A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.

Abstract

Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2‐terminal ‘leader’ peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26‐K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti‐DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti‐DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell‐free system and incubated post‐translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post‐translational import of a chimeric precursor protein by mitochondria.

Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein

“…fntroduction Most nuclear-encoded precursors of mitochondrial proteins contain amino-terminal presequences (for reviews see Pfanner and Neupert, 1987;Nicholson and Neupert, 3988). These presequences are required for the precursors to enter the mitochondrial matrix, where they are proteolytically removed (Hurt et al, 1984;Horwich et al, 1985Horwich et al, , 1986Emr et al, 1986;Keng et al, 1986;Grivell et al, 1987;Vassarotti et al, 1987a). This cleavage is not essential for completing import but is necessary for further assembly of the newly imported polypeptides into functional complexes (Zwizinski and Neupert, 1983;Lewin and Norman, 1983;Ou et al, 1986;Hart1 et al, 1986Hart1 et al, , 1987.…”

Synthesis and Assembly of Mitochondrial Proteins

“…The amino-terminal prepiece of both rat and human Ornithine carbamoyltransferase (OTC), which is 32 amino acids long and directs the protein to the matrix, can also target nonmitochondrial passenger proteins to the matrix in vitro (Horwich et al, 1985b;Nguyen et al, 1986). In contrast to the case of COX IV, the targeting information does not appear to reside in the very amino terminus of the prepiece since deletion studies in which either the amino or carboxy terminus of the prepiece were removed produced proteins which were still import competent.…”

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

“…First, completed polypeptide chains are released as precursors, most of which carry amino-terminal peptide extensions, from cytoplasmic ribosomes into precursor pools in the cytosol (Hallermayer et al, 1977;Schatz, 1979). Second, precursors are targeted to mitochondria by specific signals contained in the presequences (Hurt et al, 1984a(Hurt et al, , 1984bHorwich et al. 1985;Horwich et al, 1986;van Loon and Young, 1986) and by binding of precursors to proteinaceous receptors on the mitochondrial surface (Zwizinski et al, 1984).…”