Transport of proteins across mitochondrial membranes

The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of preproteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c 1 heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c 1 preproteins, and the mitochondrial processing peptidase which cleaves signal sequence after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle. are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c 1 heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c 1 preproteins, and the mitochondrial processing peptidase which cleaves signal sequences after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle

Transport of proteins into mitochondria

Translocational intermediates of precursor proteins of ATPase F1β subunit and cytochrome c1 across mitochondrial membranes were analyzed using two different approaches, transport at low temperature and transport after binding of precursor proteins to antibodies. Under both conditions precursors were partially transported into mitochondria in an energy-dependent manner. They were processed by the metalloprotease in the matrix but a major proportion of the polypeptide chains was still present at the outer face of the outer mitochondrial membrane. We conclude that transfer of precursors into the inner membrane or matrix space occurs through “translocation contact sites”; precursor polypeptides to F1β and cytochrome c1 enter the matrix space with the amino terminus first; and a membrane potential is required for the transmembrane movement on an amino-terminal “domain-like” structure but not for completing translocation of the major part of the polypeptides

High-affinity binding sites involved in the import of porin into mitochondria

The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane

Biogenesis of Glyoxysomes

Biosynthesis of isocitrate lyase, a tetrameric enzyme of the glyoxysomal matrix, was studied in Neurospora crassa, in which the formation of glyoxysomes was induced by a substitution of sucrose medium by acetate medium. * 1. Translation of Neurospora mRNA in reticulocyte lysates yields a product which has the same apparent molecular weight as the subunit of the functional enzyme. Using N-formyl[35S]methionyl tRNAMetf as a label, the translation product shows the same apparent size which indicates that the amino terminus has no additional 'signal'-type sequence. * 2. Read-out systems employing free and membrane-bound polysomes show that only free ribosomes are active in the synthesis of isocitrate lyase. * 3. Isocitrate lyase synthesized in reticulocyte lysate is released into the supernatant and is soluble in a monomeric form. It interacts with Triton X-100 to form mixed micells in contrast to the functional tetrameric form. * 4. Transfer of isocitrate lyase synthesized in vitro into isolated glyoxysomes is suggested by results of experiments in which supernatants from reticulocyte lysates are incubated with a particle fraction isolated from acetate-grown cells. No transfer occurs when particles from non-induced cells are employed. Resistance to added proteinase is used as a criterion for transmembrane transfer. The data support a post-translational transfer mechanism for isocitrate lyase. They suggest that isocitrate lyase passes through a cytosolic precurscr pool as a monomer and is transferred into glyoxysomes

Mitochondrial protein import

The transport of nuclear-encoded proteins from the cytosol into mitochondria is mediated by targeting (signal) sequences present on precursor forms. Most precursors of the mitochondrial matrix possess amino-terminal signals which characteristically contain hydroxylated and basic amino acids and lack acidic residues. With a minority of precursor proteins, internal sequence motifs can direct proteins to the mitochondria (Pfanner, N., Hoeben, P., Tropschug, M. and Neupert, W. (1987) J. Biol. Chem. 262, 14851–14854). The presence of a mitochondrial targeting sequence alone, however, is not sufficient for specific targeting to the organelle and further to the various subcompartments. There is the need for components which recognise the targeting sequences and others which keep the precursor protein in a translocation-competent form. Beyond the recognition step, components are required which mediate translocation across the mitochondrial membranes. Mitochondria possess two translocation machineries, one in the outer membrane and one in the inner membrane. The matrix space harbors a number of factors which participate in the import of proteins, in their unfolding and folding. Energy is required at several steps of these processes

Distinct steps in the import of ADP/ATP carrier into mitochondria

Transport of the precursor to the ADP/ATP carrier from the cytosol into the mitochondrial inner membrane was resolved into several consecutive steps. The precursor protein was trapped at distinct stages of the import pathway and subsequently chased to the mature form. In a first reaction, the precursor interacts with a protease-sensitive component on the mitochondrial surface. It then reaches intermediate sites in the outer membrane which are saturable and where it is protected against proteases. This translocation intermediate can be extracted at alkaline pH. We suggest that it is anchored to the membrane by a so far unknown proteinaceous component. The membrane potential delta psi-dependent entrance of the ADP/ATP carrier into the inner membrane takes place at contact sites between outer and inner membranes. Completion of translocation into the inner membrane can occur in the absence of delta psi. A cytosolic component which is present in reticulocyte lysate and which interacts with isolated mitochondria is required for the specific binding of the precursor to mitochondria