ADP-ATP carrier of Saccharomyces cerevisiae contains a mitochondrial import signal between amino acids 72 and 111.
The ADP-ATP carrier (also referred to as the adenine nucleotide translocator) of Saccharomyces cerevisiae is encoded by a nuclear gene, translated in the cytosol, and imported into the mitochondrial inner membrane. In order to study the determinants of mitochondrial import, a series of fusion proteins, consisting of the first 21, 72, and 111 amino acids of the ADP-ATP carrier, joined to mouse dihydrofolate reductase were generated. Dihydrofate reductase is a cytoslic protein that does not bind mitochondria. The reticulocyte lysate reaction containing the 35S-methionine-labeled protein was incubated with mitochondria in a buffer containing 3% BSA. Following incubation for import, the reactions were treated with 1 mM PMSF or 25 micrograms/ml proteinase K; mitochondria were reisolated and analyzed by gel electrophoresis. The 21 and 72 amino acid hybrid proteins showed a low level of binding to mitochondria: the bound form was entirely protease accessible. The 111 amino acid hybrid protein was imported to a protease-protected location within mitochondria. It is concluded that the first 72 amino acids of the ADP-ATP carrier do not suffice to import the protein into mitochondria and that the region between amino acids 72 and 111, a region that contains a transmembrane-spanning domain, constitutes at least part of the mitochondrial import signal.
Smagula
C S
CS
Department of Biochemistry, University of Texas Health Science Center, Dallas 75235.
Douglas
M G
MG
D000596:Amino Acids / Q000032:analysis; D007425:Intracellular Membranes / Q000201:enzymology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D000596:Amino Acids; D008565:Membrane Proteins; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases