Characterization of ATP11 and detection of the encoded protein in mitochondria of Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, expression of functional F1-ATPase requires two proteins encoded by the ATP11 and ATP12 genes. Mutations in either gene block some crucial late step in assembly of F1, causing the alpha and beta subunits to accumulate in mitochondria as inactive aggregates (Ackerman, S. H., and Tzagoloff, A. (1991) Proc. Natl. Acad. Sci. U.S.A. 87, 4986-4990). In the present study we have cloned and determined the sequence of ATP11. The encoded product is protein of 37 kDa with no obvious homology to any known protein. In vitro import assays of ATP11 precursor and immunochemical evidence indicate that the protein is located in mitochondria. A fusion was made between ATP11 and a short sequence coding for 78 amino acids with the biotination signal of bacterial transcarboxylase. The protein expressed from this construct complements atp11 mutants, indicating that the addition of the extra 78 amino acids at the carboxyl terminus of the ATP11 protein does not compromise its function. The hybrid protein is detected in mitochondria with antibodies and with peroxidase-conjugated avidin. Biotinated ATP11 protein can be partially purified by affinity chromatography on monomeric or tetrameric avidin coupled to Sepharose. A fraction eluted from the avidin column and enriched for the biotinated ATP11 protein also contains the alpha and beta subunits of F1-ATPase.
Ackerman
S H
SH
Department of Biological Sciences, Columbia University, New York, New York 10027.
Martin
J
J
Tzagoloff
A
A
D000595:Amino Acid Sequence; D001360:Avidin / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012689:Sequence Homology, Nucleic Acid
C434343:Atp11 protein, S pombe; D004271:DNA, Fungal; D005656:Fungal Proteins; D018832:Molecular Chaperones; D011498:Protein Precursors; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D001360:Avidin; D001710:Biotin; C098664:ATP11 protein, S cerevisiae; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases