Ubiquitin conjugation to cytochromes c. Structure of the yeast iso-1 conjugate and possible recognition determinants

• Published in: The Journal of biological chemistry 1992-01, Vol.267 (2), p.1067-1071
• Main Authors: Sokolik, C W, Cohen, R E
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Autoradiography; Biological and medical sciences; Cytochrome c Group - genetics; Cytochrome c Group - metabolism; Cytochromes c; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Hemoproteins; Metalloproteins; Molecular Sequence Data; Protein Conformation; Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins; Substrate Specificity; ubiquitin; Ubiquitins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)48396-2

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.