chains have to translocate through narrow channels, the diameters of which are too small to accept folded protein domains. Therefore machineries for protein translocation and degradation have activities to induce unfolding of substrate proteins when they are folded (1).The eukaryotic cell is subdivided into functionally distinct compartments or organelles that contain unique sets of proteins. Among them, mitochondria are essential organelles that are bounded by two membranes, the outer and inner membranes, and contain two aqueous compartments, the intermembrane space and the matrix. Most mitochondrial-matrix proteins and some inner mitochondrial-membrane proteins are synthesized in the cytosol as precursors with an N-terminal presequence that contains targeting information for mitochondria. They are translocated across the outer and inner membranes through the translocators in the two membranes, the TOM40 complex and TIM23 complex, respectively, with the aid of mitochondrial Hsp70 (mtHsp70) and mtHsp70-associated motor and chaperone proteins (2-5). Accumulated evidence suggests that precursor proteins that folded before import into mitochondria can be actively unfolded by the translocators to thread into their protein-conducting channels (6-9).In vitro mitochondrial import of precursor proteins containing presequences of varying lengths showed that the import rates are higher for precursor proteins with a long presequence (Ͼ70 residues) than for those with a short presequence (Ͻ70 residues) (8), indicating that these two distinct situations are to be considered for unfolding of presequence-containing precursor proteins upon import into mitochondria. Because presequences do not take ordered structures in solution, the N terminus of a long presequence can reach the matr...

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.

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.

Normal mitochondrial functions rely on optimized composition of their resident proteins, and proteins mistargeted to mitochondria need to be efficiently removed. Msp1, an AAA-ATPase in the mitochondrial outer membrane (OM), facilitates degradation of tailanchored (TA) proteins mistargeted to the OM, yet how Msp1 cooperates with other factors to conduct this process was unclear. Here, we show that Msp1 recognizes substrate TA proteins and facilitates their transfer to the endoplasmic reticulum (ER). Doa10 in the ER membrane then ubiquitinates them with Ubc6 and Ubc7. Ubiquitinated substrates are extracted from the ER membrane by another AAA-ATPase in the cytosol, Cdc48, with Ufd1 and Npl4 for proteasomal degradation in the cytosol. Thus, Msp1 functions as an extractase that mediates clearance of mistargeted TA proteins by facilitating their transfer to the ER for protein quality control.(D) prd5D cells expressing 3xFLAG-Pex15D30 under the control of the GAL1 promoter were grown in SCD at 30 C and then in SCGalSuc for 4 h at 30 C and treated with DMSO or 50 mM MG132 for 30 min at 30 C. Then, 100 mg/mL CHX was added, and the amount of 3xFLAG-Pex15D30 was analyzed as in (B). Data are representative of three independent experiments (n = 3), and values are expressed as mean ± SEM. (E) WT, atg8D, and atg32D cells expressing 3xFLAG-Pex15D30 under the control of the GAL1 promoter were grown in SCD at 30 C and then in SCGalSuc for 4 h at 30 C. Degradation of 3xFLAG-Pex15D30 was monitored by CHX chase experiments as in (B). Data are representative of three independent experiments (n = 3), and values are expressed as mean ± SEM.