Cloning and expression of the alpha subunit of succinyl-CoA synthetase from Dictyostelium discoideum.
We have isolated and characterized a genomic fragment and a cDNA clone for the alpha subunit of succinyl-CoA synthetase (SCS) from Dictyostelium discoideum. The coding region predicts a protein of 310 amino acids. Direct amino acid sequence data indicate that the first 16 amino acids encoded by the cDNA comprise a mitochondrial targeting sequence. The predicted molecular mass of the mature protein is 31 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the alpha subunit from D. discoideum shows 75% identity to that from rat liver, which represents a GTP-utilizing form of SCS, and 62% identity to the Escherichia coli protein, which is an ATP-utilizing form. We have previously shown that the enzyme purified from growing D. discoideum is a GTP-utilizing enzyme. In D. discoideum, the alpha subunit is a single copy gene that is developmentally regulated. Highest levels of its mRNA are seen in growing cells, which is followed by a relatively dramatic decline at the time cells complete their aggregation program. In contrast, the level of the protein, as determined by Western blots, remains unchanged throughout development. This indicates that any developmental changes in SCS activity reflect changes in the availability of its substrates or effectors and not changes in the level of enzyme.
Birney
M A
MA
St Louis University School of Medicine, E. A. Doisy Department of Biochemistry and Molecular Biology, Missouri 63104, USA.
Klein
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013389:Succinate-CoA Ligases / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D016054:DNA, Protozoan; D012333:RNA, Messenger; D013389:Succinate-CoA Ligases