title: The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences.
abstract: 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 (delta psi) and subsequent import after reestablishment of a delta psi. 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.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Universität Freiburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Moczko
authors.forename: U
authors.initials: U
authors.lastname: Bömer
authors.forename: M
authors.initials: M
authors.lastname: Kübrich
authors.forename: N
authors.initials: N
authors.lastname: Zufall
authors.forename: A
authors.initials: A
authors.lastname: Hönlinger
authors.forename: N
authors.initials: N
authors.lastname: Pfanner
mesh_terms: D001692:Biological Transport; D002451:Cell Compartmentation; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D018892:Proton-Motive Force; D011956:Receptors, Cell Surface; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D029701:Saccharomyces cerevisiae Proteins; C090857:TOM22 protein, S cerevisiae; D013762:Tetrahydrofolate Dehydrogenase