Tim50, a novel component of the TIM23 preprotein translocase of mitochondria

Abstract

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be crosslinked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

“…Saturation of mouse liver mitochondria TOM and TIM23 complexes with cold SU9-DHFR (dihydrofolate reductase) and B2Δ19-DHFR inhibited in a dose-dependent manner the import of 35 S-Met-labeled SU9-DHFR 23,[32][33][34][35] used throughout this work as a positive control (Supplementary Figure 2A). This inhibition was even more pronounced when cold SU9-DHFR and B2Δ19-DHFR were stabilized with methotrexate (MTX) 23 (Supplementary Figure 2B). However, SU9-DHFR did not inhibit GB import, while B2Δ19 did so to a minor level at the highest dose (Supplementary Figure 2C); however, MTX stabilization completely prevented B2Δ19-DHFR inhibition of GB import (Supplementary Figure 2D).…”

“…The mitochondrial nucleoid encodes only for 13 structural proteins, while the rest of the mitochondrial proteome is nuclear-encoded and transported to the mitochondria through a sophisticated protein import machinery. [19][20][21][22][23][24][25][26][27][28] The translocase of the outer mitochondrial membrane (TOM), the entry gate to the mitochondria, 20 passes on the cargos to the sorting and assembly machinery (SAM) complex, to the mitochondrial intermembrane space assembly (MIA) complex and to the translocases of the inner mitochondrial membrane TIM22 or TIM23 complexes for delivery to the outer membrane, the intermembrane space, the inner membrane or the matrix, respectively. [19][20][21][22]25,26,28 Unexpectedly, we found that GB mitochondrial entry is independent of the TOM-TIM23 complexes, but requires instead the Sam50 and Tim22 channels and mitochondrial heat-shock protein 70 (mtHsp70).…”

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

“…At this stage of translocation, no cross-links of translocating precursors to any other TIM23 subunit were observed so far. 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 deletion strain of TIM50 in a diploid W303 background was described before (Mokranjac et al, 2003a). It was transformed with a pVT-U plasmid (Vernet et al, 1987) carrying a full-length wild-type Tim50 and subsequently sporulated to obtain a haploid yeast strain with a chromosomal deletion of TIM50 and a wild-type copy of Tim50 on the URA plasmid.…”

“…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.…”