Joachim Ostermann

Successive translocation into and out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a bipartite signal peptide

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.

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.

The processing peptidase of yeast mitochondria: the two co-operating components MPP and PEP are structurally related

Two proteins co‐operate in the proteolytic cleavage of mitochondrial precursor proteins: the mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP). In order to understand the structure and function of this novel peptidase, we have isolated mutants of Saccharomyces cerevisiae which were temperature sensitive in the processing of mitochondrial precursor proteins. Here we report on the mif2 mutation which is deficient in MPP. Mitochondria from the mif2 mutant were able to import precursor proteins, but not to cleave the presequences. The MPP gene was isolated. MPP is a hydrophilic protein consisting of 482 amino acids. Notably, MPP exhibits remarkable sequence similarity to PEP. We speculate that PEP and MPP have a common origin and have evolved into two components with different but mutually complementing functions in processing of precursor proteins.

Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix

We previously reported that hsp70 in the mitochondrial matrix (mt‐hsp70 = Ssc1p) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt‐hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt‐hsp70 for import, suggesting a general role of mt‐hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co‐precipitated with antibodies directed against mt‐hsp70. We conclude that mt‐hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.

Stress proteins and mitochondrial protein import

Many proteins have to cross membranes to get from their site of synthesis, usually the cytosol, to their functional destination in various intracellular compartments (the organelles) (Wickner and Lodish, 1985). Recent research is directed towards elucidating the molecular mechanisms of translocation of proteins across membranes that are naturally impermeable to macromolecules. We thereby have focused on the biogenesis of mitochondrial proteins. Mitochondria import the vast majority of their proteins from the cytosol (Attardi and Schatz, 1988; Hartl and Neupert, 1990; Pfanner and Neupert, 1990). Precursor proteins are recognized by receptors on the mitochondrial surface and are imported at sites of close contact between outer and inner membranes. In the inner mitochondrial subcompartment, the matrix, the amino-terminal signal sequences (presequences) of the precursors are proteolytically removed by the enzyme processing peptidase. The process of membrane translocation of mitochondrial precursor proteins is not really understood. We know that various energy sources, ATP in the cytosol and matrix and the electrical membrane potential across the inner membrane, are required. Moreover, precursor proteins are unfolded prior to translocation and are refolded in the matrix. Several stress proteins (heat shock proteins, hsps) seem to play critical roles with regard to translocation and modifying the conformation of precursor proteins.

Import of Cytochromes b2 and c1 into Mitochondria is Dependent on Both Membrane Potential and Nucleoside Triphosphates

Import of precursors of cytochromes b2 and c1 into mitochondria requires a mitochondrial membrane potential. We show here that in addition ΔΨ, nucleoside triphosphates (NTPs) are necessary for protein translocation. At low concentrations of NTPs, intermediate-sized cytochrome b2 was accumulated spanning the outer and inner membranes at contact sites. For complete translocation into mitochondria, higher concentrations of NTPs were necessary. We conclude that different levels of NTPs are required for distinct steps in the import pathway.

Import of Cytochrome c1 and Cytochrome b2 into Mitochondria

We have investigated the import pathways of the two mitochondrial proteins, cytochrome c1 (Neurospora crassa) and b2 (yeast). These proteins are synthesized in the cytosol as larger precursor molecules, posttranslationally imported into mitochondria and processed in two steps to their respective mature sized forms (1). Cytochrome c1 is a protein of the inner mitochondrial membrane with a large hydrophilic domain exposed to the intermembrane space. Cytochrome b2 is a soluble protein of the intermembrane space. Both proteins enter mitochondria via translocation contact sites (2).

NADH: A Common Requirement for the Import and Maturation of Cytochromes c and c1

The covalent attachment of heme to apocytochrome c, which is catalyzed by the mitochondrial enzyme cytochrome c heme lyase, was dependent on NADH. In addition, a cofactor present in reticulocyte lysate or a Neurospora crassa cytosol fraction was required for the NADH-dependent step. In the absence of NADH, apocytochrome c was bound to the mitochondrial surface and remained accessible to externally added proteases. In the presence of NADH, covalent attachment of heme occurred with concomitant translocation of cytochrome c across the outer mitochondrial membrane to a protease-resistant location. Both heme attachment and translocation were inhibited by the heme analogue deuterohemin.