The TIM machinery of the
mitochondrial inner membrane is required for the import of mitochondrial
proteins across the inner membrane. In addition to the well-characterized Tim23p/Tim17p complex for mitochondrial precursors
carrying an N-terminal targeting presequence, a second TIM complex that
imports mitochondrial carriers into the inner membrane has been
identified. Components of this complex are the inner membrane proteins,
Tim22p and Tim54p, and intermembrane space proteins, Tim10p and Tim12p;
all are essential for viability. Conditional mutants of Tim10p and
Tim12p were generated to assess the roles of these proteins in the
import pathway. In vitro import of mitochondrial carriers and Tim22p in
tim10 and tim12 temperature-sensitive (ts) mutants is
defective; import of precursor proteins into the matrix and
intermembrane space occurs at wild-type rates, demonstrating the
selectivity of this second TIM complex. From biochemical analysis of
import intermediates, Tim10p is required to transport carrier precursors
across the outer membrane whereas Tim12p and Tim22p mediate their
insertion into the inner membrane. The identification of TIM22 as
a multicopy suppressor of the tim12 ts mutant suggests an
interaction between Tim12p and Tim22p. Indeed, both proteins co-purify
with a hexahistidine-tagged version of Tim10p from detergent-solubilized
mitochondria, indicating that at least these three proteins form a
complex in the intermembrane space. We presently are defining the
mechanism by which this complex mediates import of carrier proteins.
Both Tim10p and Tim12p may function as chaperone-like components for the
highly insoluble carrier proteins in the intermembrane space.
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