An amino terminal prosequence is required for efficient synthesis of S. cerevisiae a-factor.
The Saccharomyces cerevisiae a-mating pheromones are 12 amino acid lipopeptides whose secretion is dependent on the ABC transporter, Ste6p. The pheromones are synthesized as 36 and 38 amino acid precursors that terminate in a CaaX box (C is Cys, a is an aliphatic residue, and X is the C-terminal amino acid). Posttranslational processing of the a-factor precursors includes at least 5 events. C-terminal processing of the CaaX box includes farnesylation of Cys, removal of the -aaX residues, and methylation of the cysteine alpha-carboxyl group. The N-terminal steps involve proteolytic cleavages that remove the prosequences. In this report, we have investigated the role of posttranslational modification in the generation of functional a-factor. Wild type, mutant and chimeric forms of a-factor have been expressed in yeast and assessed for their abilities to serve as sources of functional a-factor. We have found that although modification of the CaaX box is necessary, it is not sufficient to generate bioactive a-factor. The amino terminal prosequences are also required. Deletion of these sequences reduces intracellular levels of a-factor resulting in sterility. Glutathione-S-transferase (GST)-a-factor fusions undergo CaaX box processing and membrane localization, but are not substrates for the N-terminal proteases and fail to interact with Ste6p. These results suggest that the amino terminal precursor sequences play a direct role in the generation of functional a-factor.
Quinby
G E
GE
Department of Biochemistry, University of Iowa, Iowa City 52242, USA.
Deschenes
R J
RJ
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000378:metabolism; D006023:Glycoproteins; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry; D008745:Methylation; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010675:Pheromones; D010957:Plasmids; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; D006023:Glycoproteins; D008074:Lipoproteins; C095063:MFA2 protein, S cerevisiae; D010675:Pheromones; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase