The small Tim proteins in the mitochondrial intermembrane space participate in the TIM22 import pathway for assembly of the inner membrane. Assembly of the small TIM complexes requires the conserved "twin CX 3 C" motif that forms juxtapositional intramolecular disulfide bonds. Here we identify a new intermembrane space protein, Hot13p, as the first component of a pathway that mediates assembly of the small TIM complexes. The small Tim proteins require Hot13p for assembly into a 70-kDa complex in the intermembrane space. Once assembled the small TIM complexes escort hydrophobic inner membrane proteins en route to the TIM22 complex. The mechanism by which the small Tim proteins bind and release substrate is not understood, and we investigated the affect of oxidant/reductant treatment on the TIM22 import pathway. With in organello import studies, oxidizing agents arrest the ADP/ATP carrier (AAC) bound to the Tim9p-Tim10p complex in the intermembrane space; this productive intermediate can be chased into the inner membrane upon subsequent treatment with reductant. Moreover, AAC import is markedly decreased by oxidant treatment in ⌬hot13 mitochondria and improved when Hot13p is overexpressed, suggesting Hot13p may function to remodel the small TIM complexes during import. Together these results suggest that the small TIM complexes have a specialized assembly pathway in the intermembrane space and that the local redox state of the TIM complexes may mediate translocation of inner membrane proteins.Mitochondrial inner membrane biogenesis is an essential process in all eukaryotes. The Translocase of the Inner Membrane (TIM) 1 22 complex is dedicated to the insertion of nuclear-coded polytopic inner membrane proteins, including the mitochondrial carrier family and import components Tim22p and Tim23p (1-3). The 300-kDa membrane complex consists of a pore-forming subunit Tim22p, in addition to Tim18p and Tim54p, that plays an unknown function and a fraction of the small Tim proteins Tim9p, Tim10p, and Tim12p (1-3). Substrates are escorted from the Translocase of the Outer Membrane (TOM) complex to the TIM22 translocon by two distinct 70-kDa chaperone-like complexes in the intermembrane space: Tim9p-Tim10p and Tim8p-Tim13p (4 -7). Insertion into the inner membrane requires a membrane potential (⌬⌿), but the events by which a substrate is transported from the TOM complex to the inner membrane complex remain obscure. The carrier proteins and Tim23p are targeted to the Tom70p receptor on the outer membrane (7, 8) and then pass through the TOM complex as a loop (4,5,8). The small Tim proteins subsequently bind to hydrophobic domains in the substrates and chaperone them to the 300-kDa insertion complex (4, 5, 9). The substrate is transferred to the insertion complex and inserted into the inner membrane (10). Cryoelectron microscopy and reconstitution studies have shown that Tim22p forms a twin-pore translocase that is voltagegated (10, 11).The small Tim proteins contain the "twin CX 3 C" motif that is required for assembly of the...
Tim54p, a component of the inner membrane TIM22 complex, does not directly mediate the import of inner membrane substrates but is required for assembly/stability of the 300-kD TIM22 complex. In addition, Δtim54 yeast exhibit a petite-negative phenotype (also observed in yeast harboring mutations in the F1Fo ATPase, the ADP/ATP carrier, mitochondrial morphology components, or the i–AAA protease, Yme1p). Interestingly, other import mutants in our strain background are not petite-negative. We report that Tim54p is not involved in maintenance of mitochondrial DNA or mitochondrial morphology. Rather, Tim54p mediates assembly of an active Yme1p complex, after Yme1p is imported via the TIM23 pathway. Defective Yme1p assembly is likely the major contributing factor for the petite-negativity in strains lacking functional Tim54p. Thus, Tim54p has two independent functions: scaffolding/stability for the TIM22 membrane complex and assembly of Yme1p into a proteolytically active complex. As such, Tim54p links protein import, assembly, and turnover pathways in the mitochondrion.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.