The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44

Abstract

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70–Tim44 driving system. By blue native electrophoresis, we identify an ∼90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence‐containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

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“…The integral membrane proteins Tim17 and Tim23, which are homologous to one another, are both essential for cell viability 13,14,[17][18][19] . They are present in equimolar amounts and stably associated in the 90 kDa core complex of the translocase 12,20 , compatible with the hypothesis that these two proteins together form the import channel. However, this model does not fit the observation that yeast mitochondria containing overexpressed Tim23 import significantly more matrix-targeted preprotein than do control mitochondria 21 .…”

“…Sample buffer (5 µl) was added (5% (w/v) Coomassie brilliant blue G-250, 100 mM Bis-Tris, pH 7.0 and 500 mM 6-aminocaproic acid) and the samples were then separated on 6-16.5% polyacrylamide gradient gels at 4 °C (ref. 20).…”

“…To assess whether Tim23 could form a channel in the mitochondrial inner membrane in the absence of a stable association with Tim17, we used a mutant form, Tim23-2, with a single amino acid substitution in the membrane domain (G112E) that destabilizes the 90 kDa TIM23 complex 20 . Upon lysis of tim23-2 mitochondria with digitonin and separation by blue native polyacrylamide electrophoresis (BN-PAGE), Tim23 and Tim17 were lost from the 90 kDa complex (Fig.…”

“…The tim23-2 mitochondria specifically processed and imported the protein in a membrane potentialdependent manner (Fig. 5a, lanes [16][17][18][19][20] with an efficiency of 50-70% relative to wild type mitochondria. Similar import efficiencies were seen with purified inner membrane vesicles (not shown).…”

“…We conclude that Tim23 is responsible for forming the presequence-sensitive channel of the inner membrane. This conclusion, combined with the fact that the TIM channels are four-fold less abundant than the TOM channels and, thus, limiting for the import of presequence-containing preproteins 20 , explains the observation that overexpression of only Tim23 strongly stimulates the import of these preproteins into mitochondria 21 .…”