Import Pathways of Precursor Proteins into Mitochondria: Multiple Receptor Sites Are Followed by a Common Membrane Insertion Site

The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bctcomplex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit 13 of the F,-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments. N 'UCLEAR-coded mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria (for review see Pfanner and Neupert, 1987a; Nicholson and Neupert, 1988). Most precursors contain positively charged peptide extensions (presequences) at their amino terminus. The presequences and the amino-terminal portions of uncleaved precursors have been shown to contain information for targeting to mitochondria (for review see Hurt and van Loon, 1986). In addition, it was recently demonstrated that other portions of precursor proteins can carry specific import information (Pfanner et al., 1987b,c). Nucleoside triphosphates are required for translocation-competent folding ("unfolding") of the precursor proteins in the cytosol (for review see Eilers and Schatz, 1988; Pfanner and Neupert, 1988). The precursors interact with proteins of the mitochondrial outer membrane that are proposed to perform the function of import receptors. The precursors are then translocated into or across the mitochondrial membranes. In most cases, import occurs at contact sites between outer and inner membranes (Schleyer and Neupert, 1985; Hartl et al., 1986, Pfanner and Neupert, 1987b; Pfanner et al., 1987a,d; Schwaiger et al., 1987). Transfer into and across the inner membrane requires the electrical potential (A"P) across the inner membrane (Pfanner and Neupert, 1985). The presequences are proteolytically cleaved by the processing peptidase of the mitochondrial matrix (B6hni et al., 1980, 1983; Conboy et al., 1982; McAda and Douglas, 1982; Miura et al., 1982; Zwizinski and Neupert, 1983; Schmidt et al., 1984; Hawlitschek et al., 1988). Several precursors destined for the intermembrane space or the outside of the inner membrane are retranslocated from the matrix back across the inner membrane (Hartl et al., 1986, 1987). The translocation of cytochrome c into the intermembrane space differs in several respects from the general import mechanism as it does not involve the action of the membrane potential or proteolytic cleavage (Zimmermann et al., 1981). The following observations suggested that proteinaceous binding sites in the outer membrane are involved in protein import into mitochondria. (a) Pretreatment of isolated mitochondria with proteases inhibited subsequent import of precursor proteins (Gasser et al., 1982; Argan et al., 1983; Hennig et al., 1983; Riezman et al., 1983; Zwizinski et al., 1984; Schmidt et al., 1985; Hartl et al., 1986; Kleene et al., 1987; Ohba and Schatz, 1987a,b; Pfaller and Neupert, 1987; Pfanner and Neupert, 1987b; Pfanner et al., 1987b,c; Schwaiger et al., 1987). (b) Precursor proteins could be trapped at the level of binding to mitochondria by either lowering the temperature of the import reaction in the case of the outer membrane protein porin (Kleene et al., 1987; Pfaller and Neupert, 1987); dissipation of the membrane potential in the case of the inner membrane proteins ADP/ATP carrier (AAC), ~ subunit 9 of the F0-ATPase (F09), and the intermembrane space protein cytochrome b2 (Zwizinski et al., 1983; Riezman et al., 1983; Pfanner and Neupert, 1985, 1987b; Schmidt et al., 1985; Pfanner et al., 1987b, c,d); or, in the case of the intermembrane space protein cytochrome c, inhibition of cytochrome c heme lyase Nikolaus Pfanner's present address is Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544. 1. Abbreviations used in this paper: AAC, ADP/ATP carrier; Ft~, F r ATPase subunit 13; F09, F0-ATPase subunit 9; GIP, general insertion protein; TMPD, N,N,N',N'-tetramethylphenylendiamine; ws-porin, watersoluble porin. © The Rockefeller University Press, 0021-9525/88/12/2483/8 $2.00 The Journal of Cell Biology, Volume 107, (No. 6, Pt. 2) Dec. 1988 2483-249