Abstract. Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix.Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy ,x,l% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to ~7 % of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Abstract. With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria . Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain . Thereby, three salient features of mitochondrial protein up-ROTEIN translocation across biological membranes is a key step in the biogenesis of cell organelles (Wickner and Lodish, 1985). The basic problem ofhow cy tosolically synthesized precursor proteins traverse organelle membranes was studied predominantly in cell-free systems. These systems consisted of isolated organelles, such as microsomes (ER), mitochondria, chloroplasts or peroxisomes, and precursor proteins that were synthesized in vitro or expressed in Escherichia coli and subsequently purified. With regard to mitochondria, the following steps ofprotein import have been established (for reviews see Attardi and Schatz, 1988 ;Hard and Neupert, 1990; Pfanner and Neupert,1990) . Nuclear-encoded precursor proteins are synthesized on cytosolic polysomes and targeted to specific receptors on the outer mitochondrial membrane. A loosely folded ("unfolded") conformation of a precursor protein is a prerequisite for its translocation across the mitochondrial membranes (Schleyer and Neupert, 1985;Eilers and Schatz, 1986; Chenand Douglas, 1987a) . Most precursors are translocated through contact sites between outer and inner mitochondrial membranes (translocation contact sites) . The membrane potential across the inner membrane is required for the initial entrance of a precursor into this membrane, while the completion of translocation does not depend on A0. During or after membrane translocation, the amino-terminal targeting sequence (presequence) is proteolytically cleaved off by the processing peptidase in the mitochondrial matrix .Though posttranslational translocation of unfolded precursor proteins through mitochondrial contact sites is well established in the isolated system, little is known about protein transport into mitochondria in intact cells . In early studies performed at low temperature, cytoplasmic pools of mitochondrial precursor proteins were found in Neurospora

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Transfection of foreign DNA into eukaryotic cells has become an important tool in molecular biology. Based on the results of previous studies of the core structure of human adenoviruses, we have developed a novel transfection method. The procedure involves the in vitro reconstitution of foreign DNA-of viral or other origins-with the major core protein VII of adenovirus type 2 (Ad2) or protamine from salmon sperm. Both proteins are rich in basic amino acids and appear to share structural features. The DNA-protein complexes are added directly to the medium of eukaryotic cells. The in vitro formation of specific DNA-protein complexes can be assessed by gel electrophoretic analyses. Bovine serum albumin does not enter into specific complexes with DNA. Transfection of DNA-protein VII or DNA-protamine complexes results in their rapid transport into the cell nuclei. About 2-4 hr after transfection, up to 40% of the DNA added to cell cultures in complexes can be found in the nucleus, as compared with less than 10% of the DNA when other transfection methods are applied or when naked DNA is added to cell cultures. DNAs transfected by the new method into mammalian or insect cells retain their characteristic restriction patterns at least 48 hr after transfection and are expressed efficiently. Supercoiled circular plasmid DNAs are converted to open circular or linear DNA. Expression has been measured both for transiently expressed genes (chloramphenicol acetyltransferase gene, Ad2 DNA in human HeLa cells) and for genes that have been integrated into the host genome and are expressed permanently, such as the gene for neomycin phosphotransferase in hamster BHK21 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryProtein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes(l-". In particular we will focus on recently identified components of the mitochondrial import apparatus.