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.     

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.     

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.     

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.     

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.     

Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p

Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.     

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.     

Topological and Mutational Analysis of Saccharomyces cerevisiae Ste14p, Founding Member of the Isoprenylcysteine Carboxyl Methyltransferase Family

Eukaryotic proteins that terminate in a CaaX motif undergo three processing events: isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. In Saccharomyces cerevisiae, the latter step is mediated by Ste14p, an integral endoplasmic reticulum membrane protein. Ste14p is the founding member of the isoprenylcysteine carboxyl methyltransferase (ICMT) family, whose members share significant sequence homology. Because the physiological substrates of Ste14p, such as Ras and the yeast a-factor precursor, are isoprenylated and reside on the cytosolic side of membranes, the Ste14p residues involved in enzymatic activity are predicted to be cytosolically disposed. In this study, we have investigated the topology of Ste14p by analyzing the protease protection of epitope-tagged versions of Ste14p and the glycosylation status of Ste14p-Suc2p fusions. Our data lead to a topology model in which Ste14p contains six membrane spans, two of which form a helical hairpin. According to this model most of the Ste14p hydrophilic regions are located in the cytosol. We have also generated ste14 mutants by random and site-directed mutagenesis to identify residues of Ste14p that are important for activity. Notably, four of the five loss-of-function mutations arising from random mutagenesis alter residues that are highly conserved among the ICMT family. Finally, we have identified a novel tripartite consensus motif in the C-terminal region of Ste14p. This region is similar among all ICMT family members, two phospholipid methyltransferases, several ergosterol biosynthetic enzymes, and a group of bacterial open reading frames of unknown function. Site-directed and random mutations demonstrate that residues in this region play a critical role in the function of Ste14p.     

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.     

Role for the Ubiquitin-Proteasome System in the Vacuolar Degradation of Ste6p, the a-Factor Transporter in Saccharomyces cerevisiae

Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.     

AtFACE-2, a functional prenylated protein protease from Arabidopsis thaliana related to mammalian Ras-converting enzymes

Eukaryotic proteins containing a CAAX (A is aliphatic amino acid) C-terminal tetrapeptide sequence generally undergo a lipid modification, the addition of a prenyl group. Proteins that are modified by prenylation, such as Ras GTPases, can be subsequently modified by a proteolytic event that removes a C-terminal tripeptide (AAX). Two distinct proteases have been identified that are involved in the CAAX proteolytic step, FACE-1/Ste24 and FACE-2/Rce1. These proteases have different enzymatic properties, substrate specificities, and biological functions. However, a proposal has been made that plants lack a FACE-2/Rce1-type protease. Here, we describe the isolation of a cDNA from Arabidopsis thaliana that encodes a 311-aa protein with characteristics that are similar to the FACE-2/Rce1 group of enzymes. Northern blot analysis demonstrates widespread expression of this gene in plant tissues. Heterologous expression of the A. thaliana cDNA in yeast restores CAAX proteolytic activity to yeast lacking native CAAX proteases. The recombinant protein produced in this system displays an in vivo substrate specificity profile distinct from AtSte24 and cleaves a farnesylated CAAX tetrapeptide in vitro. These results provide evidence for the existence of a previously unsuspected plant FACE-2/Rce1 ortholog and support the evolutionary conservation of dual CAAX proteolytic systems in eukaryotes.     

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.     

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.     

Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein

The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.     

Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl ketones toward the CaaX proteases

Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A ‘warhead’ free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.     

A membrane-associated metalloprotease of Taenia solium metacestode structurally related to the FACE-1/Ste24p protease family

The CaaX proteases are intimately involved in the post-translational modification of prenylated proteins and play a critical role in the activation/stabilization of membrane-bound or secreted molecules constituting the CAAX protein family. In this study, we have isolated a full-length cDNA putatively encoding a type I CaaX protease of the Taenia solium metacestode (TsM), which an agent causative of human neurocysticercosis. The cDNA, designated TsSte24p, comprised 1,505 bp and coded for an open reading frame of 472 amino acids with predicted Mr 54.5 kDa. This monoexonic TsSte24p gene existed as a single copy within the TsM genome and constantly expressed in the parasite from metacestode to adult stages. The TsSte24p exhibited the typical CaaX protease topology, including seven transmembrane domains and a metalloprotease segment with a zinc-binding motif. It shared a significant degree of sequence identity with the type I CaaX proteases such as Saccharomyces cerevisiae Ste24p and Caenorhabditis elegans CeFACE-1. A comparative phylogenetic analysis demonstrated that this protein family is tightly conserved across taxa, from bacteria to mammals. The bacterially expressed recombinant TsSte24p showed proteolytic activity, with an optimal pH of 7.5. The enzyme activity was significantly inhibited by EDTA. Its activity was increased in the presence of low concentrations of the Zn2+(0.001-0.01 mM); but was reversibly down-regulated at high doses (over 0.1 mM). The native TsSte24p appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bond. The protein was localized in the bladder wall and scolex with differential patterns of distribution. Our results indicated that TsSte24p is a zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.     

The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants.

We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life > 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.     

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.     

The Arabidopsis AtSTE24 is a CAAX protease with broad substrate specificity

Following prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting. First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety. An Arabidopsis homologue of the yeast CAAX protease STE24 (AFC1) was cloned and expressed in rce1 Delta ste24 Delta mutant yeast to demonstrate functional complementation. The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box. Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24. In addition, AtSTE24 promoted plasma membrane association of the GFP-Rac fusion protein, which terminates with a CLLM CAAX box. Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data. Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established. Transient expression of GFPAtSTE24 in leaf epidermal cells of Nicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation.     

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.