Biogenesis of the Saccharomyces cerevisiae Pheromone a-Factor,from Yeast Mating to Human Disease

Summary: The mating pheromone a-factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a-factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a-factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a-factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a-factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a-factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a-factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila. Thus, additional prenylated signaling molecules resembling a-factor, with as-yet-unknown roles in metazoan biology, may await discovery.     

Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis

The yeast mating pheromone a-factor precursor contains an N-terminal extension and a C-terminal CAAX motif within which multiple posttranslational processing events occur. A recently discovered component in a-factor processing is Ste24p/Afc1p, a multispanning endoplasmic reticulum membrane protein that contains an HEXXH metalloprotease motif. Our in vivo genetic characterization of this protein has demonstrated roles for Ste24p in both the N-terminal and C-terminal proteolytic processing of the a-factor precursor. Here, we present evidence that the N-terminal proteolysis of the a-factor precursor P1 can be accurately reconstituted in vitro using yeast membranes. We show that this activity is dependent on Ste24p and is abolished by mutation of the Ste24p HEXXH metalloprotease motif or by mutation of the a-factor P1 substrate at a residue adjacent to the N-terminal P1 cleavage site. We also demonstrate that N-terminal proteolysis of the P1 a-factor precursor requires Zn(2+) as a co-factor and can be inhibited by the addition of the metalloprotease inhibitor 1,10-orthophenanthroline. Our results are consistent with Ste24p itself being the P1-->P2 a-factor protease or a limiting activator of this activity. Interestingly, we also show that the human Ste24 homolog expressed in yeast can efficiently promote the N-terminal processing of a-factor in vivo and in vitro, thus establishing a-factor as a surrogate substrate in the absence of known human substrates. The results reported here, together with the previously reported in vitro reconstitution of Ste24p-dependent CAAX processing, provide a system for examining the potential bifunctional roles of yeast Ste24p and its homologs.     

The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor

Saccharomyces cerevisiae Ste24p is a multispanning membrane protein implicated in the CAAX proteolysis step that occurs during biogenesis of the prenylated a-factor mating pheromone. Whether Ste24p acts directly as a CAAX protease or indirectly to activate a downstream protease has not yet been established. In this study, we demonstrate that purified, detergent-solubilized Ste24p directly mediates CAAX proteolysis in a zinc-dependent manner. We also show that Ste24p mediates a separate proteolytic step, the first NH(2)-terminal cleavage in a-factor maturation. These results establish that Ste24p functions both as a bona fide COOH-terminal CAAX protease and as an a-factor NH(2)-terminal protease. Importantly, this study is the first to directly demonstrate that a eukaryotic multispanning membrane protein can possess intrinsic proteolytic activity.     

Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH₂-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p is necessary for the first NH₂-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271–285). In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and J. Rine. 1997. Science. 275:1796–1800). Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Δ rce1Δ) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1Δ single mutant, the ste24Δ single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH₂-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing. Second, by using a novel set of ubiquitin–a-factor fusions to separate the NH₂- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24Δ rce1Δ) strain, implying that like yeast Ste24p, Hs Ste24p can mediate multiple types of proteolytic events.     

A homolog of Ste6, the a-factor transporter in Saccharomyces cerevisiae, is required for mating but not for monokaryotic fruiting in Cryptococcus neoformans

Fungal pheromones function during the initial recognition stage of the mating process. One type of peptide pheromone identified in ascomycetes and basidiomycetes terminates in a conserved CAAX motif and requires extensive posttranslational modifications to become mature and active. A well-studied representative is the a-factor of Saccharomyces cerevisiae. Unlike the typical secretory pathway utilized by most peptides, an alternative mechanism involving the ATP-binding cassette transporter Ste6 is used for the export of mature a-factor. Cryptococcus neoformans, a bipolar human pathogenic basidiomycete, produces CAAX motif-containing lipopeptide pheromones in both MATa and MATalpha cells. Virulence studies with a congenic pair of C. neoformans serotype D strains have shown that MATalpha cells are more virulent than MATa cells. Characterization of the MATalpha pheromones indicated that an autocrine signaling loop may contribute to the differentiation and virulence of MATalpha cells. To further address the role of pheromones in the signaling loop, we identified a STE6 homolog in the C. neoformans genome and determined its function by gene disruption. The ste6 mutants in either mating-type background showed partially impaired mating functions, and mating was completely abolished in a bilateral mutant cross. Surprisingly, the MATalpha ste6 mutant does not exhibit a defect in monokaryotic fruiting, suggesting that the activation of the autocrine signaling loop by the pheromone is via a Ste6-independent mechanism. MFalpha pheromone itself is essential for this process and could induce the signaling response intracellularly in MATalpha cells. Our data demonstrate that Ste6 is evolutionarily conserved for mating and is not required for monokaryotic fruiting in C. neoformans.     

Biogenesis of the Saccharomyces cerevisiae Mating Pheromone a-Factor

The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification, NH₂-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDSPAGE. We determined the biochemical composition of each by defining their NH₂-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH₂-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH₂-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH₂-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.In Saccharomyces cerevisiae, the peptide mating pheromones a-factor and α-factor function to promote conjugation between cells of the opposite mating type, MATa and MATα (Marsh et al., 1991; Sprague and Thorner, 1992). Like the peptide hormones secreted by higher eukaryotes, the yeast mating pheromones are initially synthesized as larger precursors that undergo posttranslational modification and proteolytic processing before their export from the cell. Despite their functional equivalence as signaling molecules, the a-factor and α-factor pheromones are structurally quite dissimilar and exemplify distinct paradigms for biogenesis. The maturation of α-factor is well characterized and involves the “classical” secretory pathway (ER→ Golgi→ secretory vesicles; Julius et al., 1984). Subsequent to its translocation across the ER membrane, the α-factor precursor undergoes signal sequence cleavage, glycosylation, a series of proteolytic processing steps in the lumenal compartments of the secretory pathway, and then exits the cell via exocytosis (Fuller et al., 1986; Sprague and Thorner, 1992). In contrast to our extensive understanding of α-factor maturation, our view of the events involved in a-factor biogenesis is still incomplete. An important difference between the two pheromones is that secretion of a-factor is mediated by a “nonclassical” export mechanism (Kuchler et al., 1989; McGrath and Varshavsky, 1989; Michaelis, 1993). The purpose of the present study is to delineate the steps of a-factor biogenesis that occur before its export, by the identification and characterization of a-factor biosynthetic intermediates.Mature bioactive a-factor is a prenylated and methylated dodecapeptide, derived by the posttranslational maturation of a precursor encoded by the similar and functionally redundant genes MFA1 and MFA2 (Brake et al., 1985; Michaelis and Herskowitz, 1988). The structures of the precursor and mature forms of a-factor derived from MFA1 are shown in Fig. ​Fig.1.1. The a-factor precursor can be subdivided into three functional segments: (a) the mature portion (shaded in Fig. ​Fig.1),1), which is ultimately secreted; (b) the NH₂-terminal extension; and (c) the COOH-terminal CAAX motif (C is cysteine, A is aliphatic, and X is one of many residues). As shown here, and also suggested by our previous studies, the biogenesis of a-factor occurs by an ordered series of events involving first COOH-terminal CAAX modification, then NH₂-terminal processing, and finally export from the cell (He et al., 1991; Michaelis, 1993; Sapperstein et al., 1994). Open in a separate windowFigure 1Structure of precursor and mature forms of a-factor encoded by MFA1. The a-factor precursor encoded by MFA1 is shown with the NH₂-terminal extension, COOH-terminal CAAX motif, and mature portion (shaded gray) indicated. Every fifth residue is numbered. Mature a-factor derived from this precursor is modified on its COOH-terminal cysteine residue by a farnesyl moiety and a carboxyl methyl group, as indicated.The COOH-terminal maturation of the a-factor precursor is directed by its CAAX sequence. The CAAX motif is present at the COOH terminus of numerous eukaryotic proteins, most notably the Ras proteins, and is known to signal a triplet of posttranslational modifications. These include prenylation of the cysteine residue, proteolysis of the COOH terminal AAX residues (VIA for a-factor), and methylation of the newly exposed cysteine carboxyl group (Clarke, 1992; Zhang and Casey, 1996). The yeast enzymes that mediate the modification of CAAX-terminating proteins are known from genetic and biochemical studies. RAM1 and RAM2 encode the subunits of the cytosolic farnesyltransferase enzyme (Fujiyama et al., 1987; He et al., 1991; Powers et al., 1986; Schafer et al., 1990). An “AAX” endoprotease has been detected as a membrane-associated activity in yeast extracts, although the corresponding gene(s) remains elusive (Ashby et al., 1992; Hrycyna and Clarke, 1992). STE14 encodes the prenylcysteine-dependent carboxyl methyltransferase that mediates methylation, the final step in modification of CAAX proteins; Ste14p is also membrane associated (Hrycyna and Clarke, 1990; Hrycyna et al., 1991; Marr et al., 1990; Sapperstein et al., 1994). In mutants (ram1, ram2, and ste14) defective in CAAX modification, biologically active a-factor is not produced.The events involved in the NH₂-terminal proteolytic processing of the a-factor precursor are less well-defined than those of COOH-terminal maturation. It was recently shown that a protease encoded by the AXL1 gene is required for one step of the NH₂-terminal processing of a-factor (Adames et al., 1995). Axl1p belongs to the insulin-degrading enzyme (IDE)¹ subfamily of proteases; an AXL1 homologue, Ste23p, was also found to perform a role at least partially redundant to that of Axl1p in a-factor processing (Adames et al., 1995). Recently, we have identified another gene, STE24, whose product participates in the NH₂-terminal processing of the a-factor precursor in a manner distinct from Axl1p and Ste23p (Fujimura-Kamada and Michaelis, 1997). Based on a priori inspection of the precursor and mature forms of a-factor (Fig. ​(Fig.1),1), a single NH₂-terminal proteolytic cleavage event (between residues N21 and Y22) might have been predicted; however, we provide evidence in the present study that the proteolytic processing of the NH₂terminal extension of the a-factor precursor occurs in two distinct steps.The final event in a-factor biogenesis is the export of the fully matured pheromone from the cell. The absence of a canonical NH₂-terminal signal sequence in the MFA1 and MFA2 sequences, as well as the lack of effect upon a-factor secretion of sec mutants blocked at various steps in the classical secretory pathway, led to the suggestion of a nonclassical export mechanism for a-factor export (McGrath and Varshavsky, 1989; Sterne, 1989). Indeed, a-factor export is now known to be mediated by Ste6p, a member of the ATP-binding cassette (ABC) superfamily of proteins (Kuchler et al., 1989; McGrath and Varshavsky, 1989). ABC proteins carry out the ATP-dependent membrane translocation of a variety of compounds, including small peptides, hydrophobic drugs, and even prenylcysteine derivatives, by an uncharacterized mechanism (Gottesman and Pastan, 1993; Zhang et al., 1994). It is notable that a-factor undergoes COOH-terminal modification and NH₂-terminal proteolytic maturation before Ste6p-mediated membrane translocation. This order of events contrasts with those of the biogenesis of the α-factor precursor and other classical secretory substrates, which undergo ER membrane translocation first and are matured only subsequently.In the present study, we aimed to elucidate the events that occur during a-factor biogenesis, before its export from the cell. Our approach was to identify a-factor biosynthetic intermediates, determine their chemical composition and localization properties, and examine the efficiency of their formation and the effects of an a-factor CAAX mutation on their formation. In addition to identifying the biosynthetic intermediates we expected, which include the unmodified a-factor precursor (P0), the COOHterminally modified a-factor precursor (P1), and mature a-factor (M), we unexpectedly uncovered a novel and unanticipated intermediate. This species, designated P2, is fully COOH-terminally modified and has had only a segment of its NH₂-terminal extension proteolytically removed. The existence of the P2 intermediate provides evidence that an additional unpredicted step occurs during the NH₂-terminal processing of the a-factor precursor. The biosynthetic intermediates we identify here, considered together with known a-factor biogenesis components, are presented in terms of a comprehensive model for the a-factor biogenesis pathway.     

Biochemical studies of Zmpste24-deficient mice

Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif. Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p. Ste24p also cleaves the amino terminus of a-factor. The mouse genome contains orthologues for both yeast RCE1 and STE24. We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins. In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast. Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice. Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile. Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice. We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor. Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility.     

A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor

Many secreted bioactive signaling molecules, including the yeast mating pheromones a-factor and α-factor, are initially synthesized as precursors requiring multiple intracellular processing enzymes to generate their mature forms. To identify new gene products involved in the biogenesis of a-factor in Saccharomyces cerevisiae, we carried out a screen for MATa-specific, mating-defective mutants. We have identified a new mutant, ste24, in addition to previously known sterile mutants. During its biogenesis in a wild-type strain, the a-factor precursor undergoes a series of COOH-terminal CAAX modifications, two sequential NH₂-terminal cleavage events, and export from the cell. Identification of the a-factor biosynthetic intermediate that accumulates in the ste24 mutant revealed that STE24 is required for the first NH₂-terminal proteolytic processing event within the a-factor precursor, which takes place after COOH-terminal CAAX modification is complete. The STE24 gene product contains multiple predicted membrane spans, a zinc metalloprotease motif (HEXXH), and a COOH-terminal ER retrieval signal (KKXX). The HEXXH protease motif is critical for STE24 activity, since STE24 fails to function when conserved residues within this motif are mutated. The identification of Ste24p homologues in a diverse group of organisms, including Escherichia coli, Schizosaccharomyces pombe, Haemophilus influenzae, and Homo sapiens, indicates that Ste24p has been highly conserved throughout evolution. Ste24p and the proteins related to it define a new subfamily of proteins that are likely to function as intracellular, membrane-associated zinc metalloproteases.     

Mutations in the yeast Hsp40 chaperone protein Ydj1 cause defects in Axl1 biogenesis and pro-a-factor processing

The heat shock protein (Hsp) 70/Hsp40 chaperone system plays an essential role in cell physiology, but few of its in vivo functions are known. We report that biogenesis of Axl1p, an insulinase-like endoprotease from yeast, is dependent upon the cytosolic Hsp40 protein Ydj1p. Axl1 is responsible for cleavage of the P2 processing intermediate of pro-a-factor, a mating pheromone, to its mature form. Mutant ydj1 strains exhibited a severe mating defect, which correlated with a 90% reduction in a-factor secretion. Reduced levels of a-factor export were caused by defects in the endoproteolytic processing of P2, which led to its intracellular accumulation. Defective P2 processing correlated with the reduction in the steady state level of active Axl1p. Two mechanisms were uncovered to explain why Axl1p activity was diminished in ydj1 strains. First, AXL1 mRNA levels were reduced ydj1 strains. Second, the half-life of newly synthesized Axl1p was greatly diminished in ydj1 strains. Collectively, these data indicate Ydj1p functions to promote AXL1 mRNA accumulation and in addition appears to facilitate the proper folding of nascent Axl1p. This study is the first to suggest a role for Ydj1p in RNA metabolism and identifies Axl1p as an in vivo substrate of the Hsp70/Ydj1p chaperone system.     

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.     

Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.     

Mutations within the first LSGGQ motif of Ste6p cause defects in a-factor transport and mating in Saccharomyces cerevisiae.

Mating between the two haploid cell types (a and alpha) of the yeast Saccharomyces cerevisiae depends upon the efficient secretion and delivery of the a- and alpha-factor pheromones to their respective target cells. However, a quantitative correlation between the level of transported a-factor and mating efficiency has never been determined. a-Factor is transported by Ste6p, a member of the ATP-binding cassette (ABC) family of transporter proteins. In this study, several missense mutations were introduced in or near the conserved LSGGQ motif within the first nucleotide-binding domain of Ste6p. Quantitation of extracellular a-factor levels indicated that these mutations caused a broad range of a-factor transport defects, and those directly within the LSGGQ motif caused the most severe defects. Overall, we observed a strong correlation between the level of transported a-factor and the mating efficiency of these strains, consistent with the role of Ste6p as the a-factor transporter. The LSGGQ mutations did not cause either a significant alteration in the steady-state level of Ste6p or a detectable change in its subcellular localization. Thus, it appears that these mutations interfere with the ability of Ste6p to transport a-factor out of the MATa cell. The possible involvement of the LSGGQ motif in transporter function is consistent with the strong conservation of this sequence motif throughout the ABC transporter superfamily.     

Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.     

Role of the ABC transporter Ste6 in cell fusion during yeast conjugation

Though early stages of yeast conjugation are well-mimicked by treatment with pheromones, the final degradation of the cell wall and membrane fusion of mating that leads to cytoplasmic mixing may require separate signals. Mutations that blocked cell fusion during mating in Saccharomyces cerevisiae were identified in a multipartite screen. The three tightest mutations proved to be partial-function alleles of the ABC-transporter gene STE6 required for transport of a-factor. The ste6(cefl-1) allele was recovered and sequenced. The ste6(cefl-1) allele contained a stop codon predicted to truncate Ste6 at amino acid residue 862 (of 1290). The ste6(cef) mutations reduced, but did not eliminate, expression of a-factor. Light and electron microscopy revealed that unlike ste6 null mutations which block mating before the formation of mating pairs, the ste6(cef) (cell fusion) alleles permitted early steps in mating to proceed normally but blocked at a late stage in conjugation where mating partners were encased by a single cell wall and separated by only a thin layer of cell wall material we term the fusion wall. Morphologically the prezygotes appeared symmetrical with successful cell wall fusion at the periphery of the region of cell contact. Responses to a-factor were efficiently induced in partner cells under mating conditions as expected given the symmetric appearance of the prezygotes. A strain expressing a ste6(K1093A) mutation that conferred export of a twofold to fourfold higher level of a-factor than ste6(cef) did not accumulate prezygotes during mating which could indicate a tight threshold of a-factor signaling required for mating. However, mating to an sst2 partner which has a greatly increased sensitivity to a-factor did not suppress the fusion defect of a ste6(cef) strain. Overexpression of the structural gene for a-factor also did not suppress the fusion defect. It is possible that a-factor or STE6 play more complex roles in cell fusion.     

Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.     

Yeast as a tractable genetic system for functional studies of the insulin-degrading enzyme

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.     

Ubc2, an ortholog of the yeast Ste50p adaptor, possesses a basidiomycete-specific carboxy terminal extension essential for pathogenicity independent of pheromone response

Proteins involved in the mitogen-activated protein (MAP) kinase pathway controlling mating, morphogenesis, and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing sterile alpha motif (SAM) and ras association (RA) domains typical of Ste50-like adaptor proteins found in the fungal phylum Ascomycota, Ubc2 also contains two C-terminal SH3 domains. Yeast two-hybrid assays indicated that Ubc2 interacts with the MAP kinase-kinase kinase Ubc4 via the SAM domains at each of their respective N-termini. Site-directed mutagenesis of ubc2 and complementation analyses revealed that the SAM and RA domains of Ubc2 are essential for filamentous growth. These data support a role for the ascomycete-like N-terminus of Ubc2 in regulating pheromone-responsive mating and morphogenesis analogous to the role of Ste50p in Saccharomyces cerevisiae. In contrast, C-terminal deletion mutants were fully capable of filamentous growth and mating. However, surprisingly, these strains were nonpathogenic. Further, directed mutagenesis of the C-terminus revealed that both SH3 domains are required for pathogenicity. These results suggest that the Basidiomycota have retained the mating and morphogenetic functions of Ste50-type proteins in the N-terminal half of their Ubc2-type adaptors but, additionally, have integrated C-terminal SH3 domains that are critical for additional signal transduction mechanisms, including those that lead to pathogenesis.     

The Saccharomyces cerevisiae Prenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.     

Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.

In eukaryotes small secreted peptides are often proteolytically cleaved from larger precursors. In Saccharomyces cerevisiae multiple proteolytic processing steps are required for production of mature 12-amino-acid a-factor from its 36-amino-acid precursor. This study provides additional genetic data supporting a direct role for Afc1p in cleavage of the carboxyl-terminal tripeptide from the CAAX motif of the prenylated a-factor precursor. In addition, Afc1p had a second role in a-factor processing that was independent of, and in addition to, its role in the carboxyl-terminal processing in vivo. Using ubiquitin-a-factor fusions we confirmed that the pro-region of the a-factor precursor was not required for production of the mature pheromone. However, the pro-region of the a-factor precursor contributed quantitatively to a-factor production.