Abstract
Mitochondria prepared from the yeast nuclear pet mutant N9-84 lack a detectable F1-ATPase activity. Genetic complementation of this mutant with a pool of yeast genomic DNA in the yeast Escherichia coli shuttle vector YEp13 restored its growth on a nonfermentable carbon source. Mitochondria prepared from the transformed host contained an 8-fold higher than normal level of the F1 alpha-subunit and restored ATPase activity to 50% that of the wild-type strain. Deletion and nucleotide sequence analysis of the complementing DNA on the plasmid revealed a coding sequence designated ATP1 for a protein of 544 amino acids which exhibits 60 and 54% direct protein sequence homology with the proton-translocating ATPase alpha-subunits from tobacco chloroplast and E. coli, respectively. In vitro expression and mitochondrial import experiments using this ATP1 sequence showed that additional amino-terminal sequences not present in the comparable plant and bacterial subunits function as transient sequences for import.
Highlights
From the Departmentof Biochemistry, Southwestern Graduateschool of Biomedical Sciences, University of Texas Health Science Center, D a h s, Texas 75235
Mitochondria prepared from the yeast nuclear pet subunits in the catalysis of ATP synthesisor breakdown mutant N9-84 lack a detectable F1-ATPase activity. suggest that the catalytic sites within the catalytic core are Genetic complementation otfhis mutant with apool of reversible and that cooperative interactions occurbetween yeast genomicDNA in the yeastEscherichia colishut- these sites(Cross, 1981).Independent data indicate that there tle vector YEpl3 restoredits growth on a nonferment- are three exchangeable nucleotide sites in F, which interact able carbon source
The ATPase complexof the mitochondrial inner membrane consists of protein subunits synthesized in the mitochondrial matrix andcytoplasm (Dujon, 1981).The mitochondrial complex, like that characterized from other sources, appears to be provides the probes necessary for a detailed structure-function analysis of the different subunits in energy transduction and the construction of mutants which testcurrent models of subunit cooperativity
Summary
The yeast nuclear pet mutant (N9-84) has been previously analyzed. Analysis of plasmids rescued from these transformcharacterized (Tzagoloff et al, 1975).This mutantwas shown ants which exhibited cotransformation of the Leu+ and Gly’. Additional analyses (not strain (see “Experimental Procedures”T).he resulting mutant shown) which utilized antiserum specific for the %-subunit (XJY12) was transformed with a pool of wild-type yeast confirmed the presence of reduced and increased levels of the genomic RNAcarried in the yeast E. coli shuttle vehicle a-subunit in themutantandtransformant mitochondria, YEpl (see ”ExperimentalProcedures”). The Fl-ATPase a-subunit is still expressed end of the complementing yeast DNA fragment is shown in in the mutant and most likely co-assembles with the wild- Fig. 5 (Appendix). Within this sequence of2385 bp is contype copy to yield a “hybrid” ATPase complex. Analysis of the E. coli and chloroplast are synthesized and assembled in
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