Ubiquitin conjugation to cytochromes c. Structure of the yeast iso-1 conjugate and possible recognition determinants.
Saccharomyces cerevisiae iso-1-cytochrome c was conjugated with ubiquitin (Ub) in vitro in a rabbit reticulocyte extract (Fraction II). By N-terminal protein sequencing, it was found for both the mono- and diubiquitinated products that the major Ub attachment site is on Lys4 (residue 9) of the cytochrome c. Thus, the residue ubiquitinated in iso-1-cytochrome c is identical with that previously determined for the yeast iso-2 form (Sokolik, C. W., and Cohen, R. E. (1991) J. Biol. Chem. 266, 9100-9107). For both cytochromes c, the proportions of diubiquitinated and higher order conjugates are drastically reduced when Ub is replaced with a Lys48----Arg variant, suggesting that the Ub-Ub moieties are linked predominantly through Lys48. Despite close similarities in structure and ubiquitination sites, conjugation to iso-2-cytochrome c is approximately 5-fold faster than for the iso-1 form; vertebrate cytochromes c are even poorer substrates, being ubiquitinated at only approximately 5% of the rate of the iso-2 protein. Comparison of several cytochrome c variants excludes alpha-N-acetylation or the identity of the N-terminal amino acid as the important recognition determinants in these reactions. The results, which include the finding that ferro and ferri-iso-2-cytochromes c are ubiquitinated equally, also are evidence against a simple correlation between ubiquitination efficiency and thermodynamic stability. Rather, the presence of a pair of lysines (Lys4-Lys5) within the relatively unstructured N-terminal extension of the yeast cytochromes c may be responsible for their preferential ubiquitination.
Sokolik
C W
CW
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Cohen
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism*
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C032139:iso-2-cytochrome C; D045304:Cytochromes c