Littérature scientifique sur le sujet « Mitochondrial targeting sequence »

IDE (insulin-degrading enzyme) is a widely expressed zinc-metallopeptidase that has been shown to regulate both cerebral amyloid β-peptide and plasma insulin levels in vivo. Genetic linkage and allelic association have been reported between the IDE gene locus and both late-onset Alzheimer's disease and Type II diabetes mellitus, suggesting that altered IDE function may contribute to some cases of these highly prevalent disorders. Despite the potentially great importance of this peptidase to health and disease, many fundamental aspects of IDE biology remain unresolved. Here we identify a previously undescribed mitochondrial isoform of IDE generated by translation at an in-frame initiation codon 123 nucleotides upstream of the canonical translation start site, which results in the addition of a 41-amino-acid N-terminal mitochondrial targeting sequence. Fusion of this sequence to the N-terminus of green fluorescent protein directed this normally cytosolic protein to mitochondria, and full-length IDE constructs containing this sequence were also directed to mitochondria, as revealed by immuno-electron microscopy. Endogenous IDE protein was detected in purified mitochondria, where it was protected from digestion by trypsin and migrated at a size consistent with the predicted removal of the N-terminal targeting sequence upon transport into the mitochondrion. Functionally, we provide evidence that IDE can degrade cleaved mitochondrial targeting sequences. Our results identify new mechanisms regulating the subcellular localization of IDE and suggest previously unrecognized roles for IDE within mitochondria.

A mitochondrion is a cellular organelle able to produce cellular energy in the form of adenosine triphosphate (ATP). As in the nucleus, mitochondria contain their own genome: the mitochondrial DNA (mtDNA). This genome is particularly susceptible to mutations that are at the basis of a multitude of disorders, especially those affecting the heart, the central nervous system and muscles. Conventional clinical practice applied to mitochondrial diseases is very limited and ineffective; a clear need for innovative therapies is demonstrated. Gene therapy seems to be a promising approach. The use of mitochondrial DNA as a therapeutic, optimized by peptide-based complexes with mitochondrial targeting, can be seen as a powerful tool in the reestablishment of normal mitochondrial function. In line with this requirement, in this work and for the first time, a mitochondrial-targeting sequence (MTS) has been incorporated into previously researched peptides, to confer on them a targeting ability. These peptides were then considered to complex a plasmid DNA (pDNA) which contains the mitochondrial gene ND1 (mitochondrially encoded NADH dehydrogenase 1 protein), aiming at the formation of peptide-based nanoparticles. Currently, the ND1 plasmid is one of the most advanced bioengineered vectors for conducting research on mitochondrial gene expression. The formed complexes were characterized in terms of pDNA complexation capacity, morphology, size, surface charge and cytotoxic profile. These data revealed that the developed carriers possess suitable properties for pDNA delivery. Furthermore, in vitro studies illustrated the mitochondrial targeting ability of the novel peptide/pDNA complexes. A comparison between the different complexes revealed the most promising ones that complex pDNA and target mitochondria. This may contribute to the optimization of peptide-based non-viral systems to target mitochondria, instigating progress in mitochondrial gene therapy.

Abstract Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.

It is assumed that the survival factors Bcl-2 and Bcl-xL are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-xL is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-xL requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-xL specifically functions on the MOM.

The amino acid sequence of human hepatic peroxisomal L-alanine: glyoxylate aminotransferase 1 (AGTI) deduced from cDNA shows 78% sequence identity with that of rat mitochondrial AGTI, but lacks the N-terminal 22 amino acids (the putative mitochondrial targeting signal). In humans this signal appears to have been deleted during evolution by a point mutation of the initiation codon ATG to ATA. These data suggest that the targeting defect in primary hyperoxaluria type 1, in which AGT1 is diverted from the peroxisomes to the mitochondria, could be due to a point mutation that reintroduces all or part of the mitochondrial signal sequence.

Succinyl-CoA synthetase functions in the mitochondrial matrix as an αβ-dimer. Its constitutive subunits are thus expected to be encoded in the nucleus and synthesized in the cytoplasm as precursors containing signal sequences for mitochondrial translocation. We have previously reported the isolation and sequence of a rat liver cDNA clone (λSCS19) that apparently encodes the cytoplasmic precursor to the α-subunit. Here we report the preparation of mRNA transcripts of this cDNA insert and their in vitro translation to produce labeled protein that can be translocated across the membranes of subsequently added rat liver mitochondria. Translocation is accompanied by proteolytic processing to convert the 34.5-kilodalton precursor to the 32-kilodalton mature form of the subunit. The N-terminal sequence of the mature α-subunit from the GTP-specific isozyme has been determined by sequential Edman degradation and compared with the amino acid sequence deduced from the cDNA. This confirms that the cloned sequence encodes the GTP-specific α-subunit, and establishes that the point of cleavage is between histidyl and glycyl residues and that the signal sequence consists of 27 residues. The signal sequence shares characteristics of other mitochondrial targeting sequences that have been elucidated (largely of yeast mitochondrial precursors), including the potential to form an amphiphilic helix. Import is dependent upon the presence of ATP and is inhibited by compounds that diminish mitochondrial membrane potential. Translocation of the precursor is effective for precursor produced by the reticulocyte translation system, but is not seen for the product that is translated by a wheat germ extract, indicating that the latter may lack a factor or component that is necessary for the targeting and import process.Key words: succinyl-CoA synthetase, mitochondria, protein translocation, signal sequence.

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.

Mitochondria are ATP-generating organelles in eukaryotic cells that produce reactive oxygen species (ROS) during oxidative phosphorylation (OXPHOS). Mitochondrial DNA (mtDNA) is packaged within nucleoids and, due to its close proximity to ROS production, endures oxidative base damage. This damage can be repaired by base excision repair (BER) within the mitochondria, or it can be degraded via exonucleases or mitophagy. Persistent mtDNA damage may drive the production of dysfunctional OXPHOS components that generate increased ROS, or OXPHOS components may be directly damaged by ROS, which then can cause more mtDNA damage and create a vicious cycle of ROS production and mitochondrial dysfunction. If mtDNA damage is left unrepaired, mtDNA mutations including deletions can result. The accumulation of mtDNA mutations has been associated with conditions ranging from the aging process to cancer and neurodegenerative conditions, but the sequence of events leading to mtDNA mutations and deletions is yet unknown. Researchers have utilized many systems and agents for generating ROS in mitochondria to observe the downstream effects on mtDNA, ROS, and mitochondrial function; yet, there are various drawbacks to these methodologies that limit their precision. Here, we describe a novel chemoptogenetic approach to target oxidative damage to mitochondria and mtDNA with a high spatial and temporal resolution so that the downstream effects of ROS-induced damage can be measured with a high precision in order to better understand the mechanism of mitochondrial dysfunction in aging, cancer, and neurodegenerative diseases.

Increased ROS proto-oncogene 1 (ROS1) expression has been implicated in the invasiveness of human oral squamous cell carcinoma (OSCC). The cellular distribution of ROS1 has long-been assumed at the plasma membrane. However, a previous work reported a differential cellular distribution of mutant ROS1 derived from chromosomal translocation, resulting in increased carcinogenesis. We thus hypothesized that cellular distribution of upregulated ROS1 in OSCC may correlate with invasiveness. We found that ROS1 can localize to mitochondria in the highly invasive OSCC and identified a mitochondria-targeting signal sequence in ROS1. We also demonstrated that ROS1 targeting to mitochondria is required for mitochondrial fission phenotype in the highly invasive OSCC cells. OSCC cells expressing high levels of ROS1 consumed more oxygen and had increased levels of cellular ATP levels. Our results also revealed that ROS1 regulates mitochondrial biogenesis and cellular metabolic plasticity. Together, these findings demonstrate that ROS1 targeting to mitochondria enhances OSCC invasion through regulating mitochondrial morphogenesis and cellular respiratory.

Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the mitosomes of the human intestinal parasite Entamoeba histolytica are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged EhAS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between EhAS and DvAS in amoebic cells, we identified the ITSs responsible for mitosome targeting of EhAS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins.

Le résidu situé en position 2 des protéines, suivant leur méthionine initiatrice, est un signal clé pour le recrutement co-traductionnel de diverses enzymes de modification qui impactent précocement leur destinée cellulaire (adressage, repliement, temps de demi-vie). Bien que l’importance de ce résidu soit établie pour quelques protéines, son rôle à l’échelle globale du protéome et la nature des pressions sélectives auxquelles il pourrait être soumis demeurent à ce jour inexplorés. Durant ma thèse, j’ai pour la première fois utilisé des méthodologies d’analyse globale pour réaliser une étude fonctionnelle et évolutive du résidu situé en position 2 des protéines. J’ai mis au profit de cette étude deux approches complémentaires, développées in silico chez la levure modèle Saccharomyces cerevisiae. La première approche utilisée consiste en l’étude d’enzymes de modification dont le recrutement dépend du résidu en position 2 de leurs cibles. Je me suis en particulier intéressée aux N-acétyltransférases. Celles-ci possèdent la même activité enzymatique de N-acétylation, mais ciblent des sous-ensembles distincts de protéines et leurs délétions sont associées à des phénotypes différents, ce qui pose la question du rôle spécifique de chacune dans la physiologie cellulaire. Grâce à l’analyse de données expérimentales relatives à ces enzymes, j’ai caractérisé leur sélectivité globale in vivo et j’ai formellement démontré qu’elles ont effectivement des rôles physiologiques différentiels. La seconde approche utilisée est l’étude de la distribution des acides aminés en position 2 dans le protéome et dans les groupes fonctionnels de protéines définis par la Gene Ontology. Alors que les outils actuels utilisés pour réaliser des analyses de Gene Ontology ne tiennent pas compte de la structure hiérarchique de cette ressource, j’ai développé un algorithme permettant de synthétiser et de visualiser les résultats obtenus par une telle analyse pour en faciliter l’interprétation. Cette approche a permis l’identification de groupes fonctionnels de protéines présentant en position 2 une utilisation d’acides aminés distincte de celle observée dans le protéome à cette position. Ces deux méthodes d’analyse globale ont convergé vers un même résultat, à savoir que les précurseurs mitochondriaux possédant une séquence N-terminale d’adressage (MTS pour mitochondrial targeting sequence) présentent en position 2 une sur-représentation de larges résidus hydrophobes, critique pour leur import à la mitochondrie et permettant leur reconnaissance par la N-acétyltransférase NatC. Le biais d’acides aminés en position 2 des MTS est très conservé dans la lignée des Saccharomycotina et a partiellement évolué chez l’Homme et la plante Arabidopsis thaliana. J’ai de plus mis en évidence l’existence de plusieurs catégories de MTS en fonction de la nature du résidu qu’ils portent en position 2, ce qui peut indiquer une co-évolution de la position 2 des MTS et de leur composition globale et pose la question des propriétés optimales de ces séquences. Enfin, j’ai montré que les peptides signaux de la levure et la séquence N-terminale d’adressage au chloroplaste d’Arabidopsis thaliana présentent également des biais d’acides aminés en position 2, suggérant que le résidu à cette position pourrait jouer un rôle clé dans la reconnaissance de ces séquences par les systèmes d’adressage et d’import associés
The residue located at position 2 of proteins, following their initiator methionine, is a key signal for the co-translational recruitment of various modification enzymes that early impact their cellular fate (addressing, folding, half-life). Although the importance of this residue is established for a few proteins, its role at the global scale of the proteome and the nature of the selective pressures it may be subject to remain unexplored to this day. During my thesis, I used for the first time global analysis methodologies to conduct a functional and evolutionary study of the residue located at position 2 of proteins. I used two complementary in silico approaches developed in the model yeast Saccharomyces cerevisiae. The first approach I used is the study of modification enzymes whose recruitment depends on the residue at position 2 of their targets. I focused in particular on N-acetyltransferases. These enzymes have the same enzymatic activity of N-acetylation but target distinct subsets of proteins, and their deletions are associated with different phenotypes, raising the question of the specific role of each enzyme in cellular physiology. Through the analysis of experimental data related to these enzymes, I characterized their global selectivity in vivo and formally demonstrated that they indeed have differential physiological roles. The second approach I used is the study of the distribution of amino acids at position 2 in the proteome and in functional groups of proteins defined by the Gene Ontology. While current tools used to perform Gene Ontology analyses do not take into account the hierarchical structure of this resource, I developed an algorithm to synthesize and visualize the results obtained by such analyses to facilitate their interpretation. This approach allowed the identification of groups of proteins that present a distinct amino acid usage at position 2 compared to that observed in the proteome at this position. These two global analysis methods converged toward the same result, namely that mitochondrial precursors possessing an N-terminal addressing sequence (MTS for mitochondrial targeting sequence) exhibit at position 2 an overrepresentation of large hydrophobic residues, critical for their import into mitochondria and enabling their recognition by the NatC acetyltransferase. The amino acid bias at position 2 of MTS is highly conserved in the Saccharomycotina lineage and has partially evolved in humans and the plant Arabidopsis thaliana. I also highlighted the existence of several categories of MTS depending on the nature of the residue they carry at position 2, which may indicate co-evolution of position 2 of MTS and their overall composition and raises the question of optimal properties of these sequences. Finally, I showed that yeast signal peptides and the chloroplast N-terminal addressing sequence in Arabidopsis thaliana also exhibit amino acid biases at position 2, suggesting that the residue at this position could play a key role in the recognition of these sequences by associated addressing and import systems

Les mitochondries sont des organites complexes impliquant un millier de protéines, la plupart codées dans le génome nucléaire. Leur biogenèse a nécessité au cours de l’évolution la mise en place de systèmes efficaces d’adressage et d’import protéique, et des défaillances de ces systèmes sont associées à des pathologies graves, neuropathies, troubles cardiovasculaires, myopathies, maladies neurodégénératives ainsi que cancers. De nombreuses protéines mitochondriales possèdent en N-terminal une séquence d’adressage appelée MTS (Mitochondrial Targeting sequence) qui forme une hélice alpha amphiphile essentielle pour leur import mitochondrial. La séquence des divers MTSs est néanmoins très variables et leur caractéristiques critiques ne sont pas encore bien comprises. Le point de départ de ma thèse est la découverte, chez les levures, d’une surreprésentation en position 2 des séquences MTSs de 4 acides aminés hydrophobes (F, L, I, W). Au cours de mes années de thèse, j’ai pu confirmer, par des expériences de mutagenèse dirigée, le rôle critique de la nature du résidu en position 2 des MTSs. En effet, grâce au développement d’un système novateur de criblage des défauts d’import basé sur le sauvetage fonctionnel de la toxicité d’une protéine mitochondriale, j’ai pu observer que seuls les résidus surreprésentés en position 2 des protéines mitochondriales permettaient un import efficace. Mon travail a ainsi démontré l'existence de fortes contraintes évolutives s’exerçant au niveau de la position 2 des MTSs dont la compréhension pourrait à terme être utile pour optimiser l’adressage mitochondrial de protéines thérapeutiques chez des patients atteints de maladies mitochondriales
Mitochondria are complex organelles involving a thousand proteins, most of which are encoded in the nuclear genome. Their biogenesis has required the evolutionary development of efficient protein addressing and import systems, and failures of these systems are associated with serious pathologies, neuropathies, cardiovascular disorders, myopathies, neurodegenerative diseases and cancers.Many mitochondrial proteins have an N-terminal addressing sequence called MTS (Mitochondrial Targeting Sequence) which forms an amphiphilic alpha helix essential for their mitochondrial import. However, the sequence of the various MTSs is highly variable and their critical characteristics are not yet well understood. The starting point of my thesis was the discovery in yeast of an overrepresentation of 4 hydrophobic amino acids (F, L, I, W) at position 2 of the MTSs sequences. During my thesis, I was able to confirm the critical role of the nature of the residue in position 2 of the MTSs through directed mutagenesis experiments. Indeed, thanks to the development of an innovative system for screening import defects based on the functional rescue of the toxicity of a mitochondrial protein, I was able to observe that only residues overrepresented at position 2 of mitochondrial proteins allowed efficient import. My work has thus demonstrated the existence of strong evolutionary constraints at position 2 of MTSs, the understanding of which could ultimately be useful for optimising the mitochondrial addressing of therapeutic proteins in patients suffering from mitochondrial diseases

碩士
國立清華大學
分子醫學研究所
98
Oxidative phosphorylation system in mammalian cells contains five enzyme complexes. Among them, mitochondrial complex I is the biggest and the most complicated, with many undefined subunits and has no resolved complete structure. Mammalian mitochondrial complex I comprises of forty-five subunits, and seven of them are encoded by the mitochondrial genome. The remaining subunits are encoded by the nuclear genome and imported into mitochondria to perform their functions. NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is one of the nuclear-encoded mitochondrial core proteins of complex I. It contains two tetranuclear iron-sulfur clusters and plays an important role in electron transport. Mutations on NDUFS8 have been found to cause Leigh syndrome with mitochondrial complex I deficiency. In this study, RNA interference technique was used to knock down the NDUFS8 expression in T-REx293 cells to investigate the function of NDUFS8. Experimental results demonstrated that reducing expression of NDUFS8 would retard the cellular growth rate, slow down the oxygen consumption efficiency and increase the production of reactive oxygen species (ROS). Using high resolution clear native gel electrophoresis (HrCNE) for investigating the integrity of mitochondrial complex I revealed that knockdown of NDUFS8 would not affect the assembly of mitochondrial complex I but reduce its NADH oxidation activities. Restoration of NDUFS8 in a suppressed cell line improved the ability of oxygen consumption and NADH oxidation of complex I. In addition, various deletion and fusion constructs of NDUFS8 were generated to characterize the mitochondrial targeting sequence of this protein. The results revealed that the N-terminal fragment of 18 residues possessed the ability to import EGFP into mitochondria, which is shorter than the prediction of 34 amino acid residues proposed by MitoprotII program. Interestingly, there was also an unexpected result that all of the N-terminal deletion constructs of NDUFS8 protein were located in a specific region of nuclei. It was speculated that there is a nuclear localization signal hiding in NDUFS8 sequence. This study demonstrated that NDUFS8 play an essential role in complex I activity, and the mitochondrial targeting sequence of NDUFS8 existing or not will determine the subcellular localization in mitochondria or in nuclei.

Abstract The amino acid sequence deduced from the cDNA sequence of rat liver mt-GrpE (GenBank accession number U62940; Naylor et al. 1996) reveals a precursor protein of 217 amino acids (c. 24.3 kDa) with a 27 residue N-terminal targeting sequence, which upon mitochondrial protein import is proteolytically removed to generate the mature protein of 190 amino acids (c. 21.3 kDa). The nucleotide sequences of two partial cDNA clones from human white blood cells (TIGR EST#121800), normal foreskin melanocyctes (Genbank accession number N28384), together encode a precursor protein of 217 amino acids. A partial mouse cDNA clone (GenBank accession number W08216) has also been isolated.

Abstract FKBP73 of wheat is a PPIase (peptidyl-prolyl cis-trans isomerase) member of the FKBP (FK506-binding protein) family, being most homologous to mammalian FKBP52. Uniquely, the deduced amino acid sequence contains three FKBP12-like domains, a calmodulin-binding site and a putative TPR (tetratricopeptide) motif. The wheat FKBP73 is abundant in highly dividing tissues and an additional isoform of 71 kDa is associated with mature cells. The amino acid sequence deduced from the nucleotide sequence of a cDNA clone isolated from a wheat root tip cDNA library (EMBL accession number X86903) reveals an open reading frame of 559 amino acids with calculated molecular mass of 62 kDa and with electrophoresis mobility of 73 kDa on SDS-PAGE (Blecher et al., 1996). The N- terminal amino acid sequence does not reveal similarity to mitochondrial or chloroplast targeting sequences.

Abstract Over the last decade mitochondrial protein import has developed into one of the principal experimental systems for studying the general principles of intracellular protein sorting and the mechanisms involved in the translocation of proteins across membranes (for review, see references 1-5). Most mitochondrial proteins are synthesized as precursors on cytosolic polysomes and are subsequently imported into the pre-existing mitochondria which propagate by growth and division (Figure 1). The cytosolic precursors of mitochondrial proteins are maintained in a loosely folded, translocation competent conformation by the interaction with chaperone components such as cytosolic hsp70. In general, precursors carry amino-terminal pre-sequences which contain necessary and sufficient information for the targeting to mitochondria. Mitochondrial targeting signals are typically positively charged and rich in hydroxylated amino acids. Precursors interact with receptor proteins of 19 and 72 kd (in Neurospora crassa) on the surface of mitochondria. For the insertion into the outer membrane most precursors appear to use a ‘general insertion protein ‘ (GIP) in the outer membrane. Proteins of 38 kd in N. crassa and 42 kd in Saccharomyces cerevisiae have recently been identified which may constitute at least part of GIP.

Abstract The process of polypeptide translation, intracellular targeting and folding is a highly complex biochemical reaction. During translation, several events occur while the polypeptide is present as a ribosome-bound nascent chain that determine its ultimate fate in the cell. First, misfolding and aggregation of nascent chains are prevented. Secondly, the correct cellular location of the emerging nascent chain is determined based on the presence of targeting sequences such as an N-terminal signal sequence. Targeting to the correct cellular location may require translocation across intracellular membranes such as those of the endoplasmic reticulum (ER) and mitochondria. In order for this to occur, nascent polypeptides must be maintained in an unfolded state. Finally, polypeptides must be folded to their correct native state or be assembled into homo- or hetero-oligomeric complexes.

Abstract Amino acid sequences deduced from the nucleotide sequences of CPN60 cDNAs (Jindal et al., 1989; Peralta et al., 1990) (see also GenBank accession numbers M22882, M22383, X54793, X53585, X53584) reveal that mammalian Cpn60 is encoded as a 573 amino acid precursor protein (p-Cpn60) from which a typical amphipathic N-terminal targeting signal is removed upon mitochondrial import. This generates a mature protein (Cpn60) of 547 amino acids with a predicted mass of c. 57.9 kDa (Peralta etal., 1993).