Mitochondrial import of the ADP/ATP carrier protein in Saccharomyces cerevisiae. Sequences required for receptor binding and membrane translocation.
The ADP/ATP carrier of yeast (309 amino acids) is an abundant transmembrane protein of the mitochondrial inner membrane whose import involves well-defined steps (Pfanner, N., and Neupert, W. (1987) J. Biol. Chem. 262, 7528-7536). Analysis of the in vitro import of gene fusion products containing ADP/ATP carrier (AAC) sequences at the amino terminus and mouse dihydrofolate reductase (DHFR) at the carboxyl terminus indicates that the first 72 amino acids of the soluble carrier protein, a hydrophilic region of the protein, are not by themselves sufficient for initial binding to the AAC receptor on the mitochondrial surface. However, an AAC-DHFR gene fusion containing the first 111 residues of the ADP/ATP carrier protein exhibited binding to mitochondria at low temperature (2 degrees C) and internalization at 25 degrees C to a mitochondrial space protected from proteinase K in the same manner as the wild-type ADP/ATP carrier protein. The AAC-DHFR protein, in contrast to the wild-type AAC protein imported into mitochondria under optimal conditions, remained extractable at alkaline pH and appeared to be blocked at an intermediate step in the AAC import pathway. Based on its extraction properties, this AAC-DHFR hybrid is proposed to be associated with a proteinaceous component of the import apparatus within mitochondria. These data indicate that the import determinants for the AAC protein are not located at its extreme amino terminus and that protein determinants distal to the first 111 residues of the carrier may be necessary to move the protein beyond the alkali-extractable step in the biogenesis of a functional AAC protein.
Smagula
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Department of Biochemistry, Southwestern Graduate School of Biomedical Sciences, University of Texas Health Science Center, Dallas 75235.
Douglas
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MG
D003001:Cloning, Molecular; D005796:Genes; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008928:Mitochondria / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics*; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases