Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation

Biochem J. 1995 Dec 15;312 ( Pt 3)(Pt 3):817-25. doi: 10.1042/bj3120817.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Biotin / metabolism*
  • Chromosome Mapping
  • DNA, Fungal / chemistry
  • DNA, Fungal / isolation & purification
  • Escherichia coli / genetics
  • Genes, Fungal
  • Isoenzymes / chemistry
  • Isoenzymes / genetics*
  • Isoenzymes / metabolism
  • Molecular Sequence Data
  • Mutagenesis
  • Polymorphism, Genetic*
  • Pyruvate Carboxylase / chemistry
  • Pyruvate Carboxylase / genetics*
  • Pyruvate Carboxylase / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*
  • Sequence Analysis
  • Sequence Homology

Substances

  • DNA, Fungal
  • Isoenzymes
  • Biotin
  • Pyruvate Carboxylase

Associated data

  • GENBANK/U35647