Transfer of proteins into mitochondria

The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway

Proteinaceous receptors for the import of mitochondrial precursor proteins

Mild trypsin treatment of isolated Neurospora mitochondria strongly inhibits their ability to bind and import the precursors of several mitochondrial proteins. Evidence is presented for two proteins, the ADP/ATP carrier and the mitochondrial porin, that specific binding of the precursors to the outer surface of the mitochondria is affected by the protease treatment. We suggest that the receptors that mediate the import of these two precursors are proteinaceous. Treatment of mitochondria with elastase also inhibits the binding and import of the ADP/ATP carrier and the porin. In contrast the import of the precursors of subunits 2 and 9 of the mitochondrial proton-translocating ATPase was unaffected by elastase treatment at the concentrations used. We suggest that the import pathways of the latter two proteins are distinct from those of the ADP/ATP carrier and the porin

Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences

Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease- mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed

Mitochondrial Protein Import

The role of nucleoside triphosphates (NTPs) in mitochondrial protein import was investigated with the precursors of N. crassa ADP/ATP carrier, F1-ATPase subunit β, F0-ATPase subunit 9, and fusion proteins between subunit 9 and mouse dihydrofolate reductase. NTPs were necessary for the initial interaction of precursors with the mitochondria and for the completion of translocation of precursors from the mitochondrial surface into the mitochondria. Higher levels of NTPs were required for the latter reactions as compared with the early stages of import. Import of precursors having identical presequences but different mature protein parts required different levels of NTPs. The sensitivity of precursors in reticulocyte lysate to proteases was decreased by removal of NTPs and increased by their readdition. We suggest that the hydrolysis of NTPs is involved in modulating the folding state of precursors in the cytosol, thereby conferring import competence

Mitochondrial precursor proteins are imported through a hydrophilic membrane environment

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit β(F1β) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1β translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites

Apocytochrome c

The cytochrome c import pathway differs markedly from the general route taken by the majority of other imported proteins, which is characterized by the import involvement of namely, surface receptors, the general insertion protein (GIP), contact sites and by the requirement of a membrane potential (Δψ). Unique features of both the cytochrome c precursor (apocytochrome c) and of the mechanism that transports it into mitochondria, have contributed to the evolution of a distinct import pathway that is not shared by any other mitochondrial protein analysed thus far. The cytochrome c pathway is particularly unique because i) apocytochrome c appears to have spontaneous membrane insertion-activity; ii) cytochrome c heme lyase seems to act as a specific binding site in lieu of a surface receptor and; iii) covalent heme addition and the associated refolding of the polypeptide appears to provide the free energy for the translocation of the cytochrome c polypeptide across the outer mitochondrial membrane

How finicky is mitochondrial protein import?

Recently it was reported that artificial targeting signals or signals specific for organelles other than mitochondria could direct proteins into mitochondria. Here we discuss findings which suggest that specific steps of mitochondrial protein import can be bypassed. Non-specific targeting signals appear to use this bypass pathway. Such import occurs at very low rates under physiological conditions and therefore does not affect the uniqueness of mitochondrial protein composition

Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase

The Fe/S protein of complex III is encoded by a nuclear gene, synthesized in the cytoplasm as a precursor with a 32 residue amino-terminal extension, and transported to the outer surface of the inner mitochondrial membrane. Our data suggest the following transport pathway. First, the precursor is translocated via translocation contact sites into the matrix. There, cleavage to an intermediate containing an eight residue extension occurs. The intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex III. We suggest that the folding and membrane-translocation pathway in the endosymbiotic ancestor of mitochondria has been conserved during evolution of eukaryotic cells; transfer of the gene for Fe/S protein to the nucleus has led to addition of the presequence, which routes the precursor back to its “ancestral” assembly pathway

A mitochondrial import receptor for the ADP/ATP carrier

We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complementary function to that of MOM19, which acts as an import receptor for the majority of mitochondrial proteins studied so far but not for the AAC. The import pathway of the precursor of MOM72 appears to involve MOM19 as receptor

Successive translocation into and out of the mitochondrial matrix

We investigated the import and sorting pathways of cytochrome b2 and cytochrome c1, which are functionally located in the intermembrane space of mitochondria. Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import. The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix. Processing by the matrix peptidase leads to intermediate-sized forms, which are subsequently redirected across the inner membrane. The second proteolytic processing occurs in the intermembrane space. We conclude that the hydrophobic stretches in the presequences of the intermediate-sized forms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space