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Kluyveromyces lactis sexual pheromones. Gene structures and cellular responses to α-factor

Laura Ongay-Larios, Rocio Navarro-Olmos, Laura Kawasaki, Nancy Velázquez-Zavala, Edith Sánchez-Paredes, Francisco Torres-Quiroz, Gerardo Coello, Roberto Coria
DOI: http://dx.doi.org/10.1111/j.1567-1364.2007.00249.x 740-747 First published online: 1 August 2007


The Kluyveromyces lactis genes for sexual pheromones have been analyzed. The α-factor gene encodes a predicted polypeptide of 187 amino acid residues containing four tridecapeptide repeats (WSWITLRPGQPIF). A nucleotide blast search of the entire K. lactis genome sequence allowed the identification of the nonannotated putative a-pheromone gene that encodes a predicted protein of 33 residues containing one copy of the dodecapeptide a-factor (WIIPGFVWVPQC). The role of the K. lactis structural genes KlMFα1 and KlMFA1 in mating has been investigated by the construction of disruption mutations that totally eliminate gene functions. Mutants of both alleles showed sex-dependent sterility, indicating that these are single-copy genes and essential for mating. MATα, Klsst2 mutants, which, by analogy to Saccharomyces cerevisiae, are defective in Gα-GTPase activity, showed increased sensitivity to synthetic α-factor and increased capacity to mate. Additionally, Klbar1 mutants (putatively defective in α-pheromone proteolysis) showed delay in mating but sensitivity to α-pheromone. From these results, it can be deduced that the K. lactis MATa cell produces the homolog of the S. cerevisiaeα-pheromone, whereas the MATα cell produces the a-pheromone.

  • α-pheromone
  • a-pheromone
  • Kluyveromyces lactis
  • mating
  • signal transduction


Mating of haploid cells of Saccharomyces cerevisiae is initiated by the secretion of diffusible peptide pheromones that are recognized by receptors on the opposite cell type. The mating pheromones (known as α-factor and a-factor) are absolutely required to trigger the mating process; cells that cannot produce these molecules or lack their specific receptors (Ste2p for α-factor or Ste3p for a-factor) are sterile (Sprague, 1991). The binding of pheromones to their cognate receptors stimulates several responses, including changes in transcription, growth arrest, and polarized morphogenesis. A receptor G-protein-coupled mitogen-activated protein kinase cascade mediates all the responses to pheromones (Herskowitz, 1995).

The best characterized mating pheromones are those of Sa. cerevisiae. The α-factor is encoded by genes MFα1 and MFα2 (Kurjan & Herskowitz, 1982; Singh, 1983), whereas the a-factor is encoded by genes MFA1 and MFA2 (Michaelis & Herskowitz, 1988). Both pheromones are synthesized as precursors that undergo an ordered set of maturation events (Sprague & Thorner, 1992). In the case of the α-factor, the maturation process includes signal sequence cleavage, glycosylation, and proteolytic processing by three peptidases (Kex2p, Kex1p, and Ste13p) during the transit of the α-factor through the secretory pathway (Fuller, 1988). During a-factor biogenesis, the a-factor precursor undergoes prenylation, proteolytic cleavage of the universal motif AAX, carboxylmethylation in the C-terminus, and two proteolytic processing events at the N-terminus (Chen, 1997).

The mature pheromones are small peptides that interact with a binding pocket formed by the extracellular loops and the extracellular side of some transmembrane regions of the receptor (Naider & Becker, 2004). The mature α-pheromone is a 13-residue peptide composed of two variants; the most abundant is encoded by the MFα1 gene (WHWLQLKPGQPMY), and its sequence variant (N5R7) is encoded by the MFα2 gene. Both genes are expressed only in α-cells (Jarvis, 1988), and their products seem to have the same biological activity (Raths, 1986).

Two forms of the mature dodecapeptide a-factor are also present in Sa. cerevisiae, differing in one residue: YIIKGLFWDPAC, encoded by MFA1, and its variant V6, encoded by MFA2. These genes are expressed only in MATa cells (Fields, 1988).

There are significant structural homologies in genes encoding sexual pheromones in a variety of fungal species, e.g. Candida albicans (Bennett, 2003; Panwar, 2003), Schizosaccharomyces pombe (Davey, 1998), Magnaporthe grisea (Shen, 1999), and Cryptococcus neoformans (Davidson, 2000). In general, one pheromone tends to be farnesylated at its C-terminus, and the other is synthesized as a precursor that has to be cleaved by specific peptidases.

The budding yeast Kluyveromyces lactis is a unicellular, haploid organism with a conventionally organized cell cycle, and it is easily subjected to genetic analysis (Wésolowski-Louvel, 1996). MATa and MATα cells undergo mating when they are mixed together in the same medium. The Gα (Saviñón-Tejeda, 2001) and the Gβ (Kawasaki, 2005) subunits of the heterotrimeric G-protein trigger the pheromone signal to downstream effectors. Sex-specific G-Protein-coupled (GPC) receptors located at the plasma membrane are essential for this process (Torres-Quiroz, 2007). Both G-protein subunits function in the mating pathway via the KlSte12p transcription factor. This factor is thought to bind to pheromone response elements located in promoter regions of genes required for mating.

The induction of growth arrest and polarized morphogenesis by the activation of the pheromone response in K. lactis is not evident, neither by mixing MATa and MATα haploid cells nor by overexpression of the Gβ subunit (Kawasaki, 2005). However, expression of a constitutively active form of the Gα subunit in a ΔGα background is able to induce weak growth arrest (Saviñón-Tejeda, 2001).

To further characterize the mating response pathway in K. lactis, in this work we analyzed the genes encoding the sexual pheromones, constructed disruption mutants, and determined the physiologic responses to synthetic α-factor.

Materials and methods

Strains and media

The yeast strains used in this work were K. lactis 155 (MATα, ade2, his3, ura3) and 12/8 (MATa, lysA, argA, ura3). Escherichia coli strains DH5α and Gm33 (for preparation of nonmethylated DNA) were used to propagate plasmids. YPD medium consisted of 1% yeast extract, 2% Bacto peptone and 2% glucose. SD minimal medium consisted of 0.67% yeast nitrogen base without amino acids (Difco) and 2% glucose. SD minimal media were supplemented with the required amino acids and nitrogen bases (50 μg mL−1). Luria–Bertani broth plus ampicillin (100 μg mL−1) was used to propagate recombinant plasmids in bacteria.

Gene disruptions

Putative KlMFα1, KlMFA1, KlSST2 and KlBAR1 gene disruptions were achieved by homologous recombination introducing the URA3 marker. For KlMFα1, a 1087-bp fragment containing the full ORF, plus 500 bp of the 5′-untranslated region (UTR) and 30 bp of the 3′-UTR, was obtained by PCR amplification and cloned into the pGEM-T-Easy vector (Promega). The EcoRI fragment was then introduced into the YIp352 vector, and PCR amplification was performed, employing divergent primers designed at positions −87 to −103 (reverse complement) and +170 to +187 (coding strand). This generated a linear product that contained the full YIp352 plasmid flanked by 430-bp and 429-bp recombinant ends. For KlMFA1, a 510-bp fragment containing the putative full ORF, 188 bp of the 5′-UTR and 221 bp of the 3′-UTR was PCR-amplified using primers that introduce EcoRI sites at positions −174 and +312. The product was ligated into the pGEM-T-Easy vector and subcloned as an EcoRI fragment into YIp352, in which the HindIII site had been previously eliminated. This last plasmid was digested at the naturally occurring HindIII (position +18) and BclI (position +157) sites, giving a linear molecule containing the YIp352 plasmid flanked by 190 bp and 150 bp of recombinant ends. For KlBAR1, a 950-bp fragment was amplified using deoxyoligonucleotides designed from positions+230 (forward) to +1183 (reverse). This fragment was ligated into the pGEM-T-Easy vector. An 823-bp EcoRI–BamHI fragment was then ligated into the YIp352 vector prepared with the same enzymes. The YIp352–KlBAR1 plasmid was then digested with BglII and SacI, giving a linear molecule that contained the full YIp352 flanked by KlBAR1 recombinant ends of 185 bp and 220 bp. KlSST2 was amplified using primers designed at positions −20 (forward) and +2188 (reverse). The 2208-bp fragment was ligated into the pGEM-T-Easy vector. KlSST2 was then ligated as a NotI–PstI (filled in at NotI) fragment into YIp352 digested with SmaI and PstI. The resulting construct was opened at the naturally occurring BclI and SpeI sites, leaving 290 bp and 418 bp as recombinant ends.

Complementation tests

An 1087-bp EcoRI fragment containing the full KlMFα1 ORF flanked by 500 bp of the 5′-UTR and 30 bp of the 3′-UTR was obtained from the pGEM-T-Easy plasmid and was subcloned into YEpKDHis (described in Kawasaki, 2005) digested with the same enzyme. An EcoRI fragment containing the full KlMFA1 ORF, flanked by upstream and downstream sequences of 188 and 221 bp, respectively, was obtained from the pGEM-T-Easy plasmid and was subcloned into the YEpKDHis vector. These constructs were introduced into disruptant yeast strains for mating assays.

Mating assays

A patch of cells of the strain to be tested was grown on a plate of selective medium for 24 h. The tester strain was grown as a lawn on a YPD plate for 24 h. Both strains were replica-plated onto YPD plates and incubated for different times at 30°C, allowing cells to mate. Diploids were selected on SD medium by replica plating.

α-Pheromone assays

Synthetic α-pheromone was obtained from GenScrip Corp at 95.5% purity, and suspended in water. For these assays, cells were suspended in YPD medium at a density of 106 cells/100 μL, and pheromone was added to a final concentration of 100 μg mL−1. Cells were examined under phase-contrast microscopy and photographed at different times.

Other methods

All PCR products were sequenced in full at the Molecular Biology Facility, IFC, UNAM. Disruption mutants were confirmed by Southern blot. Probes for Southern analysis were labeled with [α-32P]dCTP with the Random Prime Labeling System (Rediprime II, Amersham Biosciences). Standard Southern blot analysis, recombinant DNA technology and yeast genetics procedures were also performed.

Results and Discussion

Mating in K. lactis is relatively unknown compared to that in Sa. cerevisiae, where the mating pathway has been studied in detail. The first step that triggers mating is sensing of the diffusible pheromones secreted by cells of the opposite mating type. Cells that cannot produce these molecules are sterile (Sprague, 1991). In the current study, we characterized the mating pheromone genes of K. lactis, investigated the phenotypic effects of mutations in the structural genes, and constructed mutations that were sensitive to synthetic α-factor. The genes described in this article are listed in Table 1, including those that, by sequence homology, can be predicted to participate in the pheromone maturation process. All genes were identified by blast analyses of the K. lactis genome sequence database (http://cbi.labri.fr/Genolevures/elt/KLLA).

View this table:

Proven and putative genes of the Kluyveromyces lactis pheromone response pathway

Gene nameProductMutant phenotypeRelevant mating type affectedIdentity (%)E-valueORF in the K. lactis database
KlMFα1α-PheromoneSterileMAT a461e-42KLLA0E19173g
KlKEX2KR endopeptidaseNDND570KLLA0D19811g
KlSTE13Dipeptidyl aminopeptidaseNDND390KLLA0D06919g
KlRAM1Farnesyl-transferase β subunitNDND501e-119KLLA0F07161g
KlRAM2Farnesyl-transferase α subunitNDND502e-87KLLA0E18051
KlSTE14Farnesyl cysteine carboxyl-methyltransferaseNDND538e-75KLLA0A02167g
KlSTE24Zinc metalloproteaseNDND690KLLA0D10846g
KlSTE6ATP-binding transporterNDND470KLLA0B14256g
KlBAR1α-Pheromone endopeptidaseSensitivity to α-pheromoneMATα354e-90KLLA0D15917g
KlSST2G-protein regulatorSensitivity to α-pheromoneMATα and MAT a411e-152KLLA0D10549g
  • * Phenotypes relevant to this work, particularly concerning response to α-factor.

  • Calculated identity of the precursor protein.

The gene encoding the α-factor

A blast search of the K. lactis genome database using the Sa. cerevisiae mature pheromone sequence allowed the identification of a single-copy gene in K. lactis with structural similarities to the α-mating pheromone from Sa. cerevisiae. The homologous sequence was designated KlMFα1. Analysis of the sequence revealed a 564-bp ORF that codes for four identical tridecapeptide repeats separated by spacer sequences of eight, 14, 14 and 16 amino acid residues (Fig. 1a). These tandem repeats are located in the second half of a predicted 187 amino acid polypeptide, suggesting that α-pheromone is synthesized as a precursor and cleaved to produce the mature peptide. In agreement with what is known in Sa. cerevisiae (Sprague & Thorner, 1992), the K. lactis precursor has an N-terminal hydrophobic sequence that can act as signal sequence, two N-X-T motifs for attachment of asparagine-linked carbohydrate, and a segment containing the four α-factor repeats, separated from each other by spacer sequences (Fig. 1a). Each spacer begins with the basic KR residues, which are followed by two, four or five X-A dipeptides (Fig. 1b). Although the K. lactis precursor differs in amino acid sequence from that of Sa. cerevisiae, their overall structures are well conserved. The same is true for fungal species as distant as Ca. albicans (Bennett, 2003; Panwar, 2003) and Sc. pombe (Davey, 1998). This suggests that the maturation pathway of the α-factor is also well conserved between species. In the case of the Sa. cerevisiae pheromone, this pathway includes three proteolytic events: (1) excision of the pheromone repeats by the KR endopeptidase Kex2p (Julius, 1984); (2) trimming of the generated C-termini by carboxypeptidase Kex1p (Wagner & Wolf, 1987); and (3) trimming of the N-termini by the action of the dipeptidyl aminopeptidase A, Ste13p (Julius, 1983). All of these processing steps can be predicted for the maturation of the K. lactisα-pheromone on the basis of the existence of homologous genes for the three peptidases (Table 1).


(a) Nucleotide sequence of the MFα1 gene and its deduced amino acid sequence. (b) Alignment of putative spacer sequences of α-pheromone. (c) Alignment of mature α-pheromones from Kluyveromyces lactis and Saccharomyces cerevisiae. Spacer sequences are marked in blue, and mature pheromones are marked in red. *identical amino acids; :, amino acids with similar polarity;, amino acids with different polarity.

The putative K. lactisα-pheromone is also a tridecapeptide with seven residues identical to those of the Sa. cerevisiaeα-factor encoded by the MFα1 gene (Fig. 1c). Studies involving the replacement of each residue of the mature Sa. cerevisiaeα-factor by l-alanine or d-alanine residues (Naider & Becker, 2004), along with photoaffinity labeling and site-directed mutagenesis studies, have led to the construction of a model for the structure of the peptide bound to its receptor (Ste2p). Three domains are seen in this model: the signaling domain, composed of residues 1–4; the loop domain, composed of residues 7–10; and the binding domain, composed of residues 10–13. According to this model, the nature of the putative interactions involved in receptor and pheromone recognition are conserved in K. lactis. First, the W1 and W3 present in both pheromones may contact the conserved aromatic group of the Y residue present in the interface of the sixth transmembrane region (TM6) of both receptors (Torres-Quiroz, 2007). Second, Q10 of both factors may contact the receptor's polar residues present on the outside surface of the first transmembrane segment (S and T in Sa. cerevisiae; E and N in K. lactis). Finally, the aromatic group of Y13 of the Sa. cerevisiaeα-factor might contact the aromatic F and Y residues located at the extracellular side of the fifth and sixth ScSte2p transmembrane regions (TM5 and TM6), respectively (Naider & Becker, 2004). Interestingly, position 13 of the K. lactis pheromone is F, and its putative receptor-interacting residues are Y in TM5 and Y in TM6 (Torres-Quiroz, 2007). Regardless of the moderate conservation of the primary sequence of α-factors (50%), some of the nonconserved residues could play important roles in species-specific interactions with cognate receptors, and should contribute to the sterility barrier between these two yeasts.

The gene encoding the a-factor

A tblastx search of the entire K. lactis genome sequence, using the nucleotide sequence of the ScMFA1 gene as query, allowed us to identify the putative KlMFA1 gene encoding the a-factor (Table 1). KlMFA1 is a single-copy gene located in chromosome E (reverse complement, frame 1), coordinates 108 4444–108 4545. The K. lactis active a-pheromone is thought to be generated from a 33 amino acid precursor, containing one copy of the a-factor located in its C-terminal end (Fig. 2a). The structure of the gene shows conservation with that of Sa. cerevisiae, and even with those of distant fungal species such as Sc. pombe (Davey, 1998), Cr. neoformans (Davidson, 2000), and Ustilago maydis (Spellig, 1994). The C-terminus of the a-precursor contains the universal CAAX motif (CVVA) that is characteristic of these peptides. The maturation pathway of the Sa. cerevisiaea-factor is very well known. Processing of the CAAX motif involves farnesylation via a thioether linkage of the cysteine residue (Anderegg, 1988), proteolysis of the C-terminal tripeptide (AAX), and methyl esterification of the exposed carboxyl group (Sprague & Thorner, 1992). Finally, proteolytic cleavage of the N-terminus, carried out in a two-step process, produces the mature pheromone, which is exported from the cell. All genes encoding the enzymes that participate in Sa. cerevisiaea-factor processing are present in the K. lactis genome: the KlRam1p and KlRam2p farnesyl-transferase β and α subunits respectively; the KlSte14 farnesyl cysteine carboxyl-methyltransferase; the KlSte24 zinc metalloprotease; the KlAxl1 protease; and the KlSte6 ATP-binding transporter (Table 1). The putative mature a-pheromone is a dodecapeptide with 50% identity and 56% similarity to the a-pheromone from Sa. cerevisiae (Fig. 2b).


(a) Nucleotide sequence of the KIMFA1 gene and its deduced amino acid sequence. (b) Alignment of a-pheromone precursors from Kluyveromyces lactis and Saccharomyces cerevisiae. Mature pheromones are marked in red. *identical amino acids; :, amino acids with similar polarity;, amino acids with different polarity.

KlMFα1 and KlMFA1 gene disruptions

In order to ascertain the role of KlMFα1 and KlMFA1 in mating, we constructed disruption mutants in both MATα and MATa strains. Deletion of the KlMFα1 gene from the typical K. lactis MATα cells did not affect the efficiency of mating (Fig. 3). In contrast, mating of the MATa cells in which the KlMFα1 gene had been deleted was much lower than that of the parental strain. Therefore, the KlMFα1 gene is essential for mating of MATa cells in K. lactis. On the other hand, deletion of the KlMFA1 gene solely affected mating of MATα cells, and had no effect on the mating of MATa cells (Fig. 3), indicating that the KlMFA1 gene is specifically required for the mating of MATα cells. The mating defects of disruptant MATa and MATα strains were reversed by transfection with plasmidic copies of the wild-type KlMFα1 and KlMFA1 genes (not shown). This analysis also supports the suggestion that α-factor and a-factor in K. lactis are encoded by single-copy genes, whereas in Sa. cerevisiae, each pheromone is encoded by two different genes (Kurjan & Herskowitz, 1982; Singh, 1983; Michaelis & Herskowitz, 1988). All of the above observations are in agreement with the fact that K. lactis MATα cells express the KlSte2p receptor, whereas the MATa cells express the KlSte3p receptor (Torres-Quiroz, 2007).


Effect of disruption of KIMFα1, KIMFA1, KlBAR1 and KlSST2 genes on mating. Mating was done by replica plating. Patches of strains to be tested were streaked on selective medium and replicated onto YPD plates containing a lawn of the wild-type tester strain, and this was followed by incubation overnight (a, b) or for 2 h (c) at 30°C. Diploid selection was done by replica plating onto SD. Plates were photographed 48 h later. h, haploid strains; d, diploid strains.

Biological activity of α-factor

In Sa. cerevisiae, the α-factor is known to induce growth arrest and morphologic changes (shmoo morphology) in haploid MATa cells (Kurjan & Herskowitz, 1982) that are characteristic of the mating process. The appearance of morphologically abnormal cells allows a sensitive method for the assay of α-factor activity. The tridecapeptide WSWITLRPGQPIF was synthesized in vitro and added at different concentrations to wild-type K. lactis cells of both mating types, and growth arrest and induction of shmoo morphology were looked for. Wild-type cells of both mating types failed to show any response to synthetic α-factor, even at concentrations as high as 100 μg mL−1.

These observations indicate that K. lactis wild-type cells are refractory to synthetic α-factor. An additional observation supporting the above result is that, in contrast to what happens in Sa. cerevisiae, where mating products can be obtained in a short time (2 h can be sufficient to obtain good efficiencies), efficient mating of K. lactis sexual partners is obtained after long incubation periods (over 8 h). Although it is reasonable to assume that mating partners should go through the shmoo stage during mating, microscopy examinations of mating mixtures at different times of incubation did not show obvious shmoo morphology (not shown), suggesting that this stage is quite transient in K. lactis.

The molecular basis for the pheromone-resistance phenomenon can be explained, at least in part, by two different mechanisms. First, Sa. cerevisiae cells of mating type a secrete a protease (Bar1p) that hydrolyzes the α-factor (Ciejek & Thorner, 1979). Mutants deficient in this ‘barrier activity’ (bar1 mutants) are highly sensitive to α-pheromone (Chan & Otte, 1982). This protease was thought to be involved in the recovery of yeast cells from the pheromone-induced cell cycle arrest that is part of the premating response. Second, cells exposed to mating pheromones become desensitized after a period of time by different feedback events, including negative regulation of the pheromone response by the activity of Sst2p. Sst2p belongs to the designated RGS family of proteins (regulators of G-protein signaling). Sst2p physically interacts with Gpa1, the α subunit of the heterotrimeric G-protein that is involved in the pheromone response, and induces its intrinsic GTPase activity (Dohlman, 1996). Mutants defective in Sst2p activity show high sensitivity to pheromone, and fail to resume growth after exposure to pheromone (Dohlman, 1996).

Taking into account the above observations, we reasoned that the natural resistance of K. lactis wild-type cells to α-pheromone can be suppressed by inactivating either KlBAR1 or KlSST2.

KlBar1p contains 511 amino acid residues, showing a moderate degree of identity (35%) with ScBar1p (Table 1). By analogy to what is known in Sa. cerevisiae, KlBar1p should catalyze the proteolytic cleavage of the L6–R7 bond of the K. lactisα-pheromone. KlBar1p conserves the cysteine residues characteristic of the pepsin family that allow the formation of three disulfide loops, and has a very well-conserved active site (ALLDSGSTIM) containing the aspartate residue that is necessary for catalytic activity (Dunn, 2002).

The putative KlSst2p is a predicted 715-residue polypeptide that shows overall 41% identity with ScSst2p (Table 1). Sst2p is a GTPase-accelerating protein that promotes GTP hydrolysis by the α subunit of heterotrimeric G-protein, thereby inactivating the G-protein and rapidly switching off G-protein-coupled receptor signaling pathways (De Vries, 2000). ScSst2p contains an ‘RGS box’ (or RGS domain), which is required for activity, and at least one DEP domain, required for membrane targeting (Burchett, 2000). A putative RGS domain is found in the C-terminal end of KlSst2p (residues 474–715) that shows only 36% identity with the corresponding RGS domain in ScSst2p, suggesting a high specificity of binding and activity over its cognate Gα subunit. The predicted DEP domain found in KlSst2p (residues 297–381) reaches 60% of identity with its counterpart in Sa. cerevisiae.

We cloned and disrupted the putative KlBAR1 and KlSST2 genes from K. lactis wild-type cells of both mating types, determined their mating efficiency, and assayed the biological activity of the synthetic pheromone on these mutants. Deletion of KlBAR1 did not affect the mating capacity of a and α strains, but a slight delay in mating was observed in α cells (Fig. 3). It has been reported that bar1α cells in Sa. cerevisiae also mate less efficiently with a cells in a mass mating mixture (Jackson & Hartwell, 1990). The ΔKlsst2 mutant, however, showed a significant increase in its mating capacity, seen in our experiments by the high efficiency of mating at 2 h of incubation with the mating partner (Fig. 3). In this time period, wild-type strains mated sporadically and never reached the diploid density observed with the Klsst2 mutant. The effect of Klsst2 mutation was independent of the mating type, indicating that KlSst2p plays the same role in a and α cells.

Finally, we tested the activity of the synthetic α-factor on cell suspensions of Klbar1 and Klsst2 mutants. For this, cells were suspended in YPD medium, and α-pheromone was added to a final concentration of 100 μg mL−1. We observed that only Klbar1 and Klsst2 mutants of the MATα type responded to α-factor (Fig. 4), whereas MATa mutants remain unresponsive. In this experiment, both Klbar1 and Klsst2 mutants showed enhanced agglutinability after 1 h of exposure to pheromone. This phenomenon is not observed in wild-type mating mixtures, and has not been reported so far, so we believe that it is due to the artificially high concentration of the peptide pheromone added to the cell suspension. After 4 h of treatment, most cells stopped dividing (determined by the high proportion of unbudded cells, 70–80%, compared to untreated cells, 30–40%), and produced polarized projections that closely resembled the shmoo cells induced by mating pheromones in Sa. cerevisiae (Fig. 4). Although individual polarized cells were easily observed after 8 h of treatment (Fig. 4), most cells were agglutinated, rounded and smaller in size (half-size in average) as compared with untreated cells. Prolonged treatment with α-pheromone caused a decrease in agglutination, but cell morphology remained unchanged.


Effect of synthetic α-pheromone on the morphology of MATαKlbar1 and Klsst2 mutants. Upper photographs show mutant strains without α-pheromone. α-Pheromone was added to cell suspensions at a final concentration of 100 μg mL−1, and the suspensions were observed at different times. For simplicity, only images taken at 1 h and 8 h are shown. Photographs were obtained by phase-contrast microscopy.

We have identified the genes encoding the sexual pheromones in K. lactis, and shown that mutations in KlBAR1 and KlSST2 induce sensibility to synthetic α-pheromone. The amino acid sequences of both the α-pheromone and a-pheromone are related to those of Sa. cerevisiae, and are expected to follow similar processing pathways. Further information on the mechanisms of pheromone activity can be obtained by mutagenic studies oriented towards the natural substitutions that evolution has caused in the pheromones of these two yeast species. This report supports the idea that the general characteristics of pheromone signaling have been closely conserved between yeasts.


We thank Guadalupe Codiz and Minerva Mora (Molecular Biology Unit) for technical assistance, and the staff of the Computer Unit at IFC, UNAM. We also thank Marisela Bolaños for technical assistance. This work was partially funded by grants 44178 from CONACyT, and PAPIIT IN211906 from DGAPA, UNAM.


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