SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In Saccharomyces cerevisiae, the cell surface protein, Muc1p, was shown to be critical for invasive growth and pseudohyphal differentiation. The transcription of MUC1 and of the co-regulated STA2 glucoamylase gene is controlled by the interplay of a multitude of regulators, including Ste12p, Tec1p, Flo8p, Msn1p and Mss11p. Genetic analysis suggests that Mss11p plays an essential role in this regulatory process and that it functions at the convergence of at least two signalling cascades, the filamentous growth MAPK cascade and the cAMP-PKA pathway. Despite this central role in the control of filamentous growth and starch metabolism, the exact molecular function of Mss11p is unknown. We subjected Mss11p to a detailed molecular analysis and report here on its role in transcriptional regulation, as well as on the identification of specific domains required to confer transcriptional activation in response to nutritional signals. We show that Mss11p contains two independent transactivation domains, one of which is a highly conserved sequence that is found in several proteins with unidentified function in mammalian and invertebrate organisms. We also identify conserved amino acids that are required for the activation function.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Upon nutrient limitation, cells of the yeast Saccharomyces cerevisiae undergo a transition from ovoid cells, which bud in an axial (haploid) or bipolar (diploid) fashion, to elongated cells that bud in a unipolar fashion. The daughter cells remain attached to the mother cells, which results in chains of cells referred to as pseudohyphae. These filaments can grow invasively into the agar and away from the colony and are hypothesized to be an adaptation of yeast cells to search for nutrient-rich substrates (reviewed by Kron, 1997; Madhani and Fink, 1998; Borges-Walmsley and Walmsley, 2000; Pan et al., 2000; Bauer and Pretorius, 2001; Gancedo, 2001). A large number of genes that play a role in this adaptation to changing environmental conditions have been isolated, and most were shown to participate in distinct signalling cascades that regulate the dimorphic switch from yeast to hyphal form. The best characterized of these signalling pathways are the invasive growth MAP kinase cascade (Liu et al., 1993; Cook et al., 1996; 1997; Mösch et al., 1996; Madhani et al., 1997) and the Gpa2p-cAMP-PKA pathway (Ward et al., 1995; Roberts et al., 1997; Robertson and Fink, 1998; Pan and Heitman, 1999; Rupp et al., 1999; Lorenz et al., 2000; Tamaki et al., 2000; Gagiano et al., 2002). In addition to the components of these established regulatory cascades, several other factors have also been identified for their roles in regulating pseudohyphal and invasive growth. These include Phd1p (Gimeno and Fink, 1994; Lorenz and Heitman, 1998), Sok2p (Ward et al., 1995; Pan and Heitman, 1999), Elm1p (Blacketer et al., 1993; Garret, 1997; Koehler and Myers, 1997), Msn1p and Mss11p (Gagiano et al., 1999a,b), but these factors have not been placed in the context of known signal transduction pathways, have not been characterized sufficiently or seem to function through alternative pathways.

MUC1 (also known as FLO11) is a member of the adhesin- or flocculin-encoding genes and is regulated by the signalling pathways that determine filamentous growth (Guo et al., 2000). It encodes a large, cell wall-associated, glycosylphosphatidylinositol (GPI)-anchored threonine/serine-rich protein with structural resemblance to mammalian mucins and yeast flocculins (Lambrechts et al., 1996a; Lo and Dranginis, 1996). Deletion analyses demonstrated that MUC1 is critical for pseudohyphal differentiation and invasive growth and that overexpression of this gene results in flocculating yeast strains in liquid media and pseudohyphal/invasive growth on solid media (Lambrechts et al., 1996a; Lo and Dranginis, 1996; 1998; Guo et al., 2000).

The upstream regulatory region of MUC1 is one of the largest yeast promoters identified to date, and areas as far as 2.4 kb upstream of the transcription start site have been shown to be required for the regulation of MUC1 expression (Gagiano et al., 1999a; Rupp et al., 1999). The data suggest that most, if not all, the previously mentioned signalling pathways and regulatory proteins converge on this promoter to regulate invasive growth and pseudohyphal differentiation, making this gene the most relevant target of this complex regulatory network. The entire MUC1 upstream region is almost identical to that of the STA2 gene (Gagiano et al., 1999b), which is present only in some S. cerevisiae strains and codes for an extracellular glucoamylase that enables the yeast cell to use starch as a carbon source (reviewed by Pretorius et al., 1991; Vivier et al., 1997; Gagiano et al., 2002).

Of the regulatory proteins mentioned above, Mss11p appears to play one of the most central roles in regulating filamentous growth and starch metabolism. When the MSS11 gene is present on a multiple copy plasmid, strong invasive and pseudohyphal growth is observed in all the strains tested, including those with single or multiple deletions of genes encoding other factors that activate MUC1 and STA2 transcription (Gagiano et al., 1999a,b). On the other hand, the deletion of the MSS11 locus results in the complete absence of these phenotypes, which cannot be suppressed efficiently by overexpressing any of the factors identified to date, including Ste12p, Flo8p and Msn1p (Gagiano et al., 1999a,b).

Despite a clear role in regulating filamentous growth and starch metabolism, the exact molecular function of Mss11p is unknown. Although it was shown to regulate the expression of MUC1 and STA2 at a transcriptional level (Webber et al., 1997; Gagiano et al., 1999a,b), and that this activation occurs via specific areas within the MUC1 and STA2 promoters (Gagiano et al., 1999a), it is unclear whether it confers this activation directly, i.e. by acting as a transcription activator, or indirectly, i.e. by interacting with or favouring the recruitment of other transcription factors for example. Mss11p has no significant sequence homology to any yeast protein, with the exception of some limited homology to the Flo8p transcription activator (Gagiano et al., 1999a). Mss11p, however, contains several distinctive domains (Fig. 1), including (i) a poly glutamine and (ii) a poly asparagine domain, which are similar to, but significantly larger than, similar domains observed in the repressor Ssn6p. It also contains (iii) a putative ATP- or GTP-binding domain, commonly found in ATP- or GTP-binding proteins such as the kinases, ATPases or GTPases (Saraste et al., 1990); and (iv) two short stretches (labelled H1 and H2) of amino acids with significant homology to Flo8p. The functional relevance and significance of all these domains has not yet been investigated. Furthermore, Mss11p has only been implicated in the regulation of MUC1 and STA2 transcription and, therefore, it is unknown whether any other target genes exist or whether Mss11p also plays a role in cellular processes other than filamentous growth or starch metabolism.

image

Figure 1. A diagrammatic representation of Mss11p that illustrates the position and length of the different domains. Domains H1 and H2 represent the domains with homology to S. cerevisiae Flo8p. The alignment of the second homology domain, H2, with Flo8p and proteins of unknown function from other organisms is shown. The first homology domain, H1, has no significant homology to any protein besides Flo8p. The putative ATP/GTP-binding domain (P-loop) is represented by a P. The poly glutamine and poly asparagine domains are indicated by poly Q and poly N respectively. The large domain between the poly glutamine and poly asparagine domains has no known or predicted structural features or homology to any protein identified to date. It was subdivided into three smaller domains for the functional analysis. These smaller domains were named interdomain regions 1, 2 and 3 and are indicated by ID1, ID2 and ID3 on the diagram.

Download figure to PowerPoint

In this paper, we show that Mss11p can activate transcription directly. The data suggest that Mss11p is regulated on a post-translational level in response to specific nutritional signals and that the N-terminal domain is responsible, at least in part, for this regulation. Furthermore, we delineate activation domains in Mss11p by means of domain mapping and show that these domains are sufficient for the activation of a reporter gene, as well as of the MUC1 and STA2 genes. We also identify specific amino acids that are involved in transcriptional activation and define a domain that is highly conserved in several mammalian and invertebrate proteins of unknown function.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Mss11p regulates MUC1 and STA2 mRNA levels

To determine the transcription levels of MUC1 and STA2 under different nutritional conditions and to determine the effect of multiple copies of MSS11 or the deletion of MSS11, we isolated RNA from cells grown in synthetic media containing high (2%) or low (0.1%) concentrations of glucose as carbon source and (NH4)2SO4 as nitrogen source. The wild-type strain, ISP15, was transformed with the 2 µ plasmid bearing MSS11, YEplac112-MSS11 or the unmodified vector, YEplac112, as negative control. The effect of a deletion of MSS11 was assessed using strain ISP15Δmss11 transformed with the unmodified vector, YEplac112. The results are presented in Fig. 2.

image

Figure 2. Northern blot analysis on the effect of single and multiple copies of MSS11, as well as the deletion thereof, on the transcript levels of STA2 and MUC1 under different nutritional conditions. The concentrations and components of the different media are described in detail in Table 2.

Download figure to PowerPoint

No mRNA from either STA2 or MUC1 could be detected in the Δmss11 strain, confirming the essential role played by Mss11p in the transcription of these genes. In the wild-type strain, MUC1 transcription levels were also undetectable in all media tested, including under nitrogen and carbon limitation. However, the transcription levels of STA2 in the wild-type background were clearly detectable in all media except the synthetic medium containing 2% glucose (SCD). This discrepancy in the transcription levels of the two genes with almost identical regulatory regions has been described before and can be attributed, at least in part, to the presence of two inserts in the promoter region of MUC1 that are absent from that of STA2 (Gagiano et al., 1999a).

The data clearly show that both nitrogen and carbon limitations result in increased transcription of STA2. Although no STA2 transcript was detectable in a medium with high glucose and ammonium concentration (SCD), the STA2 mRNA signal intensity reached 17%, 100% and 100% of that of the actin signal when the cells were grown in SLAD (nitrogen limited), SCLD (glucose limited) and SLALD (glucose and ammonium limited) respectively. The presence of multiple copies of MSS11 increased the transcription levels of STA2 significantly in SCD, SLAD and SCLD, reaching 50% (up from non-detectable in the wild type), 66% (up from 17%) and 149% (up from 100%) of the actin signal intensity respectively. However, the presence of the multiple copy vector alone did not result in a further increase in the STA2 transcription levels in SLALD medium.

Although the absolute transcription levels varied in the different media, the presence of MSS11 in multiple copies increased the transcription of MUC1 significantly under all conditions. The signal intensity of MUC1 reached 55%, 32%, 36% and 18% of that of the actin signal in SCD, SLAD, SCLD and SLALD respectively. Contrary to what was observed for STA2, multiple copies of MSS11 therefore resulted in a significant increase in MUC1 mRNA levels in SLALD. However, the nutritional conditions continued to exert control over the relative expression levels of MUC1 and STA2 in the presence of multiple copies of MSS11. In particular, and contrary to what was observed for STA2, the expression of MUC1 was high in media containing elevated (2%) glucose concentrations (SCD, SLAD). This observation can be explained by findings from other groups, which identified MUC1 as being downstream of the Gpr1p-Gpa2p glucose receptor that senses high glucose concentrations and transmits the signal to MUC1 via changes in intracellular cAMP levels (Lorenz et al., 2000). This pathway was shown to require glucose as well as complex media for sustained activation (Colombo et al., 1998), and a key component of the pathway, the Sch9p protein kinase, was shown to regulate MUC1 transcription in response to cAMP levels (Lorenz et al., 2000).

Mss11p is a transcription activator that regulates MUC1 and STA2 differentially in response to nutritional conditions

The Northern blot analysis clearly demonstrates that Mss11p mediates the expression levels of MUC1 and STA2. However, it does not allow assessment of whether (i) Mss11p plays a direct role as a transcription activator or acts at a different level; and (ii) whether Mss11p is required for transmitting the nutritional information.

In order to clarify the first question, we exploited the modular characteristic of transcription factors (reviewed by Triezenberg, 1995) to identify the domain(s) in Mss11p that would be required for the transcriptional activation of the target genes. A series of fusions were created between the open reading frames (ORFs) of MSS11 and the GAL4 transcription factor, of which the activation domain was deleted. The constructs included the full-length MSS11 ORF, as well as sequential deletions thereof, which were transformed into a strain containing an integrated reporter gene, lacZ, under the control of the GAL7 promoter. This promoter contains binding sites for the Gal4p transcription activator and, if Mss11p con-tained transcription activation domains, the fusion pro-tein would mediate the transcriptional activation of the reporter gene (James et al., 1996). Both liquid and plate β-galactosidase assays were used to identify such activation domains. The results of these assays are presented in Figs 3 and 4.

image

Figure 3. Identification of Mss11p as a transcriptional activator and the identification of specific activation domains. The different Mss11p fragments fused to Gal4p are represented diagrammatically, and the levels of reporter gene activity conferred in liquid SCLD media by each, as measured by β-galactosidase activity, are indicated next to the relevant construct.

Download figure to PowerPoint

image

Figure 4. Effect of variable nutrient availability on the ability of the Mss11p–Gal4p fusion protein to activate transcription and relevance of the Mss11p poly glutamine (poly Q) and poly asparagine (poly N) domains. The ability of the different Mss11p fragments, fused to the Gal4p DNA-binding domain, to activate the PGAL7lacZ reporter system was assessed in strain pJ69-4A. The Mss11p domains fused to Gal4p are represented diagrammatically, and the levels of reporter gene activity conferred by each are represented by the intensity of the colonies in the photographs. The constituents of the media used are listed in Table 2.

Download figure to PowerPoint

Figure 3 indicates that, relative to the vector containing only the Gal4p DNA-binding domain as a negative control, the fusion protein with full-length Mss11p results in an ≈ 40-fold increase in reporter gene activity in liquid SCD medium. This result clearly suggests that Mss11p can either activate transcription directly or recruit other factors required for activation.

The fusions of the MSS11–GAL4 DNA-binding domain gene are under the control of the constitutively active ADH1 promoter, resulting in high levels of transcription under all nutritional conditions. If transcriptional activation by Mss11p is constitutive, the fusion protein can be expected to result in high levels of β-galactosidase on all media. The differences in β-galactosidase levels observed on the different media (Fig. 4) would therefore suggest that the ability of Mss11p to activate transcription is regulated at a post-transcriptional level in response to specific nutritional conditions. Indeed, β-galactosidase activity is weakest on SCD medium, in which carbon and nitrogen are in sufficient supply, while being induced significantly on the three nutrient-limited media, SLAD, SCLD and SLALD. This would suggest that Mss11p-dependent activation occurs directly as a response to nutrient limitation. Interestingly, the variation in expression levels conferred by the Mss11p–Gal4p fusion protein on the reporter gene (Fig. 4) correlates well with the observed differences in STA2 mRNA levels in the presence of multiple copies of MSS11 (Fig. 2). However, the same does not hold true for the MUC1 mRNA levels detected in the Northern blots. The data therefore again suggest that other factors make a contribution to the regulation of this gene in the presence of high levels of glucose.

The conserved H2 domain and the C-terminus are required for the transcriptional activation function of Mss11p

The systematic deletions from both the N- and C-termini to identify the domains specifically required for the activation function are presented in Fig. 3.

The data suggest that there are two areas in Mss11p that are required for transcriptional activation. All constructs in which the extreme C-terminal end of the protein (amino acids 640–720) is deleted show significantly reduced transcriptional activation of the reporter gene, independent of corresponding sequential deletions from the N-terminal side of the protein. Furthermore, the C-terminal domain on its own (construct NF-OR) confers significant activation to the reporter gene, suggesting that it is able to interact with the transcription machinery and to promote transcription.

However, the constructs in which the C-terminal sections of the protein are deleted continue to generate significant, although reduced, levels of β-galactosidase activity, and activation only ceases once the 272 N-terminal amino acids are deleted concomitantly with the C-terminal section (construct QxF-NxR). The data clearly show that a second activation domain is located in a region bordered by amino acids 126 and 272 (construct H2F-QR), which confers levels of β-galactosidase activity similar to those observed with full-length Mss11p. Within this section of the protein, the 26 amino acids that constitute the H2 domain (construct H2F-H2R), which corresponds to one of the two short stretches of Mss11p with homology to Flo8p, confer strong transcriptional activation, corresponding to 50% of the values obtained for full-length Mss11p, whereas the domain composed of amino acids 152–272 is unable to activate the reporter gene. This suggests that the 26-amino-acid H2 domain acts as a transcription activation domain. This is further supported by the fact that the deletion of this domain leads in all cases to a significant drop in transcription activation by the remaining sections of the protein.

It is interesting to note that activation by Mss11p appears to be negatively regulated by the domain composed of amino acids 34–126, which immediately precedes the H2 domain and contains the second short stretch of amino acids with significant homology to Flo8p, H1, as well as a putative P-loop. All constructs containing the H1-P-loop domain show significantly lower activa-tion levels than corresponding constructs in which the sequence has been deleted (see for example H1F-ID2R compared with H2F-ID2R), whereas the truncated versions of Mss11p with deletions of these amino acids result in the highest levels of activation observed. These levels are significantly higher than those achieved with the construct containing full-length Mss11p. This negative effect of the domain appears particularly clearly when the C-terminal activation domain has been deleted (see for example H1F-QR compared with H2F-QR), suggesting that the H1-P-loop sequence may particularly influence activation by the H2-associated domain.

Analysis of the poly glutamine and poly asparagine domains of Mss11p

Glutamine-rich domains have been identified as the activation domains of transcription factors in a number of organisms, ranging in complexity from yeast (e.g. Mcm1p) to humans (e.g. Oct1 and Oct2) (Johnson et al., 1993). The difference between these prototypical glutamine-rich activation domains and the poly glutamine domain of Mss11p, however, is the dispersion of hydrophobic amino acids, such as leucine, valine and phenylalanine, between the glutamine residues (Johnson et al., 1993; Triezenberg, 1995), which were shown to be critical for the activation function of the transcription factors (Gill et al., 1994). A poly glutamine domain significantly shorter than that of Mss11p (12 glutamine residues) was also identified in the yeast protein, Pgd1, a component of the mediator complex between transcriptional activators and the RNA polymerase II complex, but its exact role has yet to be investigated (Brohl et al., 1994; Gustafsson et al., 1998; Myers et al., 1998).

The function of the poly asparagine domains in pro-teins is unknown at this stage. Asparagine-rich domains have been described for only two other S. cerevisiae proteins. S. cerevisiae Azf1p and Swh1p include short, asparagine-rich domains (Schmalix and Bandlow, 1994; Stein et al., 1998), but the relationship between the function and the presence of these domains has not been investigated.

The data presented in Fig. 3 clearly demonstrate that both the poly glutamine and the poly asparagine domains (constructs QxF-QxR and NxF-NxR respectively) are unable to activate transcription when fused to the Gal4p DNA-binding domain (Fig. 3), whereas their deletion appears not significantly to affect transcriptional activation by the remains of the truncated proteins (Fig. 4).

As repetitive coding sequences, commonly referred to as trinucleotide repeats, are known to vary in size in mammalian genomes, and recombination between these sequences may cause neurodegenerative diseases such as Huntington's disease and Friedrich's ataxia in humans (reviewed by Jakupciak and Wells, 2000; Shimohata et al., 2001), we investigated the size of the Mss11p poly glutamine and poly asparagine domains in different strains. The corresponding MSS11-encoding sequences were polymerase chain reaction (PCR) amplified from strains ISP15, ISP52, FY23 (S288C) and W303, and the resulting fragments were sequenced. There was no difference in either the size or the specific sequences between the different fragments obtained from these strains from different genetic backgrounds (results not shown).

Specific amino acids in the conserved H2 domain are critical for the Mss11p transcriptional activation function

As the H2 domain was shown to be required for the activation function of Mss11p and also to be able to stimulate transcription of the PGAL7lacZ reporter gene when fused to the Gal4p DNA-binding domain, we investigated whether the conserved amino acids identified in H2 (Fig. 1) are required for the activation function. We specifically targeted the conserved amino acid pairs Phe-133–Leu-134, Trp-137–Trp-138, Ile-140–Phe-141 and Leu-144–Phe-145 (Fig. 1). All these amino acids were mutated to glycine and alanine respectively (Table 4, Fig. 5). The effects of the mutations on the ability of Mss11p to stimulate transcription of the reporter genes were assessed through β-galactosidase liquid assays in strain Σ1278bΔmss11Δmuc1::lacZ (Fig. 5A), whereas invasive growth was assessed in strain Σ1278bΔmss11 (Fig. 5B).

Table 4. . The list of plasmids used in this work.
PlasmidRelevant genotypeSource/reference
  1. For the plasmids carrying MSS11 fragments, the encoded area is indicated in subscript, giving the first and last amino acids of the Mss11p derivative encoded by the respective insert.

PPMUC1-lacZ CEN4 URA3 PMUC1-lacZ Gagiano et al. (1999a)
PPSTA2-lacZ CEN4 URA3 PSTA2-lacZ Gagiano et al. (1999a)
YEplac1122 µTRP1 Gietz and Sugino (1988)
YEplac112-MSS112 µTRP1 MSS11 Gagiano et al. (1999a)
YEplac112-MSS11exp2 µTRP1 PMSS11 TMSS11This work
YEplac112-MSS11-OF-OR2 µTRP1 MSS111−758This work
YEplac112-MSS11-Pmut2 µTRP1 MSS11−758; G93[RIGHTWARDS ARROW]A; K94[RIGHTWARDS ARROW]RThis work
YEplac112-MSS11-ΔQ2 µTRP1 MSS111−758; Δ272−329This work
YEplac112-MSS11-ΔN2 µTRP1 MSS111−758; Δ605−640This work
YEplac112-MSS11-FL2 µTRP1 MSS111758 F133[RIGHTWARDS ARROW]G; L134[RIGHTWARDS ARROW]AThis work
YEplac112-MSS11-WW2 µTRP1 MSS111758 W137[RIGHTWARDS ARROW]G; W138[RIGHTWARDS ARROW]AThis work
YEplac112-MSS11-IF2 µTRP1 MSS111758 I140[RIGHTWARDS ARROW]G; F141[RIGHTWARDS ARROW]AThis work
YEplac112-MSS11-LF2 µTRP1 MSS111758 L144[RIGHTWARDS ARROW]G; F145[RIGHTWARDS ARROW]AThis work
image

Figure 5. Identification of critical amino acids in the H2 activation domain of Mss11p. FL (Phe–Leu), IF (Ile–Phe), LF (Leu–Phe) and WW (Trp–Trp) indicate the pairs of amino acids within the H2 domain that were mutated in the corresponding version of Mss11p. All pairs were changed to GA (Gly–Ala).

A. Impact of the mutations in the H2 domain on the ability of Mss11p to activate the chromosomal copy of MUC1 in the Σ1278bΔmss11Δmuc1::lacZ strain (lacZ integrated in chromosomal copy of MUC1) in media containing fermentable and non-fermentable carbon sources.

B. Ability of multiple copy plasmids encoding either the wild-type (wt) or H2 point mutation forms of Mss11p to induce invasive growth in a Σ1278bΔmss11 strain. The invasiveness of each strain correlates perfectly with the level of transcriptional activation.

Download figure to PowerPoint

From the results, it is clear that all the conserved pairs of amino acids are required for full activation, as all the mutations significantly reduced the ability of Mss11p to activate the lacZ reporter gene. For cells grown in SCD medium, β-galactosidase values were reduced by ≈ 50% in all the mutants in comparison with those obtained for the wild-type protein. This reduction is similar to what is observed when the entire N-terminal domain of Mss11p, including H2, is deleted (Fig. 3), suggesting that the remaining activation may result from transcriptional activation by the C-terminal domain and further strengthening the hypothesis that Mss11p indeed contains two independent activation domains. Furthermore, although the expression of wild-type Mss11p results in increased transcriptional activation on SCGE medium compared with SCD medium, the mutated Mss11p proteins result in similar activation on both media. This suggests that activation by the H2 domain is responsible, at least in part, for the glucose-dependent regulation of gene expression by Mss11p.

The reduced ability of the mutated forms of Mss11p to activate the reporter gene correlates well with their ability to induce invasive growth (Fig. 5B). On both SCD and SCGE media, the Δmss11 strains transformed by vectors encoding the H2-mutated versions of Mss11p all display phenotypes that are intermediate between the strong invasiveness of the strain transformed with wild-type MSS11 and the absence of invasion of the strain transformed with the vector alone. These data clearly demonstrate a direct correlation between the ability of Mss11p to activate transcription and the regulation of invasive growth.

The putative ATP- and/or GTP-binding domain of Mss11p appears not to affect transcriptional activation

The analysis of transcriptional activation by truncated forms of Mss11p suggested a possible regulatory function for the domain containing the H1 domain together with the putative P-loop (Gly-88–Ser-89–Ala-90–Ser-91–Gly92–Gly-93–Lys-94–Thr-95–Ser-96), an ATP- and/or GTP-binding sequence. To test whether the P-loop regulated the activation function of Mss11p, we mutated two of the critical amino acids, Gly-93 and Lys-94, to Ala-93 and Arg-94 respectively. We tested the ability of the P-loop-mutated allele of MSS11 to confer transcriptional activation in strains ISP15Δmuc1::lacZ and Σ1278b, in either a wild-type or a Δmss11 genetic background. We also fused the MSS11 ORF, carrying the P-loop mutations, to the GAL4 fragment encoding the DNA-binding domain to assess whether the encoded protein would be able to activate the PGAL7lacZ reporter gene in strain pJ69-4A on different media.

In no case could we observe significant differences between the ability of the wild type and the mutated versions of Mss11p to activate transcription (data not shown). The data suggest that the putative P-loop is either non-functional or that it regulates other aspects of Mss11p function.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this paper, we present a molecular analysis of Mss11p, a transcriptional regulator of the MUC1 and STA2 genes of S. cerevisiae. As a regulator of these two genes, it is also a major regulator of the ability of S. cerevisiae to form pseudohyphae, grow invasively and metabolize starch (Webber et al., 1997; Lorenz and Heitman, 1998; Gagiano et al., 1999a,b). The correlation between Mss11p levels, MUC1 and STA2 transcription and these phenotypes is well established (Webber et al., 1997; Lorenz and Heitman, 1998; Gagiano et al., 1999a,b). However, the impact of specific nutritional signals on this relationship has never been assessed properly. Here, we show, through Northern analyses and reporter gene expression analysis in different media, that Mss11p relates the effect of nutritional signals, specifically the glucose and nitro-gen limitation signals, to the transcription of MUC1 and STA2. These observations reaffirm previous observations on the co-regulation of MUC1 and STA2 and, consequently, on the co-regulation of the filamentous growth and starch metabolism phenotypes. The results also suggest that the effects of the different nutritional conditions on MUC1 and STA2 transcription are transmitted via Mss11p.

The molecular analysis of Mss11p presents conclu-sive evidence that Mss11p acts as a transcriptional activator. This activation function is regulated on a post-transcriptional level, as the activation efficiency is clearly dependent on the amount of nutrients present in the media, even if Mss11p is fused to the Gal4p DNA-binding domain and expressed from a heterologous promoter that is not regulated by nutrient availability. Furthermore, the regulation of the transcriptional activation is not dependent on the binding of Mss11p to its target, either DNA or other proteins, as the Mss11p fusions to the Gal4p-binding domain result in a similar pattern of transcriptional regulation to that exerted by the native protein on MUC1 and STA2. The molecular dissection of Mss11p identified two activation domains, one of which, named H2, appears to be highly conserved among several proteins of unknown function. Changes in individual amino acids within this domain significantly reduce the ability of Mss11p to activate transcription. This reduction is accompanied by a correspondingly reduced ability to induce invasive growth, confirming that the ability of Mss11p to activate transcription is directly correlated with the effects of Mss11p overexpression and deletion on invasive and pseudohyphal differentiation. Interestingly, the data strongly suggest that the H2 domain could be responsible for transmitting a carbon source-dependent signal, as the mutant versions no longer lead to higher MUC1 transcription in media containing non-fermentable carbon sources. The domain therefore plays a central role in the control of Mss11p-dependent activation. The identification of this conserved domain may provide useful insights into the role of the proteins of unknown function listed in Fig. 1.

The data presented here also suggest that the N-terminal domain of Mss11p is involved in the regulation of the activation function. Mutations resulting in the dele-tion of the H1- and P-loop-containing domain result in increased transcription. This suggests that ATP or GTP binding may be involved in the regulation of Mss11p activity. It is possible that the observed regulation results from an autoregulatory function, similar to what is observed in the Snf1p protein kinase, for example, where a regulatory domain inhibits the function of the catalytic domain in repressive conditions (Carlson, 1998; 1999).

Furthermore, we show that the unique poly glutamine and poly asparagine domains are not required for the activation function of Mss11p. The role of these domains therefore remains to be identified. Although significantly smaller, a poly glutamine domain has been identified in only one other S. cerevisiae protein, namely Pgd1p (Brohl et al., 1994). Pgd1p was shown to be a component of the mediator complex between transcriptional activators and the RNA polymerase II complex (Gustafsson et al., 1998; Myers et al., 1998). Considering that Pgd1p functions in a multicomponent protein complex, it is possible that the poly glutamine domain has a structural role or that it is required for protein–protein interactions.

The length and composition in amino acids of the Mss11p poly glutamine domain are reminiscent of the poly glutamine stretches found in mammalian proteins, such as Huntington and frataxin. The poly glutamine domains of these two (and several other) mammalian proteins are notorious, as recombination events in the repetitive coding sequences, commonly referred to as trinucleotide repeats, cause neurodegenerative diseases such as Huntington's disease and Friedrich's ataxia (reviewed by Jakupciak and Wells, 2000; Shimohata et al., 2001).

We could not detect any variation in the size of these sequences in four S. cerevisiae strains of different genetic background. This does not preclude the possibility that such variation may exist, and feral strains of S. cerevisiae in particular should be investigated.

The genetic evidence presented to date suggests that Mss11p, like Pgd1p, could also have a role as a transcriptional mediator. The results of the epistasis analyses involving MSS11 demonstrated that all the other transcription factors required for the transcriptional activation of MUC1 and STA2, i.e. Ste12p, Mss10p and Flo8p, also require Mss11p for their activation function (Gagiano et al., 1999a,b). These results could also be interpreted as evidence that Mss11p is the most downstream component of each of the different signal transduction cascades represented by these transcription factors. However, the fact that Flo8p, Ste12p and Msn1p were all identified as DNA-binding transcription factors (Estruch and Carlson, 1990; Madhani and Fink, 1997; Kobayashi et al., 1999) makes this explanation highly unlikely and points strongly towards Mss11p facilitating the transcriptional activation function of these transcription factors at the MUC1 and STA2 promoters.

The strong activation in response to specific nutritional signals suggests a direct role for Mss11p as a transcriptional activator, but the dependency of three structurally dissimilar and unrelated transcription factors, Flo8p, Msn1p and Ste12p, on Mss11p is difficult to reconcile with such a role. It is therefore possible that Mss11p is part of a complex that assists transcription factors in activating genes in response to specific signals. Considering the amount of genetic evidence that points towards MUC1 and STA2 transcription being repressed by the state of the chromatin over their promoters (Inui et al., 1989; Okimoto et al., 1989; Yoshimoto and Yamashita, 1991; Yoshimoto et al., 1991; 1992; Kuchin et al., 1993; Yamashita, 1993; Park et al., 1999), a role for Mss11p in a complex that reduces this repressive effect, such as a histone acetyltransferase complex (reviewed by Sterner and Berger, 2000), seems possible. Removing or releasing the chromatin barrier over the STA2 and MUC1 promoters in response to specific nutritional signals could therefore result in the observed activation, as it would make the promoter accessible to Flo8p, Msn1p and Ste12p, as well as to other transcription factors. It was demonstrated recently that the transcription of the PGL1 gene is regulated by the same signalling elements that regulate the transcription of MUC1 in conditions conducive for filamentous growth (Madhani et al., 1999; Gognies et al., 2001). The PGL1 gene encodes an endopolygalacturonase that enables the yeast cell to hydrolyse pectin. These observations would suggest that the co-regulation of filamentous growth and starch metabolism should be extended to include polysaccharide degradation in general.

It does not appear that Mss11p plays a role in regulating the transcription of other members of the adhesin and flocculin gene family. A microarray analysis to identify target genes of Mss11p, other than MUC1 and STA2, revealed that Mss11p is very specific in regulating the transcription of MUC1 and STA2 and failed to identify other genes with significantly increased transcription in the presence of multiple copies of MSS11 (results not shown).

Future efforts will focus on the identification of proteins that interact with Mss11p to identify a more precise role for Mss11p in regulating MUC1 and STA2 transcription.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Yeast strains, genetic methods and media

The yeast strains used in this study, along with the relevant genotypes, are listed in Table 1. Strain ISP15 has the ability to degrade starch and to grow invasively. The strains ISP15 and Σ1278b have been used extensively for the characterization of invasive growth and pseudohyphal differentiation (Gimeno et al., 1992; Lambrechts et al., 1996a,b; Webber et al., 1997; Gagiano et al., 1999a,b). Strain pJ69-4A is commonly used in the analysis of two-hybrid interactions and was generously provided by P. James (James et al., 1996). In strains ISP15Δmuc1::lacZ and Σ1278bΔmss11Δmuc1::lacZ, the ORF of MUC1 has been replaced by the ORF of the reporter gene lacZ fused to the S. cerevisiae HIS3 gene, which is used as a selection marker.

Table 1. . The yeast strains used in this study.
StrainGenotypeSource or reference
ISP15 MATa STA2 his3 thr1 trp1 leu2 ura3 Gagiano et al. (1999a)
ISP15Δmss11 MATa STA2 his3 thr1 trp1 leu2 ura3Δmss11::LEU2 Gagiano et al. (1999a)
ISP15Δmuc1::lacZ MATa STA2 his3 thr1 trp1 leu2 ura3Δmuc1::lacZ-HIS3This study
Σ1278b MATα ura3-52 trp1::hisG leu2::hisG his3::hisG H. U. Mösch
Σ1278bΔmss11 MATα ura3-52 trp1::hisG leu2::hisG his3::hisG mss11::LEU2 This study
Σ1278bΔmss11Δmuc1::lacZ MATα ura3-52 trp1::hisG leu2::hisG his3::hisG mss11::LEU2 muc1::lacZ-HIS3 This study
PJ69-4A MATa his3 trp1 leu2 ura3 gal4 gal80 LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ James et al. (1996)

The carbon and nitrogen sources used in the preparation of the different yeast media are listed in Table 2. The yeast nitrogen base that was used did not contain any amino acids or nitrogen source (Becton Dickinson). All synthetic media were supplemented with the specific amino acids required to fulfil the auxotrophic demands of each strain. Amino acids were obtained from Sigma-Aldrich and were added according to the recommended concentrations (Sherman et al., 1991; Ausubel et al., 1994). The solid media contained 2% agar (Becton Dickinson).

Table 2. . The components of the different yeast media used in this work.
MediaNitrogen sourceCarbon source (glucose)
YPD1% yeast extract, 2% peptone2%
YPLD1% yeast extract, 2% peptone0.1%
SCD1.7% yeast nitrogen base, 40 mM (NH4)2SO42%
SCLD1.7% yeast nitrogen base, 40 mM (NH4)2SO40.1%
SLAD1.7% yeast nitrogen base, 20 mM (NH4)2SO42%
SLALD1.7% yeast nitrogen base, 20 mM (NH4)2SO40.1%

Standard molecular genetic and yeast techniques were used throughout this work (Sherman et al., 1991; Ausubel et al., 1994). Yeast transformations were performed using the lithium acetate method (Ausubel et al., 1994).

Plasmid construction

Standard procedures for the isolation and manipulation of DNA were used throughout this study (Ausubel et al., 1994). All restriction enzymes, T4 DNA ligase and Expand Hi-Fidelity polymerase used in the enzymatic manipulation of DNA were obtained from Roche Diagnostics and were used according to the specifications of the supplier. Most PCR fragments generated by the PCR technique for this work were first cloned into the plasmid pGEM-T of the pGEM-T PCR cloning kit, supplied by Promega. Escherichia coli DH5α (Gibco BRL/Life Technologies) was used for the propagation of all plasmids and was grown in Luria–Bertani (LB) broth at 37°C. All E. coli transformations and the isolation of DNA were done according to Ausubel et al. (1994).

The potential functional domains in Mss11p have been described previously (Gagiano et al., 1999a). The relative sizes and positions of these domains are illustrated in Fig. 1. To identify the functional relevance of these domains, a series of plasmids was constructed that encode versions of Mss11p that are either systematically shortened from the C- or N-terminal ends or without specific, internal domains. The 2 µ plasmid, YEplac112 (Gietz and Sugino, 1986), was used to construct a base plasmid containing the promoter, start codon, stop codon and terminator region of MSS11. The resulting plasmid, YEplac112-MSS11exp, was used for all expression purposes.

The promoter region of MSS11 was PCR amplified using primers MSS11-PF and MSS11-PR, together with plasmid YEplac112-MSS11 (Gagiano et al., 1999a) as template. The reverse primer, MSS11-PR, was designed to contain an EcoRI site after the MSS11 start codon. This fragment was digested with EcoRI and ScaI and inserted into the unique EcoRI and HindII sites of plasmid YEplac112. The terminator region was PCR amplified using primers MSS11-TF and MSS11-TR, together with YEplac112-MSS11 as template. The forward primer, MSS11-TF, was designed to contain a SalI restriction site immediately 5′ to the stop codon, and the reverse primer was designed to contain a HindIII restriction site for cloning the fragment into the unique SalI and HindIII sites of plasmid YEplac112. The resulting plasmid, YEplac112-MSS11exp, therefore contained the full-length MSS11 promoter, start codon, stop codon and terminator region, as well as unique EcoRI and SalI sites for the insertion of the different MSS11 ORF fragments.

Different combinations of the primers listed in Table 3 were used to generate the truncated ORF fragments by means of PCR. Plasmid YEplac112-MSS11 was used as a template in all PCRs. All forward primers were designed to contain an EcoRI restriction site, and all reverse primers were designed to contain a SalI restriction site for cloning the different fragments in frame into plasmid YEplac112-MSS11exp, which is described above. The resulting plasmids are listed in Table 4. All plasmids were sequenced to verify that the expected deletions were correct and that no mutations were introduced through PCR.

Table 3. . A list of primers used to generate the different truncations and deletions of Mss11p for expression under its own promoter and for fusion to the Gal4p DNA-binding domain (also included are the primers used to mutate the putative ATP/GTP-binding domain and the putative activation domain, H2).
Primer namePosition relative to ORFSequence
  1. The different restriction sites generated and used for cloning purposes are indicated in underlined text. An additional nucleotide (a), indicated in italics, was inserted into the reverse primers to maintain the reading frame when ligating fragments into plasmids pGBD-C2 and YEplac112-MSS11exp. Specific nucleotide changes to introduce mutations in MSS11 are indicated in bold text. MSS11 sequences are given in capital letters. The positions relative to the ORF are given, considering the ATG as position +1 to +3 and the last nucleotide of the non-coding upstream region as position −1.

MSS11-P-F−581 to −6005′-ACAGGGCGCAATCAGCTACC-′3
MSS11-P-R+3 to −215′-cgtgaattcCATATCTTTATCATGCACCTTTTT-3′
MSS11-T-F+2275 to +23045′-atctgtcgacCTTAAAACCTATTAAACAACAAAAAGTGTTTC-3′
MSS11-T-R+2717 to +27365′-gatcaagcttTGGCCAGATAGCTTGCTTAC-3′
MSS11-OF+4 to +305′-atcgaattcGATAACACGACCAATATTAATACAAAT-3′
MSS11-OR+2250 to +22745′-gcaggtcgacaGCTATCCATTAGATCAGGAGAAAAG−3′
MSS11-H1F+103 to +1265′-gatcgaattcTTTGATGCGGATTCTCGAGTTTTC-3′
MSS11-H1R+254 to +2765′-tcaggtcgacaACCCGAAGCAGATCCGTTTATTC-3′
MSS11-H2F+376 to +3965′-gatcgaattcCTGATGGACGCTAATGACACG-3′
MSS11-H2R+421 to +4445′-tcaggtcgacaGTCTCCATTGAACAATGATTGAAA-3′
MSS11-PH2F+442 to 4655′-atggaattcGACCTAGAATCTGGGTACCAACAG-3′
MSS11-QF+988 to +10115′-atgcgaattcaCACCGTATCCTATTGTCAACCCA-3′
MSS11-QR+794 to +8165′-caggtcgacaTGCTGGTGATTGCAAATCATTGA-3′
MSS11-QxF+817 to +8375′-atggaattcCAGCCCCAGCAATCATCTCAA-3′
MSS11-QxR+961 to +9845′-gcaggtcgacaTTGCTGCTGTTGATGTTGTTGCTG-3′
MSS11-ID1R+1240 to +12605′-gatgtcgacaTTGCTGTAGTGCTTGCTGCTG-3′
MSS11-ID2F+1240 to +12605′-gatgaattcCAGCAGCAAGCACTACAGCAA-3′
MSS11-ID2R+1510 to +15305′-gatgtcgacaTAATTGCTGGTTAGCCGCCAT-3′
MSS11-ID3F+1510 to +15305′-gatgaattcATGGCGGCTAACCAGCAATTA-3′
MSS11-NF+1921 to +19445′-atggaattcACACCCACAGTATCACAACCATCA-3′
MSS11-NR+1789 to +18125′-caggtcgacaAGGCAAAGGAAAGACGGAGGTAGA-3′
MSS11-NxF+1810 to +18395′-atggaattcCCTAACAATAACAATAACAATAACAACAAC-3′
MSS11-NxR+1897 to +19265′-gcaggtcgacaGGGTGTATTATTACTATTATTATTATTATT-3′
MSS11-QReco+796 to +8165′-atcgaattcTGCTGGTGATTGCAAATCATT-3′
MSS11-NReco+1789 to +18115′-atcgaattcaGGCAAAGGAAAGACGGAGGTAGA-3′
MSS11-PloopF+247 to +2885′-TTATCTAGAATAAACGGATCTGCTTCGGGTGCGAGAACTAGC-3′
MSS11-WW-F+409 to +4325′-gaaGCCGGCGAAATTTTTCAATCATTG-3′
MSS11-WW-R+391 to +4145′-ttcGCCGGCTTCCAGTAAAAACGTGTC-3′
MSS11-IF-F+418 to +4415′-gaaGCCGGCCAATCATTGTTCAATGGA-3′
MSS11-IF-R+400 to 4235′-ttgGCCGGCTTCCCACCATTCCAGTAA-3′
MSS11-FL-F+403 to +4175′-acgGCCGGCCTGGAATGGTGGGAAATT-3′
MSS11-FL-R+379 to +4025′-cagGCCGGCCGTGTCATTAGCGTCCAT-3′
MSS11-LF-F+436 to +4535′-tcaGCCGGCAATGGAGACCTAGAATCT-3′
MSS11-LF-R+412 to +4355′-attGCCGGCTGATTGAAAAATTTCCCA-3′

The poly glutamine and poly asparagine domains were deleted by replacement with an EcoRI restriction site. Primer MSS11-OF was used in a PCR, together with primer MSS11-QReco, which is designed to contain an in frame EcoRI site. Plasmid YEplac112-MSS11 was used as template to generate a fragment stretching from the ATG initiation codon of MSS11 to before the poly glutamine domain, ending in an EcoRI site. This fragment was digested with EcoRI, ligated into plasmid YEplac112-MSS11-QF-OR and then opened with EcoRI to generate an MSS11 ORF, in which an EcoRI site replaced the area encoding the poly glutamine domain. The correct orientation was selected through restriction analysis, and the construct was sequenced for confirmation. The poly asparagine domain was deleted through a similar strategy, using YEplac112-MSS11 as template and primers MSS11-NReco and MSS11-OF in a PCR. This fragment was digested with EcoRI and ligated into plasmid YEplac112-MSS11-NF-OR, then opened with EcoRI to generate an MSS11 ORF in which an EcoRI site replaced the area encoding the poly asparagine domain.

To fuse Mss11p, as well as the different truncated and mutated derivatives, to the Gal4p DNA-binding domain, the fragments were excised from YEplac112-MSS11exp and cloned as EcoRI–Sal I fragments into the unique EcoRI and SalI sites of plasmid pGBD-C2 (James et al., 1996). The resulting plasmids are listed in Table 5.

Table 5. . The list of plasmids used to identify the activation domains of Mss11p.
PlasmidRelevant genotypeSource/reference
  1. For the plasmids carrying MSS11 fragments, the encoded area is indicated in subscript, giving the first and last amino acids of the Mss11p derivative encoded by the respective insert. The amino acids comprising the Gal4p DNA-binding domain are indicated in the same manner.

pGBD-C22 µTRP1 GAL41−147 James et al. (1996)
pGBD-C2-MSS11-OF-OR2 µTRP1 GAL41−147MSS111−758This work
pGBD-C2-MSS11-OF-NxR2 µTRP1 GAL41−147MSS111−640This work
pGBD-C2-MSS11-OF-NR2 µTRP1 GAL41−147MSS111−604This work
pGBD-C2-MSS11-OF-ID2R2 µTRP1 GAL41−147MSS111−511This work
pGBD-C2-MSS11-OF-ID1R2 µTRP1 GAL41−147MSS111−420This work
pGBD-C2-MSS11-OF-QR2 µTRP1 GAL41−147MSS111−272This work
pGBD-C2-MSS11-OF-H2R2 µTRP1 GAL41−147MSS111−168This work
pGBD-C2-MSS11-OF-H1R2 µTRP1 GAL41−147MSS111−112This work
pGBD-C2-MSS11-H1F-OR2 µTRP1 GAL41−147MSS1135−758This work
pGBD-C2-MSS11-H1F-NR2 µTRP1 GAL41−147MSS1135−604This work
pGBD-C2-MSS11-H1F-ID2R2 µTRP1 GAL41−147MSS1135−511This work
pGBD-C2-MSS11-H1F-ID1R2 µTRP1 GAL41−147MSS1135−420This work
pGBD-C2-MSS11-H1F-QR2 µTRP1 GAL41−147MSS1135−272This work
pGBD-C2-MSS11-H1F-H2R2 µTRP1 GAL41−147MSS1135−168This work
pGBD-C2-MSS11-H1F-H1R2 µTRP1 GAL41−147MSS1135−112This work
pGBD-C2-MSS11-H2F-OR2 µTRP1 GAL41−147MSS11146−758This work
pGBD-C2-MSS11-H2F-NR2 µTRP1 GAL41−147MSS11146−604This work
pGBD-C2-MSS11-H2F-ID2R2 µTRP1 GAL41−147MSS11146−511This work
pGBD-C2-MSS11-H2F-ID1R2 µTRP1 GAL41−147MSS11146−420This work
pGBD-C2-MSS11-H2F-QR2 µTRP1 GAL41−147MSS11146−272This work
pGBD-C2-MSS11-H2F-H2R2 µTRP1 GAL41−147MSS11146−168This work
pGBD-C2-MSS11-PH2F-OR2 µTRP1 GAL41−147MSS11169−758This work
pGBD-C2-MSS11-PH2F-NR2 µTRP1 GAL41−147MSS11169−604This work
pGBD-C2-MSS11-PH2F-ID2R2 µTRP1 GAL41−147MSS11169−511This work
pGBD-C2-MSS11-PH2F-QR2 µTRP1 GAL41−147MSS11169−272This work
pGBD-C2-MSS11-QxF-OR2 µTRP1 GAL41−147MSS11274−758This work
pGBD-C2-MSS11-QxF-NxR2 µTRP1 GAL41−147MSS11274−640This work
pGBD-C2-MSS11-QxF-NR2 µTRP1 GAL41−147MSS11274−604This work
pGBD-C2-MSS11-QxF-ID2R2 µTRP1 GAL41−147MSS11274−511This work
pGBD-C2-MSS11-QxF-ID1R2 µTRP1 GAL41−147MSS11274−420This work
pGBD-C2-MSS11-QxF-QxR2 µTRP1 GAL41−147MSS11274−329This work
pGBD-C2-MSS11-QF-OR2 µTRP1 GAL41−147MSS11330−758This work
pGBD-C2-MSS11-QF-ID2R2 µTRP1 GAL41−147MSS11330−511This work
pGBD-C2-MSS11-QF-ID1R2 µTRP1 GAL41−147MSS11330−420This work
pGBD-C2-MSS11-ID2F-OR2 µTRP1 GAL41−147MSS11414−758This work
pGBD-C2-MSS11-ID2F-NR2 µTRP1 GAL41−147MSS11414−604This work
pGBD-C2-MSS11-ID2F-ID2R2 µTRP1 GAL41−147MSS11414−511This work
pGBD-C2-MSS11-ID3F-OR2 µTRP1 GAL41−147MSS11504−758This work
pGBD-C2-MSS11-ID3F-NR2 µTRP1 GAL41−147MSS11504−604This work
pGBD-C2-MSS11-NxF-OR2 µTRP1 GAL41−147MSS11605−758This work
pGBD-C2-MSS11-NxF-NxR2 µTRP1 GAL41−147MSS11605−640This work
pGBD-C2-MSS11-NF-OR2 µTRP1 GAL41−147MSS11641−758This work
pGBD-C2-MSS11-ΔP2 µTRP1 GAL41−147MSS111−758; G93[RIGHTWARDS ARROW]A; K94[RIGHTWARDS ARROW]RThis work
pGBD-C2-MSS11-ΔQ2 µTRP1 GAL41−147MSS111−758; D272−329This work
pGBD-C2-MSS11-ΔN2 µTRP1 GAL41-147MSS111−758; D605−640This work
pGBD-C2-MSS11-FL2 m TRP1 GAL41−147MSS111758 F133[RIGHTWARDS ARROW]G; L134[RIGHTWARDS ARROW]AThis work
pGBD-C2-MSS11-WW2 µTRP1 GAL41−147MSS111758 W137[RIGHTWARDS ARROW]G; W138[RIGHTWARDS ARROW]AThis work
pGBD-C2-MSS11-IF2 µTRP1 GAL41−147MSS111758 I140[RIGHTWARDS ARROW]G; F141[RIGHTWARDS ARROW]AThis work
pGBD-C2-MSS11-LF2 µTRP1 GAL41−147MSS111758 L144[RIGHTWARDS ARROW]G; F145[RIGHTWARDS ARROW]AThis work

Site-directed mutagenesis

ATP- and GTP-binding proteins from a number of different organisms have been shown to carry a glycine-rich motif known as the P-loop, which is required for the binding of ATP and/or GTP and generally critical for the function of the protein (reviewed by Saraste et al., 1990). The consensus sequence of this domain was determined as Gly-1–X-2–X-3–X-4–X-5–Gly-6–Lys-7–Ser/Thr-8 by mutation analysis of a common sequence found in myosin and many other nucleotide-binding enzymes (Saraste et al., 1990). Mutation analyses of a very large number of ATP- and GTP-binding proteins suggested that the critical amino acids are indeed Gly-1, Gly-6 and Lys-7 (invariant), as well as Ser-8, which can be replaced only with a functionally equivalent Thr (Saraste et al., 1990). The putative P-loop (Gagiano et al., 1999a; Fig. 1) of Mss11p was eliminated by mutating amino acids that were shown to be critical for its function (Saraste et al., 1990), namely a glycine at position 93 and a lysine at position 94 to alanine and arginine respectively. This was achieved by designing a forward primer that contained the desired nucleotide changes. The primer was extended to span a native XbaI site in the MSS11 ORF that would aid in the cloning of the fragment. This primer, MSS11-PloopF, was used together with the reverse primer, MSS11-OR, to generate a fragment that contained the desired sequence alterations. The fragment was digested with XbaI and Sal I before ligation into plasmid MSS11-OF-OR, in which the corresponding fragment had been removed. The construct was sequenced to verify that the correct alterations were made and that no additional mutations were introduced through PCR.

A small stretch of amino acids in Mss11p was shown to have some homology to a similar sized domain in Flo8p (Gagiano et al., 1999a). This domain, dubbed H2, was subsequently found to be conserved between a number of eukaryotic proteins of unknown function. An alignment of the relevant protein sequences, with the conserved amino acids highlighted, is shown in Fig. 1. To establish whether these amino acids contribute to the functioning of Mss11p, the amino acids pairs, i.e. the isoleucine and phenylalanine, the phenylalanine and leucine, the leucine and phenylalanine, as well as the two tryptophans, were all mutated to glycine and alanine respectively. This was achieved through a PCR-based mutagenesis strategy. Forward and reverse primers containing the desired nucleotide changes were designed and, by changing the nucleotides to code for glycine and alanine, a unique Cfr10I restriction site was introduced. Using YEplac112-MSS11 as template, the different forward primers were used together with primer MSS11-OR, whereas the reverse primers were used together with primer MSS11-OF to generate fragments that contain the desired mutations. The smaller fragments, generated using primer MSS11-OF with the reverse primers, were digested with EcoRI and Cfr 10I. The larger fragments, which were generated using the forward primers together with primer MSS11-OR, were digested with Cfr10I and SalI. The fragments were ligated in the necessary combinations, together with YEplac112-MSS11exp that had been digested with EcoRI and SalI, to form full-length MSS11 fragments containing the desired mutations.

RNA isolation and Northern analysis

Colonies were inoculated from the selective media into 5 ml of liquid SCD medium and grown to an optical density, measured at 600 nm (OD600), of ≈ 1 to serve as starter cultures. These starter cultures were used to inoculate 50 ml flasks of media containing varying concentrations and types of nitrogen and carbon sources (Table 2). All media were inoculated to an initial OD600 of 0.05 and incubated on a rotary shaker to reach a final OD600 of 1.0. Total RNA from each strain was isolated and separated on a 1.2% formaldehyde agarose gel, using the Bio101 FastRNA RedKit according to the specifications of the supplier.

The RNA was transferred and fixed onto Hybond-N nylon membranes (Amersham Pharmacia Biotech), according to the specifications of the manufacturer. ACT1, MUC1 and STA2 transcripts were detected using gene-specific probes prepared with the DIG PCR labelling kit (Roche Diagnostics) according to the specifications of the manufacturer. Hybridizations were done at 42°C for 16 h in standard formaldehyde buffer containing 50% formamide.

Densitometric analysis of the results was carried out using alphaimager software version 5.5 (Alpha Innotec).

β-Galactosidase liquid and plate assays

Strains containing the lacZ reporter gene were transformed, and three independent colonies from each transformation were grown in 5 ml of selective SCD medium to an OD600 of 1.0. From each of these starter cultures, a 5 ml culture of SCD was inoculated to an OD600 of 0.05 and incubated to grow at 30°C to an OD600 of 1.0. β-Galactosidase assays were performed as described by Ausubel et al. (1994). Assays were performed on all three transformants and, in each case, the mean value is presented. The standard deviation did not exceed 15% and was usually < 8%.

For the plate assays, the strains were transformed with the different deletion and mutation constructs and with the unmodified vector, YEplac112, as negative control. Three colonies from each transformation were grown in 5 ml of selective SCD medium to an OD600 of 1.0. From each of these starter cultures, 15 µl was dropped onto solid YPD, YPLD, SCD, SCLD, SLAD and SLALD agar plates (see Table 2 for media components). These plates also contained Xgal, added according to Ausubel et al. (1994), for the optical assessment of the activity conferred by the different Mss11p derivatives on the transcription levels of the reporter genes.

Computer-aided analyses and homology searches

Homology searches with Mss11p were done using the WWW-based blastp function (Altschul et al., 1997). Optimized sequence alignments between the Mss11p domains and the domains of the proteins identified through blastp (Fig. 1) were done using the bestfit and pileup functions of the GCG Wisconsin package. Access to the software was generously provided by the South African National Bioinformatics Institute (SANBI).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The authors wish to thank P. James for providing strain pJ69-4A and plasmid pGBD-C2, the personnel of SANBI for access to Bioinformatics software (GCG), and Maryke Carstens for assistance with the Northern blot analyses. This work was supported by grants from the South African Wine Industry (Winetech) and the National Research Foundation (NRF) to I.S.P.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 33893402.
  • Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1994) Current Protocols in Molecular Biology. New York: John Wiley & Sons.
  • Bauer, F.F., and Pretorius, I.S. (2001) Pseudohyphal and invasive growth in Saccharomyces cerevisiae. Focus Biotechnol – Appl Microbiol 2: 109133.
  • Blacketer, M.J., Koehler, C.M., Coats, S.G., Myers, A.M., and Madaule, P. (1993) Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A. Mol Cell Biol 13: 55675581.
  • Borges-Walmsley, M.I., and Walmsley, A.R. (2000) cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity. Trends Microbiol 8: 133141.
  • Brohl, S., Lisowsky, T., Riemen, G., and Michaelis, G. (1994) A new nuclear suppressor system for a mitochondrial RNA polymerase mutant identifies an unusual zinc-finger protein and a polyglutamine domain protein in Saccharomyces cerevisiae. Yeast 10: 719731.
  • Carlson, M. (1998) Regulation of glucose utilization in yeast. Curr Opin Genet Dev 8: 560564.
  • Carlson, M. (1999) Glucose repression in yeast. Curr Opin Microbiol 2: 202207.
  • Colombo, S., Ma, P., Crauwenberg, L., Winderickx, J., Crauwels, M., Teunissen, A., Nauwelaers, D., et al. (1998) Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae. EMBO J 17: 33263341.
  • Cook, J.G., Bardwell, L., Kron, S.J., and Thorner, J. (1996) Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae. Genes Dev 10: 28312848.
  • Cook, J.G., Bardwell, L., and Thorner, J. (1997) Inhibitory and activating functions for MAPK Kss1 in the S. cerevisiae filamentous growth signalling pathway. Nature 390: 8588.
  • Estruch, F., and Carlson, M. (1990) Increased dosage of the MSN1 gene restores invertase expression in yeast mutants defective in the SNF1 protein kinase. Nucleic Acids Res 18: 69596964.
  • Gagiano, M., Van Dyk, D., Bauer, F.F., Lambrechts, M.G., and Pretorius, I.S. (1999a) Divergent regulation of the evolutionarily closely related promoters of the Saccharomyces cerevisiae STA2 and MUC1 genes. J Bacteriol 181: 64976508.
  • Gagiano, M., Van Dyk, D., Bauer, F.F., Lambrechts, M.G., and Pretorius, I.S. (1999b) Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Microbiol 31: 103116.
  • Gagiano, M., Bauer, F.F., and Pretorius, I.S. (2002) The sensing of nutritional status and the relationship to filamentous growth in Saccharomyces cerevisiae. FEMS Yeast Res (in press).
  • Gancedo, J.M. (2001) Control of pseudohyphae formation in Saccharomyces cerevisiae. FEMS Microbiol Rev 25: 107123.
  • Garrett, J.M. (1997) The control of morphogenesis in Saccharomyces cerevisiae by Elm1 kinase is responsive to RAS/cAMP pathway activity and tryptophane availability. Mol Microbiol 26: 809820.
  • Gietz, R.D., and Sugino, A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenised yeast genes lacking six-base pair restriction sites. Gene 74: 527534.
  • Gill, G., Pascal, E., Tseng, Z.H., and Tjian, R. (1994) A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. Proc Natl Acad Sci USA 91: 192196.
  • Gimeno, C.J., and Fink, G.R. (1994) Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development. Mol Cell Biol 14: 21002112.
  • Gimeno, C.J., Ljungdahl, P.O., Styles, C.A., and Fink, G.R. (1992) Unipolar cell divisions in the yeast Saccharomyces cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68: 10771090.
  • Gognies, S., Simon, G., and Belarbi, A. (2001) Regulation and expression of endopolygalacturonase gene PGU1 in Saccharomyces cerevisiae. Yeast 18: 423432.
  • Guo, B., Styles, C.A., Feng, Q., and Fink, G.R. (2000) A Saccharomyces cerevisiae gene family involved in invasive growth, cell-cell adhesion, and mating. Proc Natl Acad Sci USA 97: 1215812163.
  • Gustafsson, C.M., Myers, L.C., Beve, J., Spahr, H., Lui, M., Erdjument-Bromage, H., et al. (1998) Identification of new mediator subunits in the RNA polymerase II holoenzyme from Saccharomyces cerevisiae. J Biol Chem 273: 3085130854.
  • Inui, M., Fukui, S., and Yamashita, I. (1989) Genetic controls of STA1 expression in yeast. Agric Biol Chem 53: 741748.
  • Jakupciak, J.P., and Wells, R.D. (2000) Genetic instabilities of triplet repeat sequences by recombination. IUBMB Life 50: 355359.
  • James, P., Halladay, J., and Craig, E.A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144: 14251436.
  • Johnson, P.F., Sterneck, E., and Williams, S.C. (1993) Activation domains of transcriptional regulatory proteins. J Nutr Biochem 4: 386398.
  • Kobayashi, O., Harashima, S., Yoshimoto, H., and Sone, H. (1999) Genes transcriptionally regulated by the FLO8 gene in Saccharomyces cerevisiae. In Proceedings of the XIXth International Conference on Yeast Genetics and Molecular Biology, Rimini, Italy.
  • Koehler, C.M., and Myers, A.M. (1997) Serine-threonine protein kinase activity of Elm1p, a regulator of morphologic differentiation in Saccharomyces cerevisiae. FEBS Lett 408: 109114.
  • Kron, S.J. (1997) Filamentous growth in budding yeast. Trends Microbiol 5: 450454.
  • Kuchin, S.V., Kartasheva, N.N., and Benevolensky, S.V. (1993) Genes required for derepression of an extracellular glucoamylase gene, STA2, in the yeast Saccharomyces. Yeast 9: 533541.
  • Lambrechts, M.G., Bauer, F.F., Marmur, J., and Pretorius, I.S. (1996a) Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast. Proc Natl Acad Sci USA 93: 84198424.
  • Lambrechts, M.G., Sollitti, P., Marmur, J., and Pretorius, I.S. (1996b) A multicopy suppressor gene, MSS10, restores STA2 expression in Saccharomyces cerevisiae strains containing the STA10 repressor gene. Curr Genet 29: 523529.
  • Liu, H., Styles, C.A., and Fink, G.R. (1993) Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 262: 17411744.
  • Lo, W.S., and Dranginis, A.M. (1996) FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin. J Bacteriol 178: 71447151.
  • Lo, W.S., and Dranginis, A.M. (1998) The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae. Mol Biol Cell 9: 161171.
  • Lorenz, M.C., and Heitman, J. (1998) Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains. Genetics 150: 14431457.
  • Lorenz, M.C., Pan, X., Harashima, T., Cardenas, M.E., Xue, Y., Hirsch, J.P., and Heitman, J. (2000) The G-protein-coupled receptor Gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Genetics 154: 609622.
  • Madhani, H.D., and Fink, G.R. (1997) Combinatorial control required for the specificity of yeast MAPK signalling. Science 275: 13141317.
  • Madhani, H.D., and Fink, G.R. (1998) The control of filamentous differentiation and virulence in fungi. Trends Cell Biol 8: 348353.
  • Madhani, H.D., Styles, C.A., and Fink, G.R. (1997) MAP kinases with distinct inhibitory functions impart signalling specificity during yeast differentiation. Cell 91: 673684.
  • Madhani, H.D., Galitski, T., Lander, E.S., and Fink, G.R. (1999) Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants. Proc Natl Acad Sci USA 96: 1253012535.
  • Mösch, H.-U., Roberts, R.L., and Fink, G.R. (1996) Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93: 53525356.
  • Myers, L.C., Gustafsson, C.M., Bushnell, D.A., Lui, M., Erdjument-Bromage, H., Tempst, P., and Kornberg, R.D. (1998) The Medical proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain. Genes Dev 12: 4554.
  • Okimoto, Y., Yoshimoto, H., Shima, H., Akada, R., Nimi, O., and Yamashita, I. (1989) Genes required for transcription of STA1 encoding an extracellular glucoamylase in the yeast Saccharomyces. Agric Biol Chem 53: 27972800.
  • Pan, X., and Heitman, J. (1999) Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19: 48744887.
  • Pan, X., Harashima, T., and Heitman, J. (2000) Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae. Curr Opin Microbiol 3: 567572.
  • Park, S.H., Koh, S.S., Chun, J.H., Hwang, H.J., and Kang, H.S. (1999) Nrg1 is a transcriptional repressor for glucose repression of STA1 gene expression in Saccharomyces cerevisiae. Mol Cell Biol 19: 20442050.
  • Pretorius, I.S., Lambrechts, M.G., and Marmur, J. (1991) The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview. Crit Rev Biochem Mol Biol 26: 5376.
  • Roberts, R.L., Mösch, H.U., and Fink, G.R. (1997) 14-3-3 proteins are essential for RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae. Cell 89: 10551065.
  • Robertson, L.S., and Fink, G.R. (1998) The three yeast A kinases have specific signalling functions in pseudohyphal growth. Proc Natl Acad Sci USA 95: 1378313787.
  • Rupp, S., Summers, E., Lo, H.J., Madhani, H., and Fink, G.R. (1999) MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene. EMBO J 18: 12571269.
  • Saraste, M., Sibbald, P.R., and Wittinghofer, A. (1990) The P-loop – a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci 15: 430434.
  • Schmalix, W.A., and Bandlow, W. (1994) SWH1 from yeast encodes a candidate nuclear factor containing ankyrin repeats and showing homology to mammalian oxysterol-binding protein. Biochim Biophys Acta 1219: 205210.
  • Sherman, F., Fink, G.R., and Hicks, J. (1991) Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  • Shimohata, T., Onodera, O., and Tsuji, S. (2001) Expanded polyglutamine stretches lead to aberrant transcriptional regulation in polyglutamine diseases. Hum Cell 14: 1725.
  • Stein, T., Kricke, J., Becher, D., and Lisowsky, T. (1998) Azf1p is a nuclear-localized zinc-finger protein that is preferentially expressed under non-fermentative growth conditions in Saccharomyces cerevisiae. Curr Genet 34: 287296.
  • Sterner, D.E., and Berger, S.L. (2000) Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64: 435459.
  • Tamaki, H., Miwa, T., Shinozaki, M., Saito, M., Yun, C.-W., Yamamoto, K., and Kumagai, H. (2000) GPR1 regulates filamentous growth through FLO11. yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 267: 164168.
  • Triezenberg, S.J. (1995) Structure and function of transcriptional activation domains. Curr Opin Genet Dev 5: 190196.
  • Vivier, M.A., Lambrechts, M.G., and Pretorius, I.S. (1997) Co-regulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol 32: 405435.
  • Ward, M.P., Gimeno, C.J., Fink, G.R., and Garrett, S. (1995) SOK2 may regulate cyclic AMP-dependent protein kinase-stimulated growth and pseudohyphal development by repressing transcription. Mol Cell Biol 15: 68556863.
  • Webber, A.L., Lambrechts, M.G., and Pretorius, I.S. (1997) MSS11, a novel yeast gene involved in the regulation of starch metabolism. Curr Genet 32: 260266.
  • Yamashita, I. (1993) Isolation and characterization of the SUD1 gene, which encodes a global repressor of core promoter activity in Saccharomyces cerevisiae. Mol Gen Genet 241: 616626.
  • Yoshimoto, H., and Yamashita, I. (1991) The GAM1/SNF2 gene of Saccharomyces cerevisiae encodes a highly charged nuclear protein required for transcription of the STA1 gene. Mol Gen Genet 228: 270280.
  • Yoshimoto, H., Ohmae, M., and Yamashita, I. (1991) The Saccharomyces cerevisiae GAM2/SIN3 protein plays a role in both activation and repression of transcription. Mol Gen Genet 233: 327330.
  • Yoshimoto, H., Ohmae, M., and Yamashita, I. (1992) Identity of the GAM3 gene with ADR6, each required for transcription of the STA1 or ADH2 gene in Saccharomyces cerevisiae. Biosci Biotechnol Biochem 56: 527529.