No abstract

Mitochondria consist of four compartments-outer membrane, intermembrane space, inner membrane, and matrix-with crucial but distinct functions for numerous cellular processes. A comprehensive characterization of the proteome of an individual mitochondrial compartment has not been reported so far. We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. We obtained a coverage of ϳ85% based on the known outer membrane proteins. The proteome represents a rich source for the analysis of new functions of the outer membrane, including the yeast homologue (Hfd1/Ymr110c) of the human protein causing Sjö gren-Larsson syndrome. Surprisingly, a subclass of proteins known to reside in internal mitochondrial compartments were found in the outer membrane proteome. These seemingly mislocalized proteins included most top scorers of a recent genome-wide analysis for mRNAs that were targeted to mitochondria and coded for proteins of prokaryotic origin. Together with the enrichment of the precursor form of a matrix protein in the outer membrane, we conclude that the mitochondrial outer membrane not only contains resident proteins but also accumulates a conserved subclass of preproteins destined for internal mitochondrial compartments.

Mitochondrial protein import is thought to involve the sequential interaction of preproteins with binding sites on cis and trans sides of the membranes. For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) and then are translocated through a general import pore, in a process proposed to involve binding to a trans site on the intermembrane space (IMS) side. Controversial results have been reported for the role of the IMS domain of the essential outer membrane protein Tom22 in formation of the trans site. We show with different mutant mitochondria that a lack of the IMS domain only moderately reduces the direct import of preproteins with N-terminal targeting sequences. The dependence of import on the IMS domain of Tom22 is significantly enhanced by removing the cytosolic domains of import receptors or by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in the absence of a membrane potential (⌬) and subsequent import after reestablishment of a ⌬. After the removal of cytosolic receptor domains, two-step import of a cleavable preprotein strictly requires the IMS domain. In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22. We conclude that the negatively charged IMS domain of Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences, in agreement with the acid chain hypothesis of mitochondrial protein import.More than 98% of mitochondrial proteins are synthesized as precursors on cytosolic polysomes, are targeted to the mitochondrial surface, and are translocated into or across the mitochondrial outer and inner membranes (20,29,38). The majority of preproteins contain positively charged N-terminal signal sequences, termed presequences, that are removed after import (cleavable preproteins). Other preproteins contain internal targeting sequences that remain part of the mature protein. In the past, numerous components of the mitochondrial protein import machinery have been identified, yet the molecular mechanism of translocation of preproteins is poorly understood. A widely discussed model is that protein import is mediated by the sequential interaction of preproteins with binding sites on the cis and trans sides of the membranes (3,16,25,31,32,40). A molecular identification of cis and trans binding sites is thus of crucial importance for an understanding of the translocation process.A multisubunit protein complex, the translocase of the outer mitochondrial membrane (Tom), contains the receptor proteins Tom70-Tom37 and Tom20-Tom22 that recognize preproteins at the cytosolic (cis) side of the membrane (for reviews, see references 20, 29, 37, and 38). The preproteins are translocated through a general import pore and are thought to interact with a trans site located on the intermembrane space (IMS) side of the outer membrane; a candidate is the IMS domain of Tom22 (3,16,24,25). Subsequently, the preproteins are transferred to th...

The Mim1 complex imports α-helical mitochondrial outer membrane proteins with multiple transmembrane segments.