Saccharomyces cerevisiae a-Factor Mutants Reveal Residues Critical for Processing, Activity, and Export

The Saccharomyces cerevisiae mating pheromone a-factor providesa paradigm for understanding the biogenesis of prenylated fungalpheromones. 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, andSte14p, respectively); (ii) N-terminal processing, involvingtwo sequential proteolytic cleavages (by Ste24p and Axl1p);and (iii) nonclassical export (by Ste6p). Once exported, maturea-factor interacts with the Ste3p receptor on MAT ${alpha}$ cells to stimulatemating. The a-factor biogenesis machinery is well defined, asis the CAAX motif that directs C-terminal modification; however,very little is known about the sequence determinants within a-factorrequired for N-terminal processing, activity, and export. Herewe generated a large collection of a-factor mutants and identifiedresidues critical for the N-terminal processing steps mediatedby Ste24p and Axl1p. We also identified mutants that fail to supportmating but do not affect biogenesis or export, suggesting a defectiveinteraction with the Ste3p receptor. Mutants significantly impairedin export were also found, providing evidence that the Ste6p transporterrecognizes sequence determinants as well as CAAX modifications.We also performed a phenotypic analysis of the entire set ofisogenic a-factor biogenesis machinery mutants, which revealedinformation about the dependency of biogenesis steps upon one another,and demonstrated that export by Ste6p requires the completion ofall processing events. Overall, this comprehensive analysiswill provide a useful framework for the study of other fungalpheromones, as well as prenylated metazoan proteins involvedin development and aging.

INTRODUCTION

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Introduction

Most fungi secrete pheromones that play important signaling rolesin mating to stimulate cell and/or nuclear fusion. In Saccharomycescerevisiae and other ascomycetes, the mating pheromones producedby the two mating types differ, in that one is an unmodifiedpeptide, while the other is a prenylated and carboxymethylatedpeptide (i.e., ${alpha}$ -factor and a-factor, respectively) (3, 7, 21, 24, 25, 41, 49, 59, 61, 70).Basidomycetes, which can have two or even more mating types,encode exclusively the latter class of modified peptide pheromones (8, 13, 18, 26, 27, 36, 47, 56, 60).These pheromones are synthesized as precursors terminating ina C-terminal CAAX motif (where C is cysteine, A is usually aliphatic,and X is any residue). The S. cerevisiae pheromone a-factoris the best-characterized example of this class of fungal pheromonesand provides a paradigm for understanding their biogenesis andsecretion (14, 20). Studies of a-factor have also yielded criticalinsights into the processing and properties of eukaryotic CAAXproteins in general, which include Ras and other small GTP-bindingproteins, the ${gamma}$ -subunits of heterotrimeric G proteins, and thenuclear lamins A and B (30, 68, 69).

Components of the machinery required for the biogenesis of a-factorhave been defined by the identification of mating-defectivesterile (ste) mutants, and the step at which they act has been determinedby analysis of biosynthetic intermediates that accumulate in thesemutants. Mature bioactive a-factor is derived from a 36- or38-amino-acid precursor encoded by two similar and functionally redundantgenes, MFA1 and MFA2, respectively (11, 44). Studies here and elsewherehave focused on Mfa1p. A schematic of the biogenesis pathway fora-factor, with intermediates and machinery for each step, is shownin Fig. 1A. Note that the identity of these intermediates hasbeen previously established (20, 63). The first stage in maturationof the a-factor precursor (P0) is modification of the C-terminalCAAX motif. CAAX modification involves three enzymatic steps:farnesylation of the Cys residue, by the Ram1p/Ram2p heterodimericfarnesyltransferase (step 1, P0 $->$ P0*) (20, 31, 50); endoproteolytic cleavageof the AAX residues, carried out redundantly by Ste24p or Rce1p(step 2, P0* $->$ P1') (9, 10, 63); and carboxymethylation of theC-terminal Cys residue, mediated by Ste14p (step 3, P1' $->$ P1) (32, 55).The second stage in a-factor maturation involves cleavage ofthe N-terminal extension in two sequential steps mediated bySte24p (step 4, P1 $->$ P2) (28, 64) and Axl1p (step 5, P2 $->$ M) (1).Ste23p (not shown) acts redundantly with Axl1p, but its contributionto processing is minor (1, 35). Finally, export of a-factorout of the cell is mediated by an ATP binding cassette transporter,Ste6p (step 6) (5, 37, 42, 43). Importantly, all of these processingevents are predicted to occur in the cytosol or on the cytosolicface of intracellular membranes (53, 57), and mature a-factoris exported directly across the plasma membrane by Ste6p. Thus,unlike the ${alpha}$ -factor pheromone, a-factor processing completelybypasses the luminal compartments of the classical secretorypathway. Interestingly, most of the components of the a-factorprocessing machinery (Ste24p, Rce1p, Ste14p, and Ste6p) aremultispanning membrane proteins. Their catalytic properties andlocalization have been the subject of intense investigationsover the past several years (34, 48, 52, 58, 64, 66).

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FIG. 1. a-Factor biogenesis pathway. (A) Schematic of the a-factor biogenesis pathway, with intermediates and machinery shown for each step. See text for a detailed description of each step. The a-factor precursor encoded by MFA1 is shown (P0), with the N-terminal extension (consisting of two portions, shaded in gray and unshaded), the mature sequence (shaded in black), and the CAAX motif indicated. The P0 precursor is converted to mature, extracellular a-factor (M) through the indicated biosynthetic intermediates (steps 1 to 6). Farnesylation (of the C-terminal Cys) and carboxymethylation (-O-Me) are indicated. (B) Metabolic labeling analysis of a-factor in a WT strain (bottom panel) and in isogenic deletion mutants that block each step of the pathway. Cells were pulse labeled with [³⁵S]Cys for 5 min and chased with excess unlabeled Cys for the indicated times. Intracellular (I) and extracellular (E) fractions were prepared, and a-factor species were immunoprecipitated and analyzed as described in Materials and Methods. The mobility of the precursor (P0), intermediates (P0*, P1, and P2), and mature (M) a-factor are marked. Note that P0 and P0* are rapidly converted to P1 and are only visible in mutant strains that result in their accumulation (ram1 ${Delta}$ and rce1 ${Delta}$ ste24 ${Delta}$ strains, respectively). For the ste14 ${Delta}$ mutant, all the species are unmethylated and are therefore labeled as P1', P2', and M'.

While much is known about the biogenesis machinery, less isknown about critical residues within a-factor that are recognizedby this machinery. The exception to this is CAAX processing(Fig. 1A, steps 1 to 3), for which a-factor has been an importantmodel molecule in defining the sequence specificities of theprenylation and CAAX endoproteolysis enzymes (14, 16, 20, 30, 66).By contrast, systematic studies have not been performed to analyzethe sequence requirements for the N-terminal processing stepsby Ste24p and Axl1p (Fig. 1A, steps 4 and 5). Similarly, thedeterminants for Ste6p recognition are poorly defined (Fig.1A, step 6). It has been suggested that Ste6p recognition maybe solely dependent on the prenyl and methyl CAAX modificationsand not the specific residues within the mature peptide, butthere is very little evidence to support this suggestion. Studieswith synthetic peptides have demonstrated that the CAAX modificationsare required for recognition by, or access to, the a-factorreceptor (Ste3p) (40); however, little is known about the residuesin the mature peptide that may also contribute to interactionwith Ste3p.

In this study, we identified residues within a-factor criticalfor biogenesis and function through an extensive mutagenesisanalysis. This comprehensive analysis revealed, for the firsttime, distinct sequence determinants for the N-terminal processingsteps mediated by Ste24p and Axl1p. In addition, numerous mutationswere identified throughout the mature portion of a-factor thatdo not affect biogenesis or export but have a strong effecton activity and likely affect interaction with Ste3p. Interestingly,we also identified several mutants in which biogenesis is normalbut export is strongly impaired, suggesting that Ste6p may recognizespecific residues or structural features of a-factor in additionto the CAAX modifications.

We also performed an in-depth analysis of an isogenic set ofbiogenesis machinery mutants, which clearly indicated that ablock at any step in C- or N-terminal processing prevents exportof a-factor. Interestingly, whereas farnesylation (Fig. 1A, step1) is required for all downstream steps (steps 2 to 6), N-terminal processing(steps 4 and 5) was found to proceed in the absence of C-terminalproteolysis and carboxymethylation (steps 2 and 3). However, thesepartially processed intermediates were not competent for export bySte6p. Thus, only properly processed, mature a-factor is a substratefor the Ste6p transporter. Overall, the mutants generated in thisstudy form the basis for a better understanding of the stepsin a-factor biogenesis and the requirements of the machinerythat mediate those steps and provide a foundation for dissectingthe biogenesis of other lipid-modified fungal pheromones.

MATERIALS AND METHODS

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Materials and Methods

Plasmids and strains. The starting plasmid for a-factor mutagenesis was pSM478 (20).This plasmid contains the MFA1 open reading frame with 624 bpof 5' untranslated region and 833 bp of 3' untranslated regioncloned into pRS316 (CEN URA3) as an EcoRI-XbaI fragment; to facilitatesubcloning, an HpaI site at –50 and an MluI site at +130were introduced by site-directed mutagenesis. Degenerate oligonucleotidemutagenesis was used to mutagenize the MFA1 open reading framein blocks of five or six codons (oSM91, Q2 to T7; oSM90, A8to E12; oSM89, K13 to E17; oSM88, K18 to Y22; oSM73, I23 to V27;oSM68, F28 to A32). Degenerate oligonucleotides contained 12 nucleotidesflanking the 5' and 3' sides of the degenerate nucleotides.During oligonucleotide synthesis, at the sites of degeneracythe normal (wild-type) nucleotide was "spiked" with an equimolarmixture of all four nucleotides (at a ratio of 93% normal to7% mixture). Mutagenesis was performed using a Mutagene Phagemidin vitro mutagenesis kit (Bio-Rad). Pools of mutant plasmidsderived from each degenerate oligonucleotide were isolated fromEscherichia coli and transformed into the a-factorless strainSM1458 (MATa trp1 leu2 ura3 his4 can1 mfa1::LEU2 mfa2::lacZ) (20),and the transformants were screened by plate mating and halotests (44, 46) to identify mutants defective in a-factor biogenesisor activity. Mutant plasmids were recovered from yeast and sequencedto determine the site of mutation. Sequencing of random plasmidsfrom several pools indicated that the degenerate oligonucleotideprocedure yielded single or multiple mutants in about 25% ofplasmids. Deletion mutations were made by oligonucleotide-directedmutagenesis.

The strains used for this study are all derived from SM1058(Table 1). The a-factor biogenesis machinery mutant strainsused in the experiments shown in Fig. 1 and 2 are all isogenicto SM1058 and are described in Table 1. The wild-type (WT) strainused in the experiments shown in Fig. 3 is SM2331 (MATa trp1leu2 ura3 his4 can1 mfa1 ${Delta}$ mfa2 ${Delta}$ ) (20). The biogenesis machinerymutant strains shown in Fig. 3 are all derived from SM2331:SM3375(ste24::LEU2), SM3689 (rce1::TRP1), and SM3691 (ste24::LEU2 rce1::TRP1) (63).a-Factor was expressed in these strains from pSM1605 (2µURA3 MFA1-CVIA) (58) or pSM1795 (2µ URA3 MFA1-CTLM) (38).For the analyses shown in Fig. 4 to 6, the a-factor mutant plasmidswere transformed into an a-factorless strain, either SM1458(see above) or SM1229 (MATa trp1 leu2 ura3 his4 can1 mfa1::LEU2 mfa2::URA3) (44)(Table 2).

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TABLE 1. Mating of a-factor biogenesis machinery mutant strains

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FIG. 2. a-Factor halo and mating assays with isogenic biogenesis machinery mutant strains. (A) The WT strain and the indicated isogenic mutants were tested for their ability to form a halo on a lawn of MAT ${alpha}$ cells, as described in Materials and Methods. The diameter of the halo is proportional to the amount of a-factor secreted. (B) Microtiter plate mating assays were performed as described in Materials and Methods, with the dilutions indicated. (C) Quantitative mating assays were performed as described in Materials and Methods. The graph is derived from data presented in Table 1. Assays were performed in triplicate, with WT set to 100% and the mutants expressed as a percentage of WT mating efficiency. The asterisk (*) indicates mutants with a mating efficiency of <0.001%.

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FIG. 3. C-terminal CAAX endoproteolysis is not obligatory for N-terminal processing. (A) CAAX endoproteolysis of WT a-factor (terminating in CVIA; top) can be performed by both Ste24p and Rce1p, while CAAX endoproteolysis of the CTLM variant (bottom) can be performed only by Rce1p (10) (as noted by the cross-through of Ste24p). The Met residues (M) in each a-factor variant are indicated in black type. (B) Halo assays showing that CTLM a-factor halo formation is dependent on Rce1p, unlike WT (CVIA) a-factor, which can be processed by Rce1p or Ste24p. (C) Metabolic labeling of WT (CVIA) a-factor and the CTLM mutant form was carried out as described in the legend of Fig. 1 at the 10-min chase point in the indicated strains. The bands in lane 7 marked with an arrowhead reflect aberrant forms of P2 and M that retain the AAX residues. (D) The experiment is as described for panel C, except that [³⁵S]Met labeling was performed to label the Met residue in the CTLM CAAX motif. The arrowhead in lane 4 marks the aberrant P2 form that retains the AAX residues (TLM).

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FIG. 4. Random mutagenesis of a-factor reveals residues critical for P1 $->$ P2 processing. (A) Summary of mutants isolated. Mutations that result in a processing and mating defect are indicated below the a-factor sequence, while phenotypically silent mutations are indicated above the sequence. (B) Metabolic labeling of a-factor mutants was carried out as described in Materials and Methods. (C) Quantitative mating of strains expressing the indicated a-factor mutants. The graph is derived from data presented in Table 2. afrp, a-factor related peptide.

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FIG. 6. Random mutagenesis of a-factor reveals residues critical for activity and export. (A) Summary of mutants isolated. Mutations that result in an activity defect (black) or export defect (gray) are indicated below the a-factor sequence, while phenotypically silent mutations are indicated above the sequence. (B) Metabolic labeling of a-factor mutants was carried out as described in Materials and Methods. (C) Quantitative mating of strains expressing the indicated a-factor mutants. The graph is derived from data presented in Table 2. (D) Metabolic labeling and pulse-chase analysis of the most severe export-defective mutant, G26V, were carried out as described in the legend of Fig. 1B. afrp, a-factor related peptide.

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TABLE 2. a-Factor mutant strains, plasmids, and quantitative mating data

Metabolic labeling to analyze a-factor biosynthetic intermediates. Metabolic labeling experiments were performed essentially asdescribed (20). Briefly, cells were pulse labeled for 5 minwith [³⁵S]Cys (or [³⁵S]Met; see Fig. 3D) (PerkinElmer) and chasedwith excess unlabeled Cys and/or Met for the indicated times. Forexperiments with a single time point, samples were preparedafter a 10-min chase. Intracellular and extracellular fractionswere prepared, and a-factor was immunoprecipitated with anti-a-factor rabbitpolyclonal antiserum 9-497. Immunoprecipitated a-factor specieswere resolved by 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using gel conditions previously described (20), andthe a-factor species were visualized by phosphorimaging.

Halo and mating assays. Halo assays were performed essentially as described previously (44, 46).Briefly, cells were grown in the appropriate selective medium,concentrated by centrifugation, suspended in H₂O, and spotted(5 µl) onto a lawn of cells supersensitive to a-factor(SM2375: MAT ${alpha}$ sst2 rme his6 met1 can1 cyh2) (50) spread on ayeast extract-peptone-dextrose (YPD) plate containing 0.04%Triton X-100. A zone of growth inhibition (the "halo") resultsfrom a-factor-induced G₁ arrest of the supersensitive MAT ${alpha}$ cells.Plates were incubated at 30°C for 2 days and photographed.

For microtiter plate mating assays, appropriate strains of eachmating type (MATa query strain and the tester strain SM1068[MAT ${alpha}$ lys1]) were grown overnight, diluted to an optical densityat 600 nm (OD₆₀₀) of 1.0, and mixed in a 96-well plate (90 µlof MAT ${alpha}$ and 10 µl of MATa). Serial 10-fold dilutions weremade in YPD medium, and the plate was incubated for 4 h at 30°C. Theplate was centrifuged to pellet the cells, the supernatant was removed,the cells were resuspended in 100 µl of H₂O, and a 10-µlaliquot from each well was spotted on mating-selective plates.Plates were incubated at 30°C for 3 days and photographed.

Quantitative mating assays were performed as previously described (2, 46).Briefly, the MATa (query strain) and MAT ${alpha}$ (SM1068) strains weregrown overnight and diluted to an OD₆₀₀ of 1.0. For each culture,0.5 OD units was mixed and collected on a nitrocellulose filterthat was placed on a YPD plate and incubated for 4 h at 30°C.The cells were then washed off the filter and dilutions wereplated on mating-selective (synthetic medium with dextrose)and control (YPD) plates. The mating efficiency was calculatedas the ratio of colonies on the mating-selective plate relativeto the control plate and then expressed as a percentage of the amountof mating for the WT MATa strain (SM1058).

RESULTS

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Results

Phenotypes of mutants defective in the machinery required for a-factor processing and export. As a prelude to the study of mutations within a-factor, it was importantto definitively establish the phenotypes of mutants defective incomponents of the a-factor biogenesis machinery. Genes encodingthese components were originally isolated based on a-factormating and halo bioassays; however, the severity of the biogenesisdefects has not been systematically quantitated or compared inisogenic strains. Here, we have addressed this gap. To assess biogenesis,we performed metabolic labeling (Fig. 1B). We also carried out threebioassays for a-factor activity, providing an indirect measureof whether a-factor biogenesis has been completed: halo (Fig. 2A),microtiter plate mating (Fig. 2B), and quantitative filter mating(Fig. 2C). Notably, the last method provides a robust and sensitiveassay extending through five logs of activity (from 100% matingfor WT, down to 0.001% of WT mating for certain mutants) (Table 1).Below, we consider findings from these experiments.

In general, the results from all three bioassays (Fig. 2A, B, and C)show the same trend. As expected, deletions of genes known toact nonredundantly in a-factor biogenesis (ram1 ${Delta}$ , ste14 ${Delta}$ , and ste6 ${Delta}$ mutants) are completely defective for mating (<0.001% ofWT, denoted by an asterisk in Fig. 2C) (Table 1). In contrast,for the gene pairs known to function redundantly (rce1 ${Delta}$ and ste24 ${Delta}$ ;ax11 ${Delta}$ and ste23 ${Delta}$ ) (1, 9, 10, 63), only the double mutants arecompletely defective for halo and mating. The single mutants(rce1 ${Delta}$ , ste24 ${Delta}$ , and axl1 ${Delta}$ ) show residual activity (68%, 24%, and2%, respectively) (Fig. 2C). By itself, the ste23 ${Delta}$ mutant hasno phenotype because its contribution to cleavage is small comparedto Axl1p (data not shown; see references 1 and 35). An apparent inconsistencyin these assays is that the ste6 ${Delta}$ mutant is completely defectivefor mating (Fig. 2B and C) but has a significant residual halo(Fig. 2A). In the ste6 ${Delta}$ mutant, mature bioactive a-factor is producedbut is trapped inside the cell. We suspect that a small amount oflysis may occur as cells grow on the plate, allowing releaseof this intracellular a-factor and resulting in growth arrestof the MAT ${alpha}$ cells in the halo assay. However, because the lysedcells are dead and because extracellular a-factor cannot efficientlyrescue the mating defect of cells unable to produce their owna-factor, no mating occurs (12, 44). In contrast to growth onplates as in Fig. 2A, when ste6 ${Delta}$ cells are grown in logarithmicphase in liquid culture where cell lysis is minimal, no a-factoris apparent in the culture supernatant (S. Michaelis, unpublisheddata).

We also examined the isogenic a-factor biogenesis mutants bymetabolic labeling, which permits the visualization of biosyntheticintermediates (Fig. 1B) (20). The analysis shown here is thefirst time that a-factor biogenesis mutants have been testedtogether in a single experiment, permitting several importantconclusions. First, compared to WT, it is striking that no a-factor(mature or precursor) is present in the extracellular (E) fractionof any of these mutants (Fig. 1B, bottom panel of each mutantanalysis). This observation indicates that only properly processedmature a-factor is competent for export by Ste6p. Second, ingeneral each processing step appears to be dependent upon completionof the previous step. For example, in the ram1 ${Delta}$ strain in whichfarnesylation is blocked, no subsequent steps in a-factor biogenesisoccur. An important exception to this rule is the ste14 ${Delta}$ mutant. Inthis mutant, in which biosynthetic intermediates are not carboxymethylated(denoted P1', P2', and M' in Fig. 1B), the N-terminal processingof a-factor nevertheless occurs, breaking the strict dependencyrule stated above. It is notable, then, that whereas the firststep of CAAX processing, farnesylation, is required for N-terminalprocessing, the last step, carboxymethylation, is not essentialfor N-terminal processing. Importantly, the nonmethylated formof mature a-factor produced in the ste14 ${Delta}$ strain cannot be exportedby Ste6p.

CAAX processing is not required for N-terminal processing by Ste24p and Axl1p. To further clarify the dependency (or lack thereof) of processingsteps on one another, we wished to address whether the middlestep of CAAX processing, endoproteolysis (step 2), is requiredfor N-terminal processing of a-factor. However, this analysisis complicated because of the dual roles of Ste24p in the C-and N-terminal steps of a-factor biogenesis (10, 63). Thus,while an rce1 ${Delta}$ ste24 ${Delta}$ double mutant blocks CAAX endoproteolysis(step 2), N-terminal processing cannot proceed in any case,because Ste24p is also required for step 4 (Fig. 1A). To circumventthis issue, we generated a form of a-factor in which CAAX endoproteolysiswas strictly Rce1p dependent, leaving Ste24p to function solelyin N-terminal processing. This was accomplished by replacingthe CAAX motif of WT a-factor (CVIA) with an Rce1p-specificCAAX motif (CTLM) (10). These constructs are diagrammed in Fig. 3A, and the Rce1p-dependency for CAAX endoproteolysisof CTLM a-factor is evident in the halo assay shown in Fig. 3B.

In a metabolic labeling to visualize biosynthetic intermediates(Fig. 3C), the intracellular processing patterns of WT (CVIA)a-factor and CTLM a-factor were found to be essentially indistinguishable, indicatingthat CAAX endoproteolysis, like carboxymethylation, is not essentialfor N-terminal processing. Strikingly, no mature a-factor isobserved in the extracellular fraction of CTLM a-factor in rce1 ${Delta}$ (Fig. 3C, lane 7; compare this to the extracellular fractionof WT a-factor, lane 3) or by the halo assay (Fig. 3B). Theseresults are consistent with the findings discussed above forthe carboxymethyl transferase mutant, ste14 ${Delta}$ , which is not unexpected,as the unproteolyzed CTLM a-factor cannot be methylated.

While the CTLM a-factor species produced in the rce1 ${Delta}$ strainare predicted to retain the AAX residues, a mobility shift wasnot apparent in the results shown in Fig. 3C, presumably becausethe contribution of prenylation (which significantly altersthe gel mobility of proteins) obscures it. To directly determinewhether or not CAAX endoproteolysis had occurred, we performed[³⁵S]Met labeling of CTLM a-factor (Fig. 3D), which allows direct visualizationof N-terminally processed species that retain the CAAX motif(since Met is the final residue of this CAAX variant and theonly labeled residue that remains after P1 $->$ P2 cleavage) (Fig. 3A).In the rce1 ${Delta}$ mutant, a novel band is apparent for CTLM a-factor(and absent for CVIA a-factor) that corresponds to the P2 formwith its CAAX motif uncleaved (Fig. 3D, lane 4), indicating thatit retains the labeled Met residue in the CTLM CAAX motif. (The apparentlack of mature a-factor in lane 4 is a signal-strength issue,as longer exposures reveal this band [data not shown].) Thus, thisexperiment provides additional evidence that CAAX endoproteolysis isnot required for N-terminal proteolysis to proceed.

Mutational analysis defines residues important for efficient P1 $->$ P2 processing by Ste24p. Having examined the processing machinery and biogenesis stepsin detail, we next turned to a mutagenic analysis of a-factoritself. With the exception of the CAAX motif, which has beenwell studied (9, 14, 20), very few mutants that affect biogenesisor activity have been reported. Here, we randomly mutagenizedMFA1, except for the CAAX motif, in blocks of five or six codons(15 to 18 nucleotides) and screened for mutants that do notproduce mature a-factor, as assessed by mating and halo assays.Processing defects were determined by metabolic labeling, bothfor mutants compromised in activity and for several phenotypicallysilent mutants. In certain cases, deletions or specific pointmutants were also generated and analyzed. This combination of approachesyielded a large collection of mutants defective in a-factorN-terminal processing, export, or activity. The results of thisanalysis are shown in Fig. 4 to 6, and mutants are discussedaccording to the biogenesis step that is affected.

The first of the N-terminal processing events which convertsP1 a-factor to P2 (step 4) occurs between residues T7 and A8and is carried out by the protease Ste24p (20, 28, 64). Threemutants were isolated that showed a striking accumulation ofP1 a-factor and lack further processing: A8G, A8T, and A9P (Fig. 4A and B).In Fig. 4B, an intracellular species that runs slightly fasterthan mature (M) a-factor can be observed. This band has beenpreviously characterized as an a-factor related peptide (afrp)and is a prenylated and methylated 7 mer derived from MFA1,whose production is independent of Ste24p and Axl1p and thusshould not be confused with mature a-factor (19). It is variably pulleddown in immunoprecipitations and as a result does not appearin all experiments.

The mutated residues A8 and A9 lie immediately C terminal tothe P1 $->$ P2 cut site, defining these two residues as importantelements for Ste24p recognition. No mutants defective in a-factorproduction were recovered in the region N terminal to the processingsite (Fig. 4A), suggesting that this region does not containrecognition determinants for Ste24p. For example, the T5N A6Vdouble mutant that was recovered from the mutagenesis screenwas phenotypically silent. Furthermore, the ${Delta}$ QPS and ${Delta}$ QPSTAT deletions(Fig. 4B, lanes 7 and 8) had no effect on a-factor maturationor export.

These mutants were also subjected to quantitative mating assays(Fig. 4C and Table 2). The mutants with no biogenesis defect(Fig. 4B, lanes 2, 7, and 8) mated like WT, while the P1 $->$ P2processing mutants A8G, A8T, and A9P showed reduced mating.Notably, however, the mating defect was only partial, rangingfrom 45 to 60% of WT, similar to but slightly higher than thatof the ste24 ${Delta}$ strain ( $~$ 25%) (Fig. 2C). The reason for the highlevel of residual mating for these mutants is presumably the sameas we propose for the ste24 ${Delta}$ strain (i.e., a low level of directP1 $->$ P2 processing by Axl1p) (see Discussion).

It should be noted that relatively few mutants were isolatedthat are defective in the Ste24p-dependent processing step. Therandom MFA1 mutants were screened primarily for plate-matingdefects, and the high level of residual mating that occurs whenthe Ste24p processing site is blocked may explain why so few mutantswere identified. To more fully define the site will require additional,focused site-directed mutagenesis of the region. However, ourresults suggest that residues immediately C terminal to thecut site are the most important, and the mutants we have isolatedhave provided valuable tools for blocking P1 $->$ P2 processingof a-factor, independent of using a strain in which the Ste24p processingenzyme is absent.

Mutational analysis defines residues important for efficient P2 $->$ M processing by Axl1p. Axl1p-dependent processing of a-factor occurs between residuesN21 and Y22 (3, 20). Our random mutagenesis analysis revealednumerous mutations that prevent P2 $->$ M processing in residuesflanking both sides of this cleavage site (D20Y, N21I, Y22N,and Y22D) (Fig. 5A and B, lanes 11 to 14). Similar to resultsshown in Fig. 4B, the a-factor related peptide (afrp) speciesappears prominently in Fig. 5B but is distinct from M a-factor.Whereas no mutations C terminal to Y22 had an impact on Axl1pcleavage (see Fig. 6), mutations at E17, which is five residuesN terminal to the Axl1p cut site, blocked processing. Indeed,two mutants altered at E17 were found (E17K and S15R+E17Q) (Fig. 5B,lanes 6 and 7); however, because the S15R single mutant is phenotypicallysilent, the effect of the double mutant is most likely due solelyto the E17Q mutation. Interestingly, the K18A and K19A mutationsare phenotypically silent (lanes 8 and 9), even though theyare surrounded by mutations that block processing. We also generatedseveral deletions (Fig. 5A and B, lanes 16 to 18). Not surprisingly, ${Delta}$ NY, which removes the Axl1p cleavage site, blocked processing.Somewhat unexpectedly, even distant deletions within the N-terminalextension ( ${Delta}$ PKEK and ${Delta}$ TSSE) also blocked Axl1p processing, suggestingthat the structure and/or length of the N-terminal extensionis important for this cleavage step.

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FIG. 5. Random mutagenesis of a-factor reveals residues critical for P2 $->$ M processing. (A) Summary of mutants isolated. Mutations that result in a processing and mating defect are indicated below the a-factor sequence, while phenotypically silent mutations are indicated above the sequence. (B) Metabolic labeling of a-factor mutants was carried out as described in Materials and Methods. (C) Quantitative mating of strains expressing the indicated a-factor mutants. The graph is derived from data presented in Table 2. afrp, a-factor related peptide.

It is notable that all of the mutants that affected Axl1p processingwere severely defective in mating (Fig. 5C and Table 2), rangingfrom <0.1% to 10% of WT. This result is in contrast to the significantresidual mating observed with the mutants defective for P1 $->$ P2 (Ste24p) processing (Fig. 4C), suggesting that a block inP2 $->$ M processing cannot be circumvented.

Mutations in the mature a-factor sequence reveal residues essential for export and activity. Random mutagenesis of the mature a-factor sequence (residues22 to 32 of Mfa1p) was also carried out. Numerous mutationsthat resulted in mating defects were isolated throughout thesequence of the mature peptide (Fig. 6A). To determine the basisfor the mating defect, we analyzed the mutants by metaboliclabeling and SDS-PAGE (Fig. 6B). In all cases, intracellularmature a-factor was produced, indicating that all the processingsteps occurred.

The mutants fell into two classes, based on the presence orabsence of extracellular a-factor. The larger class of mutantsexhibited extracellular mature a-factor at a WT level (Fig.6B; note that this figure is a composite of multiple gels, eachwith its own WT control) but were significantly reduced in mating(40% to <0.1% of WT) (Fig. 6C and Table 2). For instance,the W29C a-factor mutant exhibited normal processing and export(Fig. 6B, lane 14) yet is completely defective for mating (<0.01%of WT) (Fig. 6C and Table 2), most likely because it is unableto productively interact with the Ste3p receptor. Other mutantsin this class included I23M, I23N, F28A, F28N, W29R, D30E, and P31A(Fig. 6A, residues in black boxes; B, lanes 2, 3, 10, 12, 15,16, and 17; and C). Complex (double and triple) mutations ina-factor at residues G26, V27, W29, D30, and A32 result in asimilar phenotype and have been previously reported by anothergroup (17). The present series of mutants greatly broadens thescope of residues that result in defective receptor interaction.

The smaller class of mutants were significantly reduced (G26C,P31Q, and A32K) (Fig. 6B, lanes 8, 18, and 20) or completelyblocked (G26V) (Fig. 6B, lane 7) in export. This class of mutantsis noted in gray in Fig. 6A to C. To confirm that the G26V mutantwas indeed export defective, we examined its biogenesis andexport over an extended time course of up to 30 min (Fig. 6D).No evidence of export was observed at any time point. Not surprisingly,the export-defective mutants were also defective for mating(Fig. 6C and Table 2); however, it is not possible to determinewhether the activity defect is due solely to the export defector if the mutation also affects interaction with the Ste3p receptor.

Taken together, we found a relatively small number of export-defectivea-factor mutants, compared to a large number of export-competentmutants (albeit with compromised activity). Thus, Ste6p recognitionof a-factor may have less to do with the sequence of a-factorand more to do with its length and C-terminal CAAX modifications(farnesylation and carboxymethylation). By contrast, recognitionof a-factor by the a-factor receptor, Ste3p, appears to be highlysequence dependent.

DISCUSSION

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Discussion

The hallmark of all fungal genes encoding lipophilic pheromonesis the C-terminal CAAX motif (8, 13, 14), which directs prenylation,endoproteolysis, and carboxymethylation. For S. cerevisiae a-factor,the best-studied member of this class of pheromones, mutagenesisof this motif has shown the importance of the CAAX modificationsfor pheromone biogenesis, stability, and bioactivity (16, 20, 55).Analysis of synthetic peptides also reflects the significanceof the CAAX modifications for pheromone activity (40, 67). However,less is known about the importance of the rest of the precursorsequence in terms of directing maturation, export, and activity.Because of the considerable sequence divergence among the lipophilicpheromones, important residues cannot be predicted by sequencecomparisons. The comprehensive mutational analysis of a-factorpresented here has provided insights into substrate recognitiondeterminants for the N-terminal processing steps of a-factormediated by Ste24p and Axl1p and for recognition by the Ste3preceptor and the Ste6p transporter. A better understanding ofhow the biogenesis machinery recognizes its substrates willbe valuable for understanding biogenesis of other fungal pheromonesas well as mammalian CAAX proteins, like prelamin A, that undergoadditional N-terminal processing similar to a-factor (4, 39, 68).

Ste24p and the P1 $->$ P2 processing step. Ste24p is a multispanning endoplasmic reticulum membrane proteinwith a zinc metalloprotease activity. It is unusual among componentsof the a-factor processing machinery in that it plays two roles:an overlapping role with Rce1p in CAAX endoproteolysis (Fig.1, step 2) (10, 63) and a unique role in the first N-terminalprocessing step (Fig. 1, step 4) (28). The sequence recognitiondeterminants for CAAX endoproteolysis by Ste24p have been defined,but little is known about determinants for P1 $->$ P2 processing.Here we report three mutants with alterations in the two residuesimmediately C terminal to the cut site that block this processingstep: A8G, A8T, and A9P. We previously reported that A8G blocksP1 $->$ P2 processing, both in vivo (28) and in vitro (58). Thesemutants indicate that Ste24p exhibits sequence specificity atthe P1 $->$ P2 processing site and that it is not simply actingas a molecular ruler and cleaving at a set distance N terminalto the farnesylcysteine (although farnesylation is clearly importantfor recognition, as Ste24p N-terminal processing does not occurin the absence of farnesylation [Fig. 1B]). It is interestingto note that the P1 $->$ P2 processing site bears no sequence resemblanceto the C-terminal CAAX endoproteolysis site (indeed, the lattermust contain a farnesylcysteine), indicating that Ste24p isunusual in having the ability to recognize at least two very differentsites. Yet in spite of this, Ste24p exhibits considerable sequencespecificity, as demonstrated by the mutants identified here (Fig.4). One possibility is that Ste24p has two active sites, onefor each cleavage event, for which there is modest evidence (22),although additional work is needed to clarify this issue.

It is notable that when the sequences of all known lipophilicfungal pheromones are aligned, no consensus sequence aroundthe N-terminal Ste24p cleavage site is apparent. Further detailedmutagenesis of A8 and A9 (as well as other residues surroundingthe cleavage site in a-factor) to define allowable residuesmay point toward a consensus sequence. Interestingly, our deletionanalysis suggests that the first part of the N-terminal extensionis dispensable, as ${Delta}$ QPSTAT, which eliminates this extension, isstill fully processed to mature a-factor. We observed similar resultswhen we replaced this region with ubiquitin, which is cleaved offby ubiquitin-specific proteases to release P2 a-factor (10, 63).Clearly this N-terminal extension needs to be removed for Axl1pprocessing to occur efficiently (see below); however, the requirementfor this N-terminal extension in the first place is unclear.

At first glance, it seems puzzling that there is considerableresidual mating in the a-factor mutants that block P1 $->$ P2 processing(Fig. 4C) and, indeed, in the ste24 ${Delta}$ mutant itself (Fig. 2).One possibility is that another protease can cleaveP1 $->$ P2 inefficiently,allowing a small amount of mature a-factor to be produced thatis below the detection limit by metabolic labeling and SDS-PAGEbut sufficient to support some mating. A more likely possibilityis that a low level of processing by Axl1p can occur despitethe absence of prior processing by Ste24p, producing maturea-factor directly from P1. Indeed, we have observed by metaboliclabeling that overexpression of Axl1p in an ste24 ${Delta}$ mutant resultsin a small but detectable amount of mature a-factor (S. Michaelis,unpublished data). The AXL1 gene is highly induced by ${alpha}$ -factor (51),so in a mating assay when MAT ${alpha}$ cells are present, Axl1p may be inducedsufficiently in the ste24 ${Delta}$ mutant to produce a low level of maturea-factor directly from P1, bypassing the requirement for P1 $->$ P2 cleavage. This amount of mature a-factor may be enough todrive some residual degree of mating. In the metabolic labelingexperiment with the ste24 ${Delta}$ strain (Fig. 1B), no MAT ${alpha}$ mating partneris present; therefore, Axl1p levels are low and no mature a-factoris observed.

Understanding the sequence requirements for the zinc metalloproteaseSte24p will be important for understanding other fungal pheromones.Interestingly, the human Ste24p ortholog, Zmpste24, can complementthe ste24 ${Delta}$ mutant for a-factor production (58, 63). In addition, mutationsin Zmpste24 lead to premature aging in mice and other laminopathiesin humans because of its role in proteolytic processing of thenuclear scaffold protein prelamin A (39, 68). Thus, further insightsinto the sequence recognition requirements for Ste24p will havesignificance in terms of human health and disease.

Axl1p and the P2 $->$ M processing step. A striking finding in this study was that we recovered manymutants defective in Axl1p cleavage (Fig. 5). These are thefirst mutants reported that block this processing step for a-factoror any fungal pheromone. The mutated residues flank the cleavagesite, indicating that recognition involves residues both N terminal(D20 and N21) and C terminal (Y22) to the Axl1p cleavage site. Interestingly,point mutations at the acidic residue E17, some distance N terminalto the processing site, also impact cleavage, although mutationsat the basic residues K18 or K19 do not affect processing. DeletionsN terminal to the processing site ( ${Delta}$ PKEK and ${Delta}$ TSSE) also blockprocessing. Together, these data suggest that the conformationand/or length of the N-terminal extension after Ste24p processingmay also be important. Notably, no mutations C terminal to Y22were found that block processing, suggesting that recognitionis directed to the cut site itself and the N-terminal regions.It is notable that the lipophilic pheromones from several otherfungi (Schizosaccharomyces pombe, Ustilago hordeii, and Cryptococcusneoformans) also have an Asn residue that immediately precedesthe predicted mature pheromone sequence (23, 26, 36), suggestingthat Asn may form part of a consensus sequence for fungal pheromone processing.

Axl1p was discovered for its role in axial bud site selectionin haploid cells and subsequently found to be required for a-factormaturation (1, 29). Axl1p is a zinc metalloprotease that shareshomology with human insulin degrading enzyme. Interestingly,mutation of its protease domain does not affect its role inbud site selection but does block a-factor processing, indicatingthat these two functions are separable (1). Similar to the ste24 ${Delta}$ deletion, some residual mating ( $~$ 2%) is also observed in an axl1 ${Delta}$ deletion. In this case, the residual mating is known to be dueto the activity of Ste23p, an Axl1p homolog (1). The contributionof Ste23p to a-factor processing is very small compared to Axl1psuch that the ste23 ${Delta}$ mutant on its own has no phenotype. Interestingly,several of the point mutants in a-factor that block Axl1p processingare mating defective to the same extent as the axl1 ${Delta}$ ste23 ${Delta}$ doublemutant, suggesting that the point mutation blocks recognitionby both proteases. Thus, the sequence recognition determinantsof Axl1p and Ste23p are likely to be similar. Similar to othergene products with a key role in mating, Axl1p is strongly ${alpha}$ -factorinduced and haploid specific (51), while Ste23p is not regulatedin a mating or mating-type specific manner (29).

Activity mutants. Our mutagenesis study has revealed that the amino acid sequenceof mature a-factor is critical for recognition by or accessto Ste3p. Numerous mutations (at residues I22, F28, W29, D30,and P31) were found that do not block maturation or export buthave a strong mating defect (Fig. 6). Another study found complex(double and triple) point mutations in mature a-factor thatalso affect activity, some of which overlap with the residuesidentified here (17). Thus, there appears to be exquisite sequencespecificity in recognition of a-factor by the Ste3p receptor,in addition to the importance of the prenyl and carboxymethylmodifications elegantly demonstrated with synthetic peptides (40).It is interesting to note that a very conservative change, namelyD30E, results in a significant reduction in activity, whilea major change like K25E (or K25A) has no functional impact.Thus, the determinants for receptor binding are likely to berather complex.

The sequence specificity required for a-factor to interact withits receptor is perhaps not surprising in light of the factthat many basidiomycetes produce multiple prenylated pheromones,in some cases with only small sequence differences, yet thesecan be specifically differentiated by particular receptors (13, 18, 27).In addition, alanine scanning with synthetic peptides of theUstilago maydis pheromone a1 revealed particular residues criticalfor biological activity (62). Synthetic peptides derived fromthe a-factor mutants reported here could be important toolsfor looking at specificity of receptor binding, for instance,if used as a basis for suppressor screens to find compensatorymutations in Ste3p. Studies with ${alpha}$ -factor and its receptor, Ste2p,have provided important insights into peptide ligand-receptorinteraction (45). Similar studies with a-factor will be of greatinterest because of the relevance to lipophilic signaling molecules;however, development of an a-factor-Ste3p binding assay hasbeen a challenge because of the inherent complications resultingfrom a lipophilic peptide binding to a membrane receptor.

Export mutants. Export of a-factor is mediated by Ste6p, a member of the ATPbinding cassette transporter superfamily whose substrates includelipophilic molecules and proteins. Of all the a-factor mutantsexamined from our screen, only one appeared to be completelyexport defective (G26V), while three others were significantlyreduced in export (G26C, P31Q, and A32K), indicating that peptidesequence is an important factor in transport. Peptide lengthmay also be a factor for Ste6p transport. For example, in machineryor a-factor mutants that block Axl1p processing (i.e., axl1 ${Delta}$ and a-factor point mutations) (Fig. 1B and 5B, respectively),no P2 a-factor is ever observed in the extracellular fraction.In addition, the ${Delta}$ PKEK and ${Delta}$ TSSE deletions that block P2 to Mprocessing are also not exported, in spite of their reduced length.

The CAAX modifications are also critical for Ste6p-mediatedexport of a-factor: mature a-factor that has not undergone CAAXendoproteolysis (Fig. 3) or carboxymethylation (Fig. 1B) isnot exported. The role of the farnesyl modification cannot beassessed in vivo because all subsequent processing depends onfarnesylation of the a-factor precursor, and an in vitro transportassay for Ste6p-mediated a-factor transport has not been developed (34, 43).

The limited number of export-defective mutants recovered fromour screen suggests that the sequence recognition determinantsfor export are not overly stringent. Several other observationssupport this conclusion. First, Ste6p ectopically expressedin S. pombe can transport M-factor, which is prenylated and carboxymethylatedbut has a different sequence than a-factor. Furthermore, whilethe basidiomycetes clearly encode many pheromones and receptors,there is no evidence for multiple transporters, suggesting thata single transporter can export pheromones with a variety ofsequences; in fact, C. neoformans Ste6 was recently shown tofunction in both mating types (33). Our most severe export-defectivemutant was G26V; interestingly, Becker and coworkers (15, 45, 71)have suggested that G26 allows mature a-factor to bend and thatthis conformation could be necessary for recognition by theSte3p receptor.Taken together, it is likely that recognitionfor export by Ste6p may be more dependent on the conformationof the mature pheromone than on its specific sequence.

Concluding comments. This study presents for the first time a direct phenotypic comparisonof mutants defective in components of the a-factor biogenesismachinery and mutations in a-factor itself that affect eachpost-CAAX processing step of its biogenesis. The machinery mutantanalysis allowed us to examine the order and dependency of theprocessing steps on each other, revealing that CAAX endoproteolysisand carboxymethylation are not required for N-terminal processingto occur and that all steps must be carried out for export toproceed. In addition, our mutational analysis of the a-factorprecursor revealed residues critical for N-terminal processingby Ste24p and Axl1p, for export by Ste6p, and for activity (i.e.,interaction with Ste3p). These results provide the basis for elucidatingthe sequence determinants for other fungal pheromones. Studieson a-factor biogenesis have already been important for understandingmammalian prelamin A processing by Zmpste24 (4, 39, 68). Interestingly, recentexperiments have suggested that prenylated signaling molecules mayplay a critical role for germ cell migration in zebrafish and Drosophila(54, 65). Thus, the relevance of understanding a-factor biogenesismay extend significantly beyond studies of fungal pheromones.

ACKNOWLEDGMENTS

This work was supported by a grant from the NIH (GM41223).

We thank J. Barrowman and P. Espenshade for helpful commentson the manuscript, E. Barrata and R. Klein for assistance withthe mutagenesis, and W. Schmidt and P. Chen for technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205. Phone: (410) 955-7274. Fax: (410) 955-4129. E-mail: michaelis{at}jhmi.edu.

REFERENCES

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