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.     

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.     

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.     

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.     

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.     

Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p

STE50 is required to sustain pheromone-induced signal transduction in?S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require G_α, G_β, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth.     

Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export

The Saccharomyces cerevisiae mating pheromone a-factor provides a paradigm for understanding the biogenesis of prenylated fungal pheromones. The biogenesis of a-factor involves multiple steps: (i) C-terminal CAAX modification (where C is cysteine, A is aliphatic, and X is any residue) which includes prenylation, proteolysis, and carboxymethylation (by Ram1p/Ram2p, Ste24p or Rce1p, and Ste14p, respectively); (ii) N-terminal processing, involving two sequential proteolytic cleavages (by Ste24p and Axl1p); and (iii) nonclassical export (by Ste6p). Once exported, mature a-factor interacts with the Ste3p receptor on MATalpha cells to stimulate mating. The a-factor biogenesis machinery is well defined, as is the CAAX motif that directs C-terminal modification; however, very little is known about the sequence determinants within a-factor required for N-terminal processing, activity, and export. Here we generated a large collection of a-factor mutants and identified residues critical for the N-terminal processing steps mediated by Ste24p and Axl1p. We also identified mutants that fail to support mating but do not affect biogenesis or export, suggesting a defective interaction with the Ste3p receptor. Mutants significantly impaired in export were also found, providing evidence that the Ste6p transporter recognizes sequence determinants as well as CAAX modifications. We also performed a phenotypic analysis of the entire set of isogenic a-factor biogenesis machinery mutants, which revealed information about the dependency of biogenesis steps upon one another, and demonstrated that export by Ste6p requires the completion of all processing events. Overall, this comprehensive analysis will provide a useful framework for the study of other fungal pheromones, as well as prenylated metazoan proteins involved in development and aging.     

A Novel Role of the Yeast CaaX Protease Ste24 in Chitin Synthesis

Ste24 is a membrane-integral CaaX metalloprotease residing in the endoplasmic reticulum (ER). In yeast, the only known substrate of Ste24 is the mating factor a precursor. A global screening for protein–protein interactions indicated that Ste24 interacts with chitin synthesis deficient (Chs)3, an enzyme required for chitin synthesis. We confirmed this interaction by yeast two-hybrid analyses and mapped the interacting cytoplasmic domains. Next, we investigated the influence of Ste24 on chitin synthesis. In sterile (ste)24Δ mutants, we observed resistance to calcofluor white (CFW), which was also apparent when the cells expressed a catalytically inactive version of Ste24. In addition, ste24Δ cells showed a decrease in chitin levels and Chs3-green fluorescent protein localized less frequently at the bud neck. Overexpression of STE24 resulted in hypersensitivity to CFW and a slight increase in chitin levels. The CFW phenotype of ste24Δ cells could be rescued by its human and insect orthologues. Although Chs3 binds to Ste24, it seems not to be a substrate for this protease. Instead, our data suggest that Chs3 and Ste24 form a complex in the ER that facilitates protease action on prenylated Chs4, a known activator of Chs3 with a C-terminal CaaX motif, leading to a more efficient localization of Chs3 at the plasma membrane.     

Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p

STE50 is required to sustain pheromone-induced signal transduction in S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require G_α, G_β, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth. Received: 20 February 1998 / Accepted: 17 March 1998     

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.     

Receptor Inhibition of Pheromone Signaling Is Mediated by the Ste4p Gβ Subunit

The pheromone response pathway of the yeast Saccharomyces cerevisiae is initiated in MATa cells by binding of α-factor to the α-factor receptor. MATa cells in which the a-factor receptor is inappropriately expressed exhibit reduced pheromone signaling, a phenomenon termed receptor inhibition. In cells undergoing receptor inhibition, activation of the signaling pathway occurs normally at early time points but decreases after prolonged exposure to pheromone. Mutations that suppress the effects of receptor inhibition were obtained in the STE4 gene, which encodes the β-subunit of the G protein that transmits the pheromone response signal. These mutations mapped to the N terminus and second WD repeat of Ste4p in regions that are not part of its G_α binding surface. A STE4 allele containing several of these mutations, called STE4^SD13, reversed the signaling defect seen at late times in cells undergoing receptor inhibition but had no effect on the basal activity of the pathway. Moreover, the signaling properties of STE4^SD13 were indistinguishable from those of STE4 in wild-type MATa and MATα cells. These results demonstrate that the effect of the STE4^SD13 allele is specific to the receptor inhibition function of STE4. STE4^SD13 suppressed the signaling defect conferred by receptor inhibition in a MATa strain containing a deletion of GPA1, the G protein α-subunit gene; however, STE4^SD13 had no effect in a MATα strain containing a GPA1 deletion. Suppression of receptor inhibition by STE4^SD13 in a MATa strain containing a GPA1 deletion was unaffected by deletion of STE2, the α-factor receptor gene. The results presented here are consistent with a model in which an a-specific gene product other than Ste2p detects the presence of the a-factor receptor and blocks signaling by inhibiting the function of Ste4p.     

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.     

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.     

Phosphorylation of the pheromone-responsive Gβ protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function

The pheromone-responsive G_β subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310–346, ste4Δ310–346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho^?) alleles of STE4: ste4-T320?A/S335A and ste4-T322 A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4Δ310–346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive G_α of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310–346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.     

A Large PEST-like Sequence Directs the Ubiquitination,Endocytosis, and Vacuolar Degradation of the Yeast a-Factor Receptor

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661–674) and both depend on sequence elements within the receptor''s regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t _1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity—no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences—a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.     

Identification of a human cDNA encoding a novel protein structurally related to the yeast membrane-associated metalloprotease, Ste24p

Recently, a novel membrane-associated metalloprotease, designated Ste24p, has been identified in Saccharomyces cerevisiae [K. Fujimura-Kamada, F.J. Nouvet, S. Michaelis, J. Cell Biol. 27 (1997) 271-285]. We cloned a human brain cDNA encoding a protein homologous to Ste24p (designated Hs Ste24p). The predicted 475-amino acid product of its open reading frame exhibited 62% similarity to Ste24p, and contained a zinc metalloprotease motif (HEXXH) and multiple predicted membrane spans. Northern blot analysis showed that this gene was expressed in most tissues. Immunofluorescence analysis of epitope-tagged Hs Ste24p constructs suggested that it is localized in the ER and possibly also in the Golgi compartment. A search of the expression sequence tag database identified a fragment of DNA encoding a segment homologous to the segment of Hs Ste24p containing the HEXXH motif in insects and nematodes. Thus, Hs Ste24p could be a member of a new family of Ste24p-like membrane-associated metalloproteases which are widely conserved in eukaryotes.     

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.     

The primary structure of cytochrome P-450d purified from rat liver microsomes: Prediction of helical regions and domain analysis

The complete amino acid sequence of rat liver microsomal cytochrome P-450d (induced by isosafrole) was deduced by microsequence analysis of the tryptic peptides after separation by reverse-phase HPLC and alignment by comparison to the cDNA sequence reported by K. Kawajiri, O. Gotoh, K. Sogawa, Y. Tagashira, M. Muramatsu, and Y. Fujii-Kuriyama (1984, Proc. Natl. Acad. Sci. USA, 81, 1649–1653). Results from the two approaches are in complete agreement with the exception of two residues, Lys-30 (Arg in cDNA) and Phe-261 (Ser in cDNA). As previously reported by us (L. H. Botelho, D. E. Ryan, P.-M. Yuan, R. Kutney, J. E. Shively, and W. Levin (1982, Biochemistry, 21, 1152–1155) the NH₂-terminal sequence of the mature protein lacks the NH₂-terminal Met residue. Comparison of the rat cytochrome P-450d sequence with the mouse cytochrome P₃-450 cDNA sequence reported by S. Kimura, F. J. Gonzalez, and D. W. Nebert (1984, Nucl. Acids Res., 12, 2917–2928) reveals a high sequence homology with a total of 32 amino acid differences including six conferring charge changes. Prediction of the secondary structure of cytochrome P-450d yields a maximum of 17 helices, two of which may be poly(Pro)-like helices adjacent to potential membrane-spanning α-helices. Four of the α-helices are sufficiently hydrophobic to traverse the endoplasmic reticulum. The remaining helices are largely amphiphilic. Analysis of the helices in reference to predicted membrane topology suggests that cytochrome P-450d either (1) has one large and one small globular domain separated by a transmembrane domain and anchored by NH₂-terminal and COOH-terminal transmembrane domains, or (2) has one large globular domain anchored at both ends by transmembrane domains.