A NOVEL A-FACTOR-RELATED PEPTIDE OF SACCHAROMYCES-CEREVISIAE THAT EXITS THE CELL BY A STE6P-INDEPENDENT MECHANISM

Many secreted signaling molecules are synthesized as precursors that u ndergo multiple maturation steps to generate their mature forms. The S accharomyces cerevisiae mating pheromone alpha-factor is a C-terminall y isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of alpha-factor occurs by a n ordered biogenesis pathway involving 1) three C-terminal modificatio n steps, 2) two N-terminal proteolytic processing events, and 3) a non classical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpe cted and abundant alpha-factor-related peptide (AFRP) exists in the cu lture fluid of MATa cells and that its biogenesis is integrally relate d to that of mature alpha-factor itself. We show by purification follo wed by mass spectrometry that AFRP corresponds to the C-terminal 7 ami no acids (VFWDPAC) of mature alpha-factor (YIIKGVFWDPAC), including bo th the farnesyl- and carboxylmethylcysteine modifications. The formati on and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl 1) that cannot produce mature alpha-factor due to an N-terminal proces sing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature alpha-factor but, instead, from the P1 form o f the alpha-factor precursor. Second, fusion constructs with foreign a mino acids substituted for authentic alpha-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds t o the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-independent but length-specific proteolytic ac tivity. Third, alpha-factor and AFRP use distinct cellular machinery f or their secretion. Whereas alpha-factor export is Ste6p-dependent, AF RP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a differen t type of transporter altogether, or possibly by diffusion. Taken toge ther, these studies indicate that the biogenesis of AFRP involves nove l mechanisms and machinery, distinct from those used to generate matur e alpha-factor. Because AFRP neither stimulates nor inhibits mating or alpha-factor halo activity, its function remains an intriguing questi on.