Protein translocation across or insertion into the inner membrane can be dissected into three distinct steps (). In the first step, the presequences of both matrix-targeted and inner membrane–sorted precursor proteins are transferred from the trans site of the TOM40 complex to Tim50 of the TIM23 complex (, 1 → 2). In the second step, the presequence enters the TIM23 channel in a ΔΨ-dependent manner (, 2 → 3). This step also requires initial trapping of the presequence by the ATP-dependent motor mtHsp70 if translocation of the presequence through the TIM23 channel requires unfolding of the tightly folded passenger domain outside the mitochondria. In the third step, translocation of matrix-targeted proteins across the inner membrane further requires the motor function of mtHsp70 (and MMC proteins), which undergoes an ATP-dependent reaction cycle of holding on and off the incoming unfolded segment of precursor polypeptides at the outlet of the TIM23 channel (, 3 → 4-1), whereas insertion of inner membrane–sorted proteins into the inner membrane is independent of the function of mtHsp70 (, 3 → 4-2).
A model of protein translocation across and insertion into the inner membrane via the TIM23 complex. Note that the interactions among some of the components are dynamic rather than static.
Here we found that the N-terminal region of Tim23 and the IMS domain of Tim50 directly interact with Tom22 in the absence of substrate precursor proteins. The Tim23–Tom22 and Tim50–Tom22 interactions may lead to efficient coupling of protein translocation through the TOM40 complex and TIM23 complex. Although a similar function was suggested for Tim21 as well, interactions between Tim21 and Tom22 were found only after solubilization of mitochondrial membranes (Chacinska et al., 2005
; Mokranjac et al., 2005
) or only for high concentrations of purified recombinant proteins (Albrecht et al., 2006
), but not in intact mitochondria (Chacinska et al., 2005
; Mokranjac et al., 2005
). We found here that the N-terminal interaction of Tim23 with the outer membrane () or the contact between Tom22 and Tim23 () was not altered by the absence of Tim21 and that depletion of Tim21 did not show synthetic growth defects with the tim23
mutants (Fig. S4, D and E). Besides, the amount of the cross-linked product of the translocation intermediate at the TOM40 complex with Tim50 was not affected by depletion of Tim21 or by wash with 150 mM KCl (unpublished data), which abolishes interactions between Tim21 and Tom22 (Albrecht et al., 2006
). Therefore, Tim21 does not appear to play a primary role in linking the TOM40 and TIM23 complexes.
Defects in the Tim23–Tim50 interactions caused by mutations in the Tim23 IMS domain are in the order of L71S > L64S >> L78S and those in the Tim50 IMS domain in the order of L279,282,286S > A352S/F355S. The normal Tim23–Tim50 interactions in the IMS appear to be important for the first step of the protein translocation because they facilitate efficient transfer of translocating proteins from the TOM40 complex to the TIM23 complex through transient interactions between the presequence and Tim50 (). Defective import of not only matrix-targeted proteins but also inner membrane–sorted proteins were observed for mitochondria containing the L71S mutation, but not the L64S or L78S mutation (); this suggests that the efficient protein transfer from the TOM40 to the TIM23 complex are impaired only when Tim23–Tim50 interactions are significantly disrupted. Because defects in Tim23–Tim50 interactions lead to the increased interaction between Tom22 and Tim50 (), Tom22 and presequences may compete with each other in interactions with Tim50. Proper Tim23–Tim50 interactions may well optimize the Tim50–Tom22 interactions so that Tim50 can stay close to Tom22 yet the presequence can efficiently substitute Tom22 to occupy Tim50 as well (, 1 → 2).
It was unexpected that changes in the Tim23–Tim50 interactions in the IMS did not affect the Tim23 dimer formation, which was proposed to reflect the channel closure, but instead affected the third step of the translocation of matrix-targeted proteins across the inner membrane. Defective motor functions of mtHsp70 usually cause retarded import of not only matrix-targeted proteins but also the inner membrane–sorted protein such as pb2
(220)-DHFR, but not pb2
(167)-DHFR (Voos et al., 1993
). This is because pb2
(220)-DHFR, not pb2
(167)-DHFR, contains a tightly folded heme-binding domain (residues 81–181) downstream of the presequence (residues 1–80) and requires mtHsp70 for the second step or initial translocation of the N terminus of the presequence across the inner membrane (Glick et al., 1993
). However, the effects of mutations L64S and L78S on the import rates of pb2
(220)-DHFR and pb2
(167)-DHFR do not markedly differ (), suggesting that the Tim23–Tim50 interactions do not affect binding of mtHsp70 in the second step of the TIM23 complex-mediated translocation. Rather, the Tim23–Tim50 interactions increase efficiency of mtHsp70 in the reaction cycle of binding to and release from the translocating protein in the third step of the inner membrane translocation. In other words, the Tim23–Tim50 interactions in the IMS facilitate turnover of mtHsp70 at the outlet of the TIM23 channel in the matrix (, 4-1). This interpretation was supported by the observation that overexpression of mtHsp70, which would hamper the mtHsp70 turnover, negatively affects the cell growth of tim23
mutants (). It is to be noted that depletion of Tim50 leads to defects in the first and third steps of the TIM23 complex-mediated translocation (Geissler et al., 2002
) like the tim23
mutants with L64S or L78S mutation rather than L71S mutation. Besides, the tim17-5
(Chacinska et al., 2005
) and tim23-76
mutant (van der Laan et al., 2007
) were reported, which showed similar phenotypes, i.e., defects in only the third step of the TIM23 complex-mediated translocation (Chacinska et al., 2005
). However, the defects in the motor function of mtHsp70 observed here are not caused by the lack of MMC proteins (Tim14 and Tim16) in the TIM23 complex because tim23-64,71,78
mitochondria contain normal amounts of Tim14 and Tim16 (). Although we cannot rule out the possibility that the observed defects in motor functions are indirect consequences of the growth defects, a more likely explanation is that tertiary and/or quaternary structural changes of the TIM23 complex caused by defective Tim23–Tim50 interactions impaired the motor functions.
Chacinska et al. (2005)
proposed that the TIM23 complex is in the equilibrium between the matrix translocation complex containing mtHsp70 and MMC proteins, but not Tim21, and the inner membrane–sorting complex containing Tim21 but not mtHsp70 or MMC proteins. However, this model was recently challenged by the observations that Tim21 was coimmunoprecipitated with MMC proteins (Tamura et al., 2006
; Popov-Čeleketić et al., 2008
). The previous observation that Tim21 was mutually exclusive with MMC proteins as a constituent of the TIM23 complex (Chacinska et al., 2005
) may be explained by the findings that Tim21 and MMC proteins occupy only a fraction of the TIM23 core complex (Tim23 and Tim17) (unpublished data) and/or may be because of the artifact of using the protein A fusion protein of Tim21 (Popov-Čeleketić et al., 2008
). The present study instead suggests that the TIM23 complex is in the two distinct states with or without activation for the successive action of the import motor mtHsp70 (). These two states of the TIM23 complex, the TIM23 complexes for motor activation (TIM23A
) and for motor resting (TIM23R
), may contain mtHsp70 and MMC proteins, but differ in their abilities to allow mtHsp70 to perform multiple rounds of holding on and off the incoming unfolded segments of translocating proteins. Conversion of the TIM23R
complex to the TIM23A
complex is caused by the Tim23–Tim50 interactions in the IMS, likely responding to the presence of precursor proteins in transit through the Tim23 channel, and is facilitated by Pam17. In other words, the IMS domains of Tim23 and Tim50 could monitor the presence of substrate polypeptide chains delivered from the TOM40 complex and transmit this information to the matrix side of the TIM23 complex, perhaps through Pam17, for on-demand activation of the motor functions without unnecessary idling. Evidently, future studies need to reveal the structural basis of the difference and of a switching mechanism between the TIM23R