N-terminal deletion of the gamma subunit affects the stabilization and activity of chloroplast ATP synthase.
Five truncation mutants of chloroplast ATP synthase gamma subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinant gamma genes were overexpressed in Escherichia coli and assembled with alphabeta subunits into a native complex. The wild-type (WT) alphabetagamma assembly i.e. alphabetagammaWT exhibited high (Mg2+)-dependent and (Ca2+)-dependent ATP hydrolytic activity. Deletions of eight residues of the gamma subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the alphabetaWT assembly. Furthermore, only approximately 6% of ATP hydrolytic activity was retained with the sequential deletions of gamma subunit up to 20 residues compared with the activity of the alphabetaWT assembly. The inhibitory effect of the epsilon subunit on ATP hydrolysis of these alphabetagamma assemblies varied to a large extent. These observations indicate that the N-terminus of the gamma subunit is very important, together with other regions of the gamma subunit, in stabilization of the enzyme complex or during cooperative catalysis. In addition, the in vitro binding assay showed that the gamma subunit N-terminus is not a crucial region in binding of the epsilon subunit.
Ni
Zhang-Lin
ZL
Shanghai Institute of Plant Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Dong
Hui
H
Wei
Jia-Mian
JM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D025222:Chloroplast Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D004795:Enzyme Stability; D008929:Mitochondria, Heart / Q000201:enzymology; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology*
D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D025222:Chloroplast Proton-Translocating ATPases; D025261:Mitochondrial Proton-Translocating ATPases