The ?-subunit of spinach chloroplast ATP synthase: isolation and characterisation of cDNA and genomic clones

Abstract

cDNA and genomic clones encoding the complete precursor polypeptide of the gamma-subunit of spinach chloroplast ATP synthase have been isolated and characterised. The longest cDNA (1320 bp), selected from a lambda gt11 spinach cDNA library, encoded a 364 amino acid residue protein (Mr 40,028) that included a putative 41 residue N-terminal transit peptide. All the gamma-subunit cDNAs analysed were derived from the same gene and Southern blot analysis of Bam HI restricted spinach DNA showed only one hybridising band (ca. 15 kb). Analysis of the cloned 15 kb genomic fragment, selected from a lambda EMBL4 spinach DNA library with a cDNA probe, confirmed there was a single gene for the gamma-subunit. Sequencing, primer extension and northern blot analysis showed the gene contains two introns, 1066 and 665 bp in length, a 173 bp 5' untranslated region and a 213 bp 3' untranslated region. A 1450 nucleotide gamma-subunit transcript was detected in RNA from light-grown and dark-grown spinach.

Import and processing of the precursor form of the gamma subunit of the chloroplast ATP synthase from tobacco

“…The CF1 complex consists of five separate subunits, cq fl, 7, 6 and e [18], of which ? and 6 are nuclear-encoded [2,6,10] and the remainder are chloroplast-encoded [3,9,28]. The ?…”

“…The 7 subunit is synthesised in the cytosol as a precursor protein [23,25,26] and is translocated across the chloroplast envelope into the stroma where it is processed to the mature form [17,22,23]. Comparison of N-terminal sequence data for the mature form of the 7 subunit from spinach [10,13] and pea [17] The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63606. with sequences derived from cDNA clones [6,10,17] indicates that the proteins are synthesised with N-terminal presequences of 41-60 amino acid residues.…”

“…Comparison of N-terminal sequence data for the mature form of the 7 subunit from spinach [10,13] and pea [17] The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63606. with sequences derived from cDNA clones [6,10,17] indicates that the proteins are synthesised with N-terminal presequences of 41-60 amino acid residues. The amino acid sequences of the mature 7 subunits, deduced from cDNA or genomic clones from spinach [ 10,13 ], Arabidopsis thaliana [6], pea [17] and Chlamydomonas [27], have been compared with sequences of the 7 subunits from mitochondria and bacteria and have revealed little overall sequence conservation except in the C-terminal region, which is important both for the assembly and the activity of the FI complex in Escherichia coli [7].…”

“…with sequences derived from cDNA clones [6,10,17] indicates that the proteins are synthesised with N-terminal presequences of 41-60 amino acid residues. The amino acid sequences of the mature 7 subunits, deduced from cDNA or genomic clones from spinach [ 10,13 ], Arabidopsis thaliana [6], pea [17] and Chlamydomonas [27], have been compared with sequences of the 7 subunits from mitochondria and bacteria and have revealed little overall sequence conservation except in the C-terminal region, which is important both for the assembly and the activity of the FI complex in Escherichia coli [7]. These comparisons have also demonstrated the presence in chloroplast 7 subunits of a block of nine amino acid residues, including two cysteine residues, which are not present in prokaryotic and mitochondrial 7 subunits [13,17,27].…”

Import and processing of the precursor of the delta subunit of tobacco chloroplast ATP synthase

“…Two genes encoding the 7 subunit of the chloroplast ATP synthase are also present in the tobacco haploid genome [ 11 ]. Single-copy genes encoding the 7 and 6 subunits of the chloroplast ATP synthase are present in the spinach genome [3,13]. The presence of two genes encoding the b subunit in tobacco could lead to heterogeneity in the 6 sub- r e s e a r c h g r a n t f r o m the U .…”