Redundant mitochondrial targeting signals in yeast adenylate kinase.
Yeast adenylate kinase (Aky2p, Adk1p) occurs simultaneously in cytoplasm and mitochondrial intermembrane space. It has no cleavable mitochondrial targeting sequence, and the signal for mitochondrial import and submitochondrial sorting is largely unknown. The extreme N terminus of Aky2p is able to direct cytoplasmic passengers to mitochondria. However, an Aky2 mutant lacking this sequence is imported with about the same efficiency as the wild type. To identify possible import-relevant information in the interior, parts of Aky2p were exchanged by homologous in vitro recombination for the respective segments of the purely cytoplasmic isozyme, Ura6p. Import studies revealed an internal region of about 40 amino acids, which was sufficient to direct the chimera to mitochondria but not for correct submitochondrial sorting. The respective Ura6p hybrid was arrested in the mitochondrial membrane at a position where it was inaccessible to protease but was released by alkaline extraction, suggesting that it had entered an import channel and passed the initial steps of recognition and uptake. Site-specific mutations within the presumptive address-specifying segment identified the amphipathic helix 5. A Ura6 mutant protein in which helix 5 had been replaced with the respective sequence from Aky2p was imported, and this address sequence cooperates with the N terminus in the respective double mutant in a synergistic fashion.
Schricker
Roland
R
Department Biologie I, Bereich Genetik, Ludwig Maximilians Universität München, Maria-Ward-Strasse 1a, D-80638 Munich, Germany.
Angermayr
Michaela
M
Strobel
Gertrud
G
Klinke
Sigrid
S
Korber
Dorothee
D
Bandlow
Wolfhard
W
D000263:Adenylate Kinase / Q000737:chemistry* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015003:Yeasts / Q000201:enzymology
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C023276:uridine monophosphate kinase; D000263:Adenylate Kinase; D009703:Nucleoside-Phosphate Kinase