Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation.
In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes designated PYC1 and PYC2. We report the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S. cerevisiae. Comparison with the previously reported sequence [Stucka, Dequin, Salmon and Gancedo (1991) Mol. Gen. Genet. 229, 307-315] revealed significant differences within the open reading frame. The most notable difference was near the 3' end, where we found a single base deletion reducing the open reading frame by 15 bases. We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-acid biotin-domain peptide. In addition, we investigated the effects of the two changes in the Pyc2 biotin domain (K1155R substitution and Q1178P/five-amino-acid extension) on the extent of biotinylation in vivo by Escherichia coli biotin ligase, and compared the biotinylation of peptides containing these changes with that of two different-length Pyc1 biotin-domain peptides. The K1155R substitution had very little effect on biotinylation, but the five-amino-acid C-terminal extension to Pyc2 and the N-terminal extension to Pycl both improved biotinylation in vivo.
Val
D L
DL
Department of Biochemistry, University of Adelaide, Australia.
Chapman-Smith
A
A
Walker
M E
ME
Cronan
J E
JE
Wallace
J C
JC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D002874:Chromosome Mapping; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011110:Polymorphism, Genetic; D011766:Pyruvate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017421:Sequence Analysis; D017385:Sequence Homology
D004271:DNA, Fungal; D007527:Isoenzymes; D001710:Biotin; D011766:Pyruvate Carboxylase