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

Abstract

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

Role of Tim50 in the Transfer of Precursor Proteins from the Outer to the Inner Membrane of Mitochondria

“…The topology of Tim50 is also in agreement with such a function as it is integrated into the inner membrane with a single transmembrane domain and exposes a conserved domain into the IMS (Geissler et al, 2002;Yamamoto et al, 2002;Mokranjac et al, 2003a). Using a yeast two-hybrid screen, pulldown experiments and crosslinking the latter domain was shown to bind to the IMS domain of Tim23 (Geissler et al, 2002;Yamamoto et al, 2002;Alder et al, 2008b). The receptor function of Tim50 remained, however, poorly characterized.…”

“…Previous work suggested Tim23 to be the binding partner of Tim50 in the TIM23 complex (Geissler et al, 2002;Yamamoto et al, 2002;Mokranjac et al, 2003a,b;Alder et al, 2008b). It remained unclear, however, whether Tim23 is the only interaction partner of Tim50 within the complex.…”

“…A receptor function in the translocase has been ascribed mainly to Tim50 because it can be cross-linked to precursors of soluble matrix proteins that were only partially translocated through the TOM complex (Yamamoto et al, 2002;Mokranjac et al, 2003a). At this stage of translocation, no cross-links of translocating precursors to any other TIM23 subunit were observed so far.…”

Role of Tim21 in Mitochondrial Translocation Contact Sites

“…The preproteins are then transferred to the TIM23 complex, which can be divided into a tightly membraneintegrated part with hydrophilic domains in the intermembrane space and the import motor. Two components of the membrane-integrated part, Tim23 and Tim17, are believed to form the preprotein-conducting channel (5,11,12), whereas Tim50 appears to exert its function in the transfer of preproteins from the TOM to the TIM23 translocase (13)(14)(15). The N-terminal hydrophilic domain of Tim23 has a role in tethering the inner membrane translocase to the outer membrane bringing both membranes in close contact (16).…”

A cryptic matrix targeting signal of the yeast ADP/ATP carrier normally inserted by the TIM22 complex is recognized by the TIM23 machinery

“…The same TOM channel that also recognizes carrier proteins allows passage of presequence containing precursors through the OM. In the trans side of the OM, translocation is mediated by the IMS segment of Tom22 [18], Tim23 [19] and Tim50 [20][21][22]. Passage across the IM then occurs through the distinct TIM23 channel and is facilitated by the import motor containing Tim44, Tim14 and Hsp70 that trap precursors and target them to the matrix [23][24][25].…”