The Assembly Pathway of the Mitochondrial Carrier Translocase Involves Four Preprotein Translocases

Abstract

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.The majority of mitochondrial proteins are nucleus encoded and imported into mitochondria through protein translocase complexes (6,7,17,25,29,33,42). The translocase of the outer membrane (TOM complex) is the general entry gate for mitochondrial precursor proteins. Two translocases of the inner membrane (TIM), the presequence translocase (TIM23 complex) and the twin-pore carrier translocase (TIM22 complex), mediate signal-selective transport of precursor proteins. While the TIM23 complex translocates the majority of substrates into the matrix and inserts only a limited number of substrates into the inner membrane (7,10,13,17,25,26,40), the TIM22 complex is dedicated to the insertion of multispanning hydrophobic proteins into the inner membrane, including a large number of metabolite carriers (7,17,25,27,29,46). The TIM22 complex is a voltage-dependent 300-kDa complex with three membrane-integral subunits, Tim18, Tim22, and Tim54. Tim22 forms the voltage-sensitive channels of the twin-pore translocase (21, 30). Tim54 was shown to play a role in the assembly of a protease complex (Yme1) of the inner membrane, yet the molecular mechanism of its action has not been elucidated (12). Thus, the molecular functions of Tim54 and Tim18 in the TIM22 complex are unknown (6,7,17,25,29).The precursors of metabolite carriers are not directly transferred from the TOM complex to the TIM22 complex, but the TIM10 translocase complex of the intermembrane space binds to the precursors and functions in a chaperone-like manner to guide them through the aqueous space between outer and inner membranes. ...

Intramolecular Disulfide Bond of Tim22 Protein Maintains Integrity of the TIM22 Complex in the Mitochondrial Inner Membrane

“…We synthesized radiolabeled wild-type Tim22 and its Cys 3 Ser mutants (C42S, C141S, and C42/141S) in vitro and imported them into mitochondria isolated from wild-type cells. As reported previously, imported wild-type Tim22 formed a Tim22 dimer and was subsequently assembled into the 300-kDa TIM22 complex in a manner dependent on the membrane potential across the IM (⌬⌿) (23) (Fig. 2A).…”

“…2B), probably due to rapid consumption of the Tim22 dimer intermediate for uptake by the TIM22 complex (23). These results suggest that the lack of the disulfide bond in Tim22 alters the quaternary structures of the TIM22 complex in such a way that newly imported wild-type Tim22 can exchange with pre-existing mutant Tim22 much faster than in wild-type mitochondria.…”

“…has accumulated that a number of proteins form disulfide bond(s) in the mitochondrial IMS, usually with the aid of the Erv1-Tim40 disulfide bond relay system (23)(24)(25)(26)(27). Tim22 is a central channel-forming component of the TIM22 complex, the insertion machinery for polytopic IM proteins.…”

“…Tim18 and Sdh3, which are homologous with each other, are involved in the assembly of the TIM22 complex on their own (19). Tim54 appears to be an adaptor protein that bridges the TIM22 core complex and small Tim proteins through direct interactions with Tim10 (22,23).…”

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

“…Yeast mitochondria purified from the parental strain YPH499 or from YPH Tim23-76ts that carry a thermosensitive allele of Tim23 unable to shuttle cargos into the mitochondrial matrix 54 were as efficiently able to import both GB (Figures 7a and b) and GA (Figures 7c and d). Unexpectedly, mitochondria from YPH-Tim22-14ts strain that carry a thermosensitive allele of Tim22 incapable of the membrane insertion of the cargos 55 were significantly reduced in their ability to import both GB (Figures 7a and b) and GA (Figures 7c and d).…”