Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases.
We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain. The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1. Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2-associated protein but not yet shown to have Met-AP activity. Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12-fold increases in Met-AP activity on different peptide substrates. The epitope-tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity. To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains. The map2 null strain, like the map1 null strain, is viable but with a slower growth rate. The map1, map2 double-null strains are nonviable. Thus, removal of N-terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone.
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104, USA.
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D015723:Gene Library; D005800:Genes, Fungal; D005804:Genes, Lethal; D016153:Genes, Suppressor; D006801:Humans; D007700:Kinetics; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D016335:Zinc Fingers
D017931:DNA Primers; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases