The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation.
The specificities of methionine aminopeptidase and amino-terminal acetylation in the yeast Saccharomyces cerevisiae were investigated in vivo by sequencing a series of altered iso-1-cytochrome c. Twenty iso-1-cytochromes c, each having a different penultimate residue in the sequence Met-Xaa-Phe-Leu-, were created by transforming yeast directly with synthetic oligonucleotides. The degree of methionine cleavage and amino-terminal acetylation was estimated from the levels of pertinent peptides separated by high performance liquid chromatography. The results confirmed our earlier hypothesis (Sherman, F., Stewart, J. W., and Tsunasawa, S. (1985) BioEssays 3, 27-31) that methionine is completely removed from penultimate residues having radii of gyration of 1.29 A or less (glycine, alanine, serine, cysteine, threonine, proline, and valine). However, only partial cleavage occurred in the sequences Met-Thr-Pro-Leu- and Met-Val-Pro-Leu-, demonstrating that proline at the third position inhibits methionine cleavage when the penultimate residue has an intermediate radius of gyration. Acetylation of the retained amino-terminal methionine occurred completely with the Ac-Met-Glu-Phe-Leu- and Ac-Met-Asp-Phe-Leu- sequences and partially with the Ac-Met-Asn-Phe-Leu-sequence. Although the consensus for acetylation of the retained amino-terminal methionine is not completely known, these results and the results of published sequences indicated that Ac-Met-Glu- and Ac-Met-Asp- (methionine followed by an acidic residue) is sufficient for amino-terminal acetylation in eukaryotes but not in prokaryotes.
Moerschell
R P
RP
Department of Biophysics, School of Medicine and Dentistry, Rochester, New York 14642.
Hosokawa
Y
Y
Tsunasawa
S
S
Sherman
F
F
D000107:Acetylation; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D008715:Methionine / Q000378:metabolism*; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014170:Transformation, Genetic
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D008715:Methionine; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases