Two Intermembrane Space Tim Complexes Interact with Different Domains of Tim23p during Its Import into Mitochondria

Abstract

Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p–Tim13p and Tim9p–Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p–Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH2-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p–Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p–Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

Juxtaposition of the Two Distal CX3C Motifs via Intrachain Disulfide Bonding Is Essential for the Folding of Tim10

“…This involves not only the TOM complex in the outer membrane (7,11) but the insertion-specific TIM22 complex in the inner membrane (8,(12)(13)(14)(15). Quite uniquely, the carrier import pathway also depends on the function of two soluble 70-kDa complexes made of Tim9 and Tim10 (the TIM10 complex) (16 -20) or Tim8 and Tim13 (21,22). The TIM10 complex appears to be a major player because it is in large excess over the Tim8-Tim13 complex, and both Tim9 and Tim10 genes are essential in yeast, whereas Tim8 and Tim13 are dispensable (18,(22)(23)(24).…”

Protein Import Into Mitochondria

“…The import signals of the carrier proteins and of the other substrates are not clearly identi ed (76,81,137,(139)(140)(141)(142). It was shown, that the bound zinc ions of the small Tim proteins are essential for the import of the carrier proteins, but the role of the zinc ngers is not clear (120).…”

“…It was shown, that the bound zinc ions of the small Tim proteins are essential for the import of the carrier proteins, but the role of the zinc ngers is not clear (120). It was supposed that the TIM9/10 complex binds to hydrophobi c sequences with only a slight sequence preference (122,131,142). It is not known, whether the transmembrane domains of the carrier precursors insert sequentially or en bloc into the inner membrane.…”

“…It interacts in a metal-dependent manner with Tim23 precursors, which are partially translocated across the outer membrane, yet it does not bind to fully imported or assembled Tim23. Tim8 and Tim13 bind to a part of the intermediate domain of Tim23 (amino acid residues 30-90) and facilitate unidirectional translocation across the outer membrane (81,142). Tim8/13 interaction maintains hydrophobi c Tim23 precursors in a translocation competent conformation, which ensures their membrane potentialdependent insertion into the inner membrane mediated by the TIM22 complex (Fig.…”

Mitochondrial ATP‐independent chaperones

“…In the case of Tim23, the two chaperones are bound to different parts of the precursor during import, showing a difference in substrate specificity between the two chaperones. TIM9.10 binds mainly to the hydrophobic C-terminal part, whereas TIM8.13 binds to the more hydrophilic N-terminal part (28,53,54) (Fig. 2B).…”