Logo of eukcellPermissionsJournals.ASM.orgJournalEC ArticleJournal InfoAuthorsReviewers
Eukaryot Cell. 2009 Sep; 8(9): 1362–1372.
Published online 2009 Jul 24. doi:  10.1128/EC.00015-09
PMCID: PMC2747823

The tRNA Modification Complex Elongator Regulates the Cdc42-Dependent Mitogen-Activated Protein Kinase Pathway That Controls Filamentous Growth in Yeast


Signal transduction pathways control multiple aspects of cellular behavior, including global changes to the cell cycle, cell polarity, and gene expression, which can result in the formation of a new cell type. In the budding yeast Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth induces a dimorphic foraging response under nutrient-limiting conditions. How nutritional cues feed into MAPK activation remains an open question. Here we report a functional connection between the elongator tRNA modification complex (ELP genes) and activity of the filamentous growth pathway. Elongator was required for filamentous growth pathway signaling, and elp mutants were defective for invasive growth, cell polarization, and MAPK-dependent mat formation. Genetic suppression analysis showed that elongator functions at the level of Msb2p, the signaling mucin that operates at the head of the pathway, which led to the finding that elongator regulates the starvation-dependent expression of the MSB2 gene. The Elp complex was not required for activation of related pathways (pheromone response or high osmolarity glycerol response) that share components with the filamentous growth pathway. Because protein translation provides a rough metric of cellular nutritional status, elongator may convey nutritional information to the filamentous growth pathway at the level of MSB2 expression.

In response to nutrient limitation, Saccharomyces cerevisiae undergoes filamentous (invasive/pseudohyphal) growth, a cellular differentiation response to nutrient limitation (28, 66). Filamentous cells have a distinct morphology and display enhanced cell-cell adhesion and cell surface properties that result from the expression of adhesion molecules such as the flocculin Flo11p (31, 49). Yeast cells also expand in connected mats or biofilms (77-79). In fungal pathogens, filamentous growth and biofilm formation are required for virulence (48, 62). Budding yeast therefore represents an attractive system to study the genetic basis for some fungal-specific behaviors. Indeed, many regulatory elements that control dimorphism are conserved across species (46, 61).

Among the pathways that control filamentous growth in yeast are the RAS-protein kinase A-cyclic AMP pathway (27, 57, 58), the target of rapamycin pathway (97), and the Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway commonly referred to as the filamentous growth pathway (10, 47, 81). The filamentous growth pathway controls Flo11p-dependent adhesion (31), enhanced cell elongation by a delay in the G2 phase of the cell cycle (42), and the reorganization of cell polarity by changes in bud site selection (14, 91). The filamentous growth pathway is also required for mat formation (78).

At the head of the filamentous growth pathway, the cell surface proteins Msb2p (10) and Sho1p (54, 55, 101) connect to the polarity control GTPase Cdc42p (37, 67). GTP-bound Cdc42p interacts with the p21-activated kinase Ste20p (43, 68) to induce a canonical MAPK pathway composed of the Ste11p, Ste7p, and Kss1p protein kinases (53, 81). Although limiting glucose and fixed nitrogen are potent inducers of filamentous growth (12, 28), it is not clear whether the cell surface proteins sense nutrient levels directly. One connection between nutrient levels and MAPK signaling is that expression of the MSB2 gene and the gene that encodes its cognate protease, YPS1, which processes Msb2p in its extracellular domain, are induced under nutrient-limiting conditions (96). The CDK Srb10p/Cdk8p has been shown to phosphorylate the transcription factor Ste12p (60), which may also connect nutritional status to filamentous growth pathway activity.

In this report, we describe an unexpected connection between a tRNA modification complex (elongator or Elp complex) (90) and the activity of the filamentous growth pathway. Elongator was required for activation of the filamentous growth pathway and functioned at the level of MSB2 expression. This regulatory feed may connect general cellular nutrition (at the level of protein translation) to MAPK signaling. Orthologs of elongator have been shown to regulate signaling networks in mammalian cells (3, 9), and our results extend the governance of tRNA modification enzymes to the regulation of MAPK pathways.


Strains, plasmids, and microbiological techniques.

The yeast strains used in this study are listed in Table Table1.1. Yeast and bacterial strains were manipulated by standard methods (82, 84). Gene disruptions and GAL1 promoter fusions were made by PCR-based methods (2, 50), including the use of antibiotic resistance markers (30) and epitope fusions (86). Integrations were confirmed by PCR analysis and phenotype. The Msb2p-GFP plasmid (GFP is green fluorescent protein) was used to measure the localization of Msb2p in live cells (96). The plasmid containing Msb2p-HA (HA is hemagglutinin) was used to examine the levels and secretion of the Msb2p protein (96). Plasmids containing filamentous growth pathway targets fused to lacZ were provided by C. Boone (80) and H. Madhani (53). Overexpression constructs were obtained from an ordered collection obtained from Open Biosystems (23). The FUS1-lacZ reporter was used to evaluate the activity of the filamentous growth pathway, which in Σ1278b strains lacking an intact mating pathway (ste4Δ) exhibits Msb2p- and Sho1p-dependent expression (10). The FUS1-HIS3 reporter was used to confirm FUS1-lacZ reporter data and was measured by spotting equal amounts of cells onto synthetic medium lacking histidine and containing the competitive inhibitor of the His3p enzyme 3-amino-1,2,4-triazole (40, 88). β-Galactosidase assays were performed as described previously (11), and the results shown represent at least two independent trials. All experiments were carried out at 30°C unless otherwise indicated.

Yeast strains used in this study

Cell biological assays.

Budding pattern was based on established methodology (5) and was confirmed for some experiments by visual inspection of connected cells. The single-cell invasive growth assay (12) and plate-washing assays (81) were performed to evaluate filamentous growth. Halo assays and the evaluation of shmoo morphologies were performed as described previously (13). Assays to evaluate mat form growth were performed as described previously (78), on low-agar yeast extract-peptone-dextrose (YEPD) medium (0.3% agar). Pectinase assays were performed based on reference 52 and are described in reference 72.

Msb2p-HA secretion by colony blot analysis.

A detailed analysis of the screen will be described elsewhere (C. Chavel, H. M. Dionne, B. Birkaya, and P. J. Cullen, submitted for publication). Briefly, haploid (MATa) mutants from an ordered deletion collection (24) were transformed with a plasmid carrying a functional epitope-tagged MSB2 gene (pMSB2-HA) by using a high-throughput microtiter plate transformation protocol (25). Transformants were pinned to synthetic defined medium without uracil (SD-URA) and, once grown, transferred to a 96-well plate containing 100 μl of water, pinned to SD-URA overlaid with a nitrocellulose disc filter (0.4 μm; HAHY08550; Millipore), and incubated for 48 h at 30°C. Filters were rinsed in distilled water to remove cells and probed with antibodies to the HA epitope (12CA5.16.4). Process/function and Gene Ontology annotations for proteins were obtained from public databases, including the Saccharomyces Genome Database (http://www.yeastgenome.org/) (7, 33). The codon adaptation index was determined according to previously published procedures (4). Codon bias in S. cerevisiae was obtained from reference 39.

Immunoblot analysis.

Immunoblot analysis was performed as described previously (96). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% precast gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes (Protran BA85). Membranes were incubated in blocking buffer (5% nonfat dry milk, 10 mM Tris-HCl [pH 8], 150 mM NaCl, 0.05% Tween 20) for 1 h at 25°C. ECL Plus immunoblotting reagents were used to detect secondary antibodies. Nitrocellulose membranes were incubated for 18 h at 4°C in blocking buffer containing a rabbit polyclonal immunoglobulin G antibody against the HA epitope.

Microscopy and protein localization.

Differential interference contrast microscopy and fluorescence microscopy were performed with an Axioplan 2 fluorescence microscope (Zeiss) with a PLAN-APOCHROMAT 100×/1.4 (oil) objective (numerical aperture 0.17). Digital images were obtained with an Axiocam MRm camera (Zeiss). Axiovision 4.4 software (Zeiss) was used for image acquisition and analysis. For protein localization experiments, cells were grown to saturation in selective medium to maintain plasmids harboring MSB2-GFP fusions. Cells were harvested by centrifugation and resuspended in YEPD medium for 4.5 h at 30°C. Cells were washed three times in water and visualized by fluorescence microscopy at a magnification of ×100.

mRNA level determination by quantitative PCR.

Total RNA was isolated from 25-ml cultures grown in yeast extract-peptone-galactose (YEP-GAL) medium for 8 h by hot acid phenol extraction. cDNA synthesis was carried out with 1 μg RNA and the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. One-tenth of the synthesized cDNA was used as the template for real-time PCR. Twenty-five-microliter real-time PCRs were performed on a Bio-Rad MyiQ Cycler with iQ SYBR green Supermix (Bio-Rad). Quantitative reverse transcription (RT)-PCR was performed by initial denaturation for 8 min at 95°C, followed by 35 × cycle 2 (denaturation for 15 s at 95°C and annealing for 1 min at 60°C). Melting curve data collection was enabled by decreasing the set point temperature after cycle 2 by 0.5°C. The specificity of amplicons was confirmed by generating the melting curve profile of all amplified products. ACT1 was used as the reference gene for normalization of mRNA levels. Gene expression was quantified as described previously (71). The primers used were based on reference 98 and were FLO11 forward (5′-GTTCAACCAGTCCAAGCGAAA-3′) and reverse (5′-GTAGTTACAGGTGTGGTAGGTGAAGTG-3′), MSB2 forward (5′-TGACCAAACTTCGACTGCTGG-3′) and reverse (5′-AGCTGCTGATGCAGTGGTAA-3′), and ACT1 forward (5′-GGCTTCTTTGACTACCTTCCAACA-3′) and reverse (5′-GATGGACCACTTTCGTCGTATTC-3′).


Identification of elongator in a screen for mutants defective for Msb2p secretion.

Components of the elongator (Elp) protein complex were identified in a screen for mutants defective for the secretion of Msb2p (Chavel et al., submitted). Elp proteins constitute an evolutionarily conserved complex (6, 32) composed of six proteins (Iki3p/Elp1p, Elp2p, Elp3p, Elp4p, Elp5p, and Elp6p) that exist in different subcomplexes (41, 45, 70, 89, 99). The iki3Δ, elp2Δ, elp3Δ, and elp4Δ mutants were identified in the screen as secretion defective (Fig. (Fig.1;1; Table Table2).2). The elp5Δ mutant is inviable (41) and was not tested (Table (Table2),2), and the elp6Δ mutant was not identified, although direct testing confirmed a role for Epl6p in Msb2p regulation (data not shown). Kti12p associates with the Elp complex and is required for Elp function (69). The kti12Δ mutant was also identified in the screen (Fig. (Fig.1;1; Table Table2).2). The elp mutants were among those that displayed the strongest defect in the screen (∼4,800 mutants tested). The Msb2p-HA secretion defect was roughly equivalent between elp mutants (Fig. (Fig.1A;1A; Table Table2),2), which is consistent with the idea that the Elp proteins work together as a complex (20). Differences in Msb2p-HA secretion between elp mutants in Fig. Fig.11 may be due to slight nutritional differences resulting from differences in processing time and the positions of the colonies on the plates (see below).

FIG. 1.
Elongator contributes to shedding of Msb2p-HA. Colony immunoblot assays are shown in which ordered deletions (24) transformed with plasmid pMSB2-HA were pinned onto SD-URA medium overlaid with a nitrocellulose filter (top panels). Colonies were incubated ...
Components of elongator and other tRNA modification proteins that influence the secretion of Msb2p-HA or MAPK activity

Elongator modifies 5-methoxycarbonylmethyl and 5-carbamoylmethyl groups at the wobble positions of uridine tRNAs (35), which represents one of many different tRNA modifications (34). Components of the Trm tRNA methyltransferase complex, including Trm1p (17) and the Urm1p ubiquitin-related modifier, which modifies some tRNA species (44, 85), were also identified in the screen (Table (Table2).2). Most of the genes that function in tRNA modification were not defective for Msb2p secretion (see Table S1 in the supplemental material), which indicates that a general defect in tRNA regulation does not underlie the secretion defect of the Msb2p protein. Similarly, many genes that function in protein translation were not identified in the screen, further suggesting a specific function for elongator in Msb2p regulation.

The Elp complex is a positive regulator of the filamentous growth pathway.

The secretion of Msb2p is tied to its function because processing and release of the N-terminal inhibitory domain of the Msb2p protein are necessary for filamentous growth pathway activity (96). Because Msb2p functions at the head of the MAPK pathway that controls filamentous growth (10), we investigated whether elongator regulates the filamentous growth pathway. The gene that encodes one of the components of elongator (Elp2p) was disrupted because loss of any member of the complex is thought to render the complex inactive (20). The ELP2 gene was disrupted in strains of the Σ1278b background, and the elp2Δ mutant was evaluated for filamentous growth and MAPK signaling. The elp2Δ mutant was defective for invasive growth (Fig. (Fig.2A),2A), as determined by the plate-washing assay (81). The invasive growth defect of the elp2Δ mutant was comparable to that of other filamentous growth pathway msb2Δ, sho1Δ, and ste11Δ mutants (Fig. (Fig.2A)2A) and less severe than that of the ste12Δ mutant (Fig. (Fig.2A),2A), which lacks the transcription factor for the pathway, rendering the pathway inoperative (51). The elp2Δ mutant colony had a smooth appearance (Fig. (Fig.2A),2A), indicative of a defect in MAPK- and Flo11p-dependent cell-cell adhesion. The elp2Δ mutant also exhibited cell elongation and distal-pole budding defects. As assessed by the single-cell invasive growth assay (12), the elp2Δ mutant failed to exhibit the elongated cell morphology characteristic of wild-type cells (Fig. (Fig.2B,2B, black arrows). The elp2Δ mutant was similarly defective in distal pole budding, and cells of the elp2Δ mutant were observed budding back toward their parents (Fig. (Fig.2B,2B, white arrow and data not shown). The polarity defect of the elp2Δ mutant was equivalent to that of other filamentous growth pathway components (Fig. (Fig.2B).2B). The elp2Δ mutant was also defective for production and release of the polygalacturonidase Pgu1p (Fig. (Fig.2C),2C), which is also regulated by the filamentous growth pathway (52).

FIG. 2.
The elp2 mutant is defective for filamentous growth and MAPK pathway activation. (A) Plate-washing assay showing the agar invasion defect of the msb2Δ (PC948), sho1Δ (PC1531), ste11Δ (PC611), and ste12Δ (PC539) filamentous ...

The elp2Δ mutant was defective for MAPK activity based on the expression of the FUS1-lacZ reporter (Fig. (Fig.2D)2D) and showed a phenotype similar to that of the msb2Δ, ste11Δ, and ste12Δ mutants (Fig. (Fig.2D).2D). The expression of other filamentous growth pathway target genes, particularly the FLO11 gene (83), was also reduced in the elp2Δ mutant (Fig. (Fig.2E).2E). Direct testing confirmed an equivalent role for Elp6p in FG pathway signaling, and although not present in the deletion collection, the KTI11 gene, which functions with elongator (21), was also required for pathway signaling (data not shown). Urm1p and Ncs2p constitute a ubiquitin-like modification system for tRNA species (44, 85) and are required for filamentous growth (29). Direct testing showed that Urm1p and Ncs2p also had a positive role in FG pathway signaling, although not to the same degree as the Elp complex (data not shown). To summarize, we conclude that elongator is required for activation of the filamentous growth pathway.

Elongator contributes to mat formation.

The filamentous growth pathway contributes to the expansion of cells in a microbial mat that occurs in a MAPK- and Flo11p-dependent manner (78, 79). Our Σ1278b strains showed a particularly strong dependency on the filamentous growth pathway for mat form growth. We tested whether the Elp complex is required for the expansion of microbial mats. The elp2Δ mutant was defective in mat expansion (Fig. (Fig.3A)3A) and exhibited a phenotype similar to but less severe than that of the flo11Δ and ste12Δ mutants (Fig. (Fig.3A).3A). The elp2 mutant also showed a reduction in the characteristic ruffling pattern of microbial mats (Fig. (Fig.3B).3B). The contribution of elongator to microbial mat expansion corroborates the conclusion that elongator is a positive regulator of the filamentous growth pathway.

FIG. 3.
Elp2p plays a role in mat expansion and patterning. (A) Wild-type (PC538, upper left) and elp2Δ (PC2763, upper right), ste12Δ (PC539, lower left), and flo11Δ (PC1029, lower right) mutant cells were spotted onto medium permissive ...

Elongator functions at the level of Msb2p.

Genetic suppression (epistasis) analysis can provide information as to where in a regulatory cascade a given protein functions. To determine at which point in the MAPK pathway elongator functions, genetic analysis was performed by examining double-mutation combinations between elp2Δ and mutations that hyperactivate the filamentous growth pathway. Overexpression of MSB2 stimulates the filamentous growth pathway (10). Overexpression of MSB2 partially bypassed the agar invasion defect of the elp2Δ mutant (Fig. (Fig.4A).4A). Likewise, overexpression of MSB2 partially bypassed the signaling defect of the elp2Δ mutant. Specifically, the activity of the growth reporter FUS1-HIS3 was partially restored in the GAL-MSB2 elp2Δ double mutant (Fig. (Fig.4A).4A). β-Galactosidase assays confirmed that FUS1-lacZ expression was partially restored in the GAL-MSB2 elp2Δ mutant (Fig. (Fig.4B).4B). The cell elongation defect of the elp2Δ mutant was not suppressed by overexpression of MSB2 (Fig. (Fig.4C),4C), indicative of partial suppression. Because MSB2 can partially bypass the defect in elongator function, elongator may function at the head of the filamentous growth pathway.

FIG. 4.
Bypass of elp2 by overexpression or hyperactivation of MSB2. (A to C) Partial suppression of the elp2Δ mutant defects in cells overexpressing MSB2. (A) Equal concentrations of cells were spotted onto plates. The wild type (PC538) and the elp2 ...

Genetic suppression analysis with a strong activated allele of MSB2 (96) bore out these findings. The hyperactive allele MSB2Δ100-818 effectively bypassed the agar invasion defect of the elp2Δ mutant (Fig. (Fig.4D).4D). The signaling and cell elongation defects were similarly suppressed by MSB2Δ100-818 (Fig. 4E and F). Activated alleles of SHO1 and STE11 (87, 96), which function below Msb2p in the pathway, also suppressed the signaling defect associated with loss of Elp function (data not shown). Together, the genetic analysis results indicate that elongator functions at or above the level of Msb2p. Because MAPK activity was not fully restored, we cannot exclude the possibility that elongator also functions at another point in the signaling cascade.

Elongator regulates starvation-dependent expression of the MSB2 gene.

Based on the fact that elongator functions at or above Msb2p in the filamentous growth pathway, we investigated different aspects of Msb2p regulation in elongator mutants. The Elp complex contributes to multiple cellular processes (90) and might potentially regulate the expression or translation of the MSB2 gene or the glycosylation, cell surface delivery, localization, processing, or shedding of the Msb2p protein. We examined the localization pattern of an Msb2p-GFP fusion protein in wild-type cells and in the elp2Δ mutant. Msb2p-GFP exhibits a diffuse localization pattern, with several punctate sites distributed throughout mother and daughter cells (Fig. (Fig.5A).5A). The localization pattern of Msb2p-GFP was indistinguishable between wild-type and elp2Δ mutant cells (Fig. (Fig.5A).5A). Other elp mutants also showed a wild-type Msb2p-GFP localization pattern (Table (Table2),2), which indicates that elongator does not regulate Msb2p localization.

FIG. 5.
Elongator promotes starvation-dependent expression of the MSB2 gene but does not influence the localization or maturation of the protein. (A) Localization of Msb2p-GFP in wild-type and elp2Δ mutant strains. DIC, differential interference contrast; ...

Elongator might regulate the delivery of Msb2p to the cell surface, particularly because elongator has an established function in exocytosis (74, 75), which involves the targeted delivery of secretory vesicles to polarized sites (22, 63, 95, 102). The role of elongator in exocytosis, however, appears to be inhibitory (74, 75), and therefore elongator mutants might be expected to show elevated secretion of Msb2p. Immunoblot analysis demonstrated that the Msb2p-HA protein was produced, glycosylated, and secreted in the elp2Δ mutant in a manner equivalent to that observed in wild-type cells, which indicates that elongator does not function in the glycosylation, trafficking, exocytosis, or processing of Msb2p (Fig. (Fig.5B5B).

Immunoblot analysis did show that the overall level of the Msb2p-HA protein was reduced in the elp2Δ mutant (Fig. (Fig.5B).5B). We therefore tested whether elongator contributes to the expression of the MSB2 gene. The expression of an MSB2-lacZ reporter was examined in wild-type cells and the elp2Δ mutant. MSB2-lacZ expression was reduced in the elp2Δ mutant (Fig. (Fig.5C).5C). This effect was subtle but was more pronounced under nutrient-limiting conditions, under which MSB2 expression is induced (S-GAL medium compared to SD medium, Fig. Fig.5C).5C). Other elp mutants also showed a reduction in MSB2-lacZ expression (Table (Table2).2). Consistent with this result, the Msb2p-HA secretion defect in elp mutants was more pronounced after 2 days (Fig. (Fig.5D;5D; Table Table2),2), presumably when environmental nutrients had become depleted. The fact that elongator acts at the level of MSB2 expression is consistent with the genetic analysis, in that overexpression or hyperactivation of Msb2p can partially bypass the signaling defect of elp mutants.

Elongator modifies tRNAs that code for glutamine (GLN) and lysine (LYS) (19, 35). We also tested whether the wobble positions of GLN and LYS codons were biased in the MSB2 gene, which might result in a translation defect of the Msb2p protein in elp mutants. The GLN codon frequency in MSB2 (73.5% CAA, 26.5% CAG) was in close agreement with the overall codon bias observed throughout the yeast genome (69% CAA, 31% CAG) (39). Similarly, the LYS codon bias in MSB2 (56% AAA, 44% AAG) resembled the overall bias throughout the genome (58% AAA, 42% AAG) (39). Thus, codon bias does not appear to account for the decrease in Msb2p protein levels in elp mutants. The fact that the expression of the MSB2 gene, not translation of the MSB2 message, is affected in elongator mutants was further supported by quantitative PCR analysis (Fig. (Fig.5E).5E). Therefore, elongator appears to function at the level of expression of the MSB2 gene.

Elongator is not required for pheromone response or high-osmolarity glycerol response (HOG) pathway activation.

The filamentous growth pathway shares components with the pheromone response pathway (1), including the p21-activated kinase Ste20p, the adaptor protein Ste50p, the MAPK kinase kinase Ste11p, the MAPK kinase Ste7p, and transcription factor Ste12p (15, 16, 18). Msb2p does not appear to be required for activation of the pheromone response pathway (10). To determine whether elongator is required for activation of the pheromone response pathway, the elp2Δ mutant was examined for defects in pheromone response. A haploid MATa strain lacking the ELP2 gene showed sensitivity to the mating pheromone α-factor equivalent to that of the wild-type MATa strain (Fig. (Fig.6A).6A). Specifically, the initial response to α-factor, assessed by shmoo formation, was equivalent between the two strains (Fig. (Fig.6A).6A). Both the number of cells that formed shmoos and the degree of polarization of shmoo tips were similar between wild-type and elp2Δ mutant cells (Fig. (Fig.6A).6A). In addition, α-factor-induced cell cycle arrest was equivalent between the two strains, as determined by halo assay (Fig. (Fig.6A,6A, right panels). Pheromone response pathway mutants did not exhibit morphological characteristics of shmoos or halo formation in response to α-factor (data not shown). Therefore, elongator is not required for pheromone response pathway activation.

FIG. 6.
Elp2p is not required for pheromone response or HOG pathway function. (A) Shmoo formation over time. Wild-type (PC313; upper panel) and elp2Δ mutant (PC2980, lower panel) cells were grown to mid-log phase in YEPD medium, washed, and resuspended ...

The filamentous growth pathway also shares components with the Sho1p branch of the HOG pathway (64, 65, 94). The Ste11p branch of the HOG pathway functions redundantly with the Sln1p branch (76, 92), so we tested for a role for elongator in mutants defective for the Sln1p branch of the HOG pathway (ssk1Δ). Cells lacking the MAPK kinase for the HOG pathway (pbs2Δ) or both upstream regulatory branches (ste11Δ ssk1Δ) were sensitive to the osmolite KCl (Fig. (Fig.6B).6B). In contrast, the elp2Δ ssk1Δ double mutant showed the same level of resistance to KCl as wild-type cells (Fig. (Fig.6B).6B). The ssk1 (PC1523), ste11 (PC611), and elp2 (PC2980) single mutants did not exhibit sensitivity to KCl (data not shown). This result demonstrates that elongator is not required for activation of the Ste11p branch of the HOG pathway. Evaluation of Elp2p function in the pheromone, filamentous growth, and HOG pathways was performed in the same background (Σ1278b). The fact that elongator regulates MSB2 expression is consistent with its failure to influence the mating and HOG pathways, given that Msb2p does not function in the mating pathway (10) and functions in the HOG pathway redundantly with the cell surface protein Hkr1p (93).


We report here a new regulatory input to the filamentous growth pathway by the tRNA modification enzyme complex elongator. Elongator is required for filamentous growth pathway activity and contributes to the established outputs of the pathway, including invasive growth, the reorganization of cell polarity, and Flo11p-dependent cell-cell adhesion of filamentous cells (Fig. (Fig.2)2) and expanding mats (Fig. (Fig.3).3). A precedent connecting tRNA metabolism to filamentous growth has been established by a GLN tRNA (tRNA-CUG), which signals nitrogen status and regulates filamentous growth (59). In that case, tRNA-CUG did not appear to function through the filamentous growth pathway. Our results support the overall notion that tRNA levels serve as an intracellular metric for cellular nutrient status by impacting the overall rate of protein translation. In this way, protein translation rate may feed into signaling networks that regulate cellular behaviors. The specific advance made by this study is the connection between nutrient regulation (at the level of tRNA modification) and the activity of a MAPK pathway that functions primarily under conditions of starvation. This discovery may begin to explain how nutrient levels feed into MAPK regulation.

We show that elongator contributes to the expression of the MSB2 gene. The MSB2 gene is induced under nutrient-limiting conditions (96), and elongator contributes to starvation-dependent induction of MSB2 expression (Fig. (Fig.5).5). Elongator may influence MSB2 expression through several different mechanisms. Elongator is a component of the RNA polymerase II holoenzyme and contributes to transcription elongation (26, 38) through a mechanism that is not entirely clear (73). Elongator also functions in chromatin remodeling (36) and has roles that overlap those of the SAGA histone acetyltransferase complex (100). It is possible that expression of the MSB2 gene is regulated by either transcription elongation or chromatin remodeling proteins or a combination of these mechanisms. Elongator may also contribute to the translation of the MSB2 mRNA. However, a specific codon bias was not detected in the coding region of the MSB2 open reading frame, which argues against this possibility. It is likely that the effect of elongator on MSB2 expression is mediated indirectly as a result of the tRNA modification function of the enzyme complex (19).

Mammalian components of elongator have been shown to regulate signal transduction networks. For example, IKAP, the mammalian homolog of Iki3p/Elp1p, has been shown to associate with the JNK kinase (56) and with IκK, the IκB kinase (3, 8, 56). StIP1, the mammalian homolog of Elp2p, binds to multiple STAT proteins and Janus kinases (9). Like Elp2p, StIP1 is thought to act as a scaffold for signaling complex assembly by virtue of its WD-40 domain. Based on the genetic suppression analysis, we cannot exclude the possibility that Elp proteins directly interact with components of the filamentous growth pathway to modulate their function. The salient finding from our study is the regulation of a MAPK pathway by a tRNA modification system through regulation of expression of the gene that functions at the pathway's head. This regulatory mechanism may occur in other systems to control signaling networks by tRNA modification enzymes.

Supplementary Material

[Supplemental material]


We thank Anders Byström for sending plasmids and Eric Phizicky for helpful discussions related to the manuscript. Thanks also to C. Boone and H. Madhani for providing plasmids. Thanks to Nadia Vadaie for assistance with the immunoblot analysis.

This work was supported by grants from the NIH (1R03DE018425-01) and the American Cancer Society (TBE-114083).


Published ahead of print on 24 July 2009.

Supplemental material for this article may be found at http://ec.asm.org/.


1. Bardwell, L. 2006. Mechanisms of MAPK signalling specificity. Biochem. Soc. Trans. 34:837-841. [PMC free article] [PubMed]
2. Baudin, A., O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, and C. Cullin. 1993. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21:3329-3330. [PMC free article] [PubMed]
3. Bouwmeester, T., A. Bauch, H. Ruffner, P. O. Angrand, G. Bergamini, K. Croughton, C. Cruciat, D. Eberhard, J. Gagneur, S. Ghidelli, C. Hopf, B. Huhse, R. Mangano, A. M. Michon, M. Schirle, J. Schlegl, M. Schwab, M. A. Stein, A. Bauer, G. Casari, G. Drewes, A. C. Gavin, D. B. Jackson, G. Joberty, G. Neubauer, J. Rick, B. Kuster, and G. Superti-Furga. 2004. A physical and functional map of the human TNF-α/NF-κB signal transduction pathway. Nat. Cell Biol. 6:97-105. [PubMed]
4. Carbone, A., A. Zinovyev, and F. Kepes. 2003. Codon adaptation index as a measure of dominating codon bias. Bioinformatics 19:2005-2015. [PubMed]
5. Chant, J., and J. R. Pringle. 1995. Patterns of bud-site selection in the yeast Saccharomyces cerevisiae. J. Cell Biol. 129:751-765. [PMC free article] [PubMed]
6. Chen, Z., H. Zhang, D. Jablonowski, X. Zhou, X. Ren, X. Hong, R. Schaffrath, J.-K. Zhu, and Z. Gong. 2006. Mutations in ABO1/ELO2, a subunit of holo-Elongator, increase abscisic acid sensitivity and drought tolerance in Arabidopsis thaliana. Mol. Cell. Biol. 26:6902-6912. [PMC free article] [PubMed]
7. Cherry, J. M., C. Adler, C. Ball, S. A. Chervitz, S. S. Dwight, E. T. Hester, Y. Jia, G. Juvik, T. Roe, M. Schroeder, S. Weng, and D. Botstein. 1998. SGD: Saccharomyces Genome Database. Nucleic Acids Res. 26:73-79. [PMC free article] [PubMed]
8. Cohen, L., W. J. Henzel, and P. A. Baeuerle. 1998. IKAP is a scaffold protein of the IκB kinase complex. Nature 395:292-296. [PubMed]
9. Collum, R. G., S. Brutsaert, G. Lee, and C. Schindler. 2000. A Stat3-interacting protein (StIP1) regulates cytokine signal transduction. Proc. Natl. Acad. Sci. USA 97:10120-10125. [PMC free article] [PubMed]
10. Cullen, P. J., W. Sabbagh, Jr., E. Graham, M. M. Irick, E. K. van Olden, C. Neal, J. Delrow, L. Bardwell, and G. F. Sprague, Jr. 2004. A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast. Genes Dev. 18:1695-1708. [PMC free article] [PubMed]
11. Cullen, P. J., J. Schultz, J. Horecka, B. J. Stevenson, Y. Jigami, and G. F. Sprague, Jr. 2000. Defects in protein glycosylation cause SHO1-dependent activation of a STE12 signaling pathway in yeast. Genetics 155:1005-1018. [PMC free article] [PubMed]
12. Cullen, P. J., and G. F. Sprague, Jr. 2000. Glucose depletion causes haploid invasive growth in yeast. Proc. Natl. Acad. Sci. USA 97:13619-13624. [PMC free article] [PubMed]
13. Cullen, P. J., and G. F. Sprague, Jr. 2002. The Glc7p-interacting protein Bud14p attenuates polarized growth, pheromone response, and filamentous growth in Saccharomyces cerevisiae. Eukaryot. Cell 1:884-894. [PMC free article] [PubMed]
14. Cullen, P. J., and G. F. Sprague, Jr. 2002. The roles of bud-site-selection proteins during haploid invasive growth in yeast. Mol. Biol. Cell 13:2990-3004. [PMC free article] [PubMed]
15. Dohlman, H. G., and J. E. Slessareva. 2006. Pheromone signaling pathways in yeast. Sci. STKE 2006:cm6. [PubMed]
16. Elion, E. A. 2000. Pheromone response, mating and cell biology. Curr. Opin. Microbiol. 3:573-581. [PubMed]
17. Ellis, S. R., M. J. Morales, J.-M. Li, A. K. Hopper, and N. C. Martin. 1986. Isolation and characterization of the TRM1 locus, a gene essential for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNA in Saccharomyces cerevisiae. J. Biol. Chem. 261:9703-9709. [PubMed]
18. Errede, B., R. M. Cade, B. M. Yashar, Y. Kamada, D. E. Levin, K. Irie, and K. Matsumoto. 1995. Dynamics and organization of MAP kinase signal pathways. Mol. Reprod Dev. 42:477-485. [PubMed]
19. Esberg, A., B. Huang, M. J. Johansson, and A. S. Bystrom. 2006. Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis. Mol. Cell 24:139-148. [PubMed]
20. Fellows, J., H. Erdjument-Bromage, P. Tempst, and J. Q. Svejstrup. 2000. The Elp2 subunit of elongator and elongating RNA polymerase II holoenzyme is a WD40 repeat protein. J. Biol. Chem. 275:12896-12899. [PubMed]
21. Fichtner, L., D. Jablonowski, A. Schierhorn, H. K. Kitamoto, M. J. Stark, and R. Schaffrath. 2003. Elongator's toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification. Mol. Microbiol. 49:1297-1307. [PubMed]
22. Finger, F. P., T. E. Hughes, and P. Novick. 1998. Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92:559-571. [PubMed]
23. Gelperin, D. M., M. A. White, M. L. Wilkinson, Y. Kon, L. A. Kung, K. J. Wise, N. Lopez-Hoyo, L. Jiang, S. Piccirillo, H. Yu, M. Gerstein, M. E. Dumont, E. M. Phizicky, M. Snyder, and E. J. Grayhack. 2005. Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 19:2816-2826. [PMC free article] [PubMed]
24. Giaever, G., A. M. Chu, L. Ni, C. Connelly, L. Riles, S. Veronneau, S. Dow, A. Lucau-Danila, K. Anderson, B. Andre, A. P. Arkin, A. Astromoff, M. El-Bakkoury, R. Bangham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss, K. Davis, A. Deutschbauer, K. D. Entian, P. Flaherty, F. Foury, D. J. Garfinkel, M. Gerstein, D. Gotte, U. Guldener, J. H. Hegemann, S. Hempel, Z. Herman, D. F. Jaramillo, D. E. Kelly, S. L. Kelly, P. Kotter, D. LaBonte, D. C. Lamb, N. Lan, H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S. L. Ooi, J. L. Revuelta, C. J. Roberts, M. Rose, P. Ross-Macdonald, B. Scherens, G. Schimmack, B. Shafer, D. D. Shoemaker, S. Sookhai-Mahadeo, R. K. Storms, J. N. Strathern, G. Valle, M. Voet, G. Volckaert, C. Y. Wang, T. R. Ward, J. Wilhelmy, E. A. Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J. D. Boeke, M. Snyder, P. Philippsen, R. W. Davis, and M. Johnston. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387-391. [PubMed]
25. Gietz, R. D., and R. H. Schiestl. 2007. Microtiter plate transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2:5-8. [PubMed]
26. Gilbert, C., A. Kristjuhan, G. S. Winkler, and J. Q. Svejstrup. 2004. Elongator interactions with nascent mRNA revealed by RNA immunoprecipitation. Mol. Cell 14:457-464. [PubMed]
27. Gimeno, C. J., and G. R. Fink. 1994. Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development. Mol. Cell. Biol. 14:2100-2112. [PMC free article] [PubMed]
28. Gimeno, C. J., P. O. Ljungdahl, C. A. Styles, and G. R. Fink. 1992. Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68:1077-1090. [PubMed]
29. Goehring, A. S., D. M. Rivers, and G. F. Sprague, Jr. 2003. Urmylation: a ubiquitin-like pathway that functions during invasive growth and budding in yeast. Mol. Biol. Cell 14:4329-4341. [PMC free article] [PubMed]
30. Goldstein, A. L., and J. H. McCusker. 1999. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541-1553. [PubMed]
31. Guo, B., C. A. Styles, Q. Feng, and G. R. Fink. 2000. A Saccharomyces gene family involved in invasive growth, cell-cell adhesion, and mating. Proc. Natl. Acad. Sci. USA 97:12158-12163. [PMC free article] [PubMed]
32. Hawkes, N. A., G. Otero, G. S. Winkler, N. Marshall, M. E. Dahmus, D. Krappmann, C. Scheidereit, C. L. Thomas, G. Schiavo, H. Erdjument-Bromage, P. Tempst, and J. Q. Svejstrup. 2002. Purification and characterization of the human elongator complex. J. Biol. Chem. 277:3047-3052. [PubMed]
33. Hong, E. L., R. Balakrishnan, Q. Dong, K. R. Christie, J. Park, G. Binkley, M. C. Costanzo, S. S. Dwight, S. R. Engel, D. G. Fisk, J. E. Hirschman, B. C. Hitz, C. J. Krieger, M. S. Livstone, S. R. Miyasato, R. S. Nash, R. Oughtred, M. S. Skrzypek, S. Weng, E. D. Wong, K. K. Zhu, K. Dolinski, D. Botstein, and J. M. Cherry. 2008. Gene ontology annotations at SGD: new data sources and annotation methods. Nucleic Acids Res. 36:D577-D581. [PMC free article] [PubMed]
34. Hopper, A. K., and E. M. Phizicky. 2003. tRNA transfers to the limelight. Genes Dev. 17:162-180. [PubMed]
35. Huang, B., M. J. Johansson, and A. S. Bystrom. 2005. An early step in wobble uridine tRNA modification requires the Elongator complex. RNA 11:424-436. [PMC free article] [PubMed]
36. Jablonowski, D., A. R. Butler, L. Fichtner, D. Gardiner, R. Schaffrath, and M. J. Stark. 2001. Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin. Genetics 159:1479-1489. [PMC free article] [PubMed]
37. Johnson, D. I. 1999. Cdc42: an essential Rho-type GTPase controlling eukaryotic cell polarity. Microbiol. Mol. Biol. Rev. 63:54-105. [PMC free article] [PubMed]
38. Jona, G., B. O. Wittschieben, J. Q. Svejstrup, and O. Gileadi. 2001. Involvement of yeast carboxy-terminal domain kinase I (CTDK-I) in transcription elongation in vivo. Gene 267:31-36. [PubMed]
39. Kliman, R. M., N. Irving, and M. Santiago. 2003. Selection conflicts, gene expression, and codon usage trends in yeast. J. Mol. Evol. 57:98-109. [PubMed]
40. Klopotowski, T., and A. Wiater. 1965. Synergism of aminotriazole and phosphate on the inhibition of yeast imidazole glycerol phosphate dehydratase. Arch. Biochem. Biophys. 112:562-566. [PubMed]
41. Krogan, N. J., and J. F. Greenblatt. 2001. Characterization of a six-subunit Holo-Elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 21:8203-8212. [PMC free article] [PubMed]
42. Kron, S. J., C. A. Styles, and G. R. Fink. 1994. Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 5:1003-1022. [PMC free article] [PubMed]
43. Leberer, E., C. Wu, T. Leeuw, A. Fourest-Lieuvin, J. E. Segall, and D. Y. Thomas. 1997. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase. EMBO J. 16:83-97. [PMC free article] [PubMed]
44. Leidel, S., P. G. Pedrioli, T. Bucher, R. Brost, M. Costanzo, A. Schmidt, R. Aebersold, C. Boone, K. Hofmann, and M. Peter. 2009. Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA. Nature 458:228-232. [PubMed]
45. Li, Y., Y. Takagi, Y. Jiang, M. Tokunaga, H. Erdjument-Bromage, P. Tempst, and R. D. Kornberg. 2001. A multiprotein complex that interacts with RNA polymerase II elongator. J. Biol. Chem. 276:29628-29631. [PubMed]
46. Liu, H. 2001. Transcriptional control of dimorphism in Candida albicans. Curr. Opin. Microbiol. 4:728-735. [PubMed]
47. Liu, H., C. A. Styles, and G. R. Fink. 1993. Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 262:1741-1744. [PubMed]
48. Lo, H. J., J. R. Kohler, B. DiDomenico, D. Loebenberg, A. Cacciapuoti, and G. R. Fink. 1997. Nonfilamentous C. albicans mutants are avirulent. Cell 90:939-949. [PubMed]
49. Lo, W.-S., and A. M. Dranginis. 1996. FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin. J. Bacteriol. 178:7144-7151. [PMC free article] [PubMed]
50. Longtine, M. S., A. McKenzie III, D. J. Demarini, N. G. Shah, A. Wach, A. Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953-961. [PubMed]
51. Madhani, H. D., and G. R. Fink. 1997. Combinatorial control required for the specificity of yeast MAPK signaling. Science 275:1314-1317. [PubMed]
52. Madhani, H. D., T. Galitski, E. S. Lander, and G. R. Fink. 1999. Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants. Proc. Natl. Acad. Sci. USA 96:12530-12535. [PMC free article] [PubMed]
53. Madhani, H. D., C. A. Styles, and G. R. Fink. 1997. MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation. Cell 91:673-684. [PubMed]
54. Maecker, H. T., S. C. Todd, and S. Levy. 1997. The tetraspanin superfamily: molecular facilitators. FASEB J. 11:428-442. [PubMed]
55. Maeda, T., M. Takekawa, and H. Saito. 1995. Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. Science 269:554-558. [PubMed]
56. Mercurio, F., B. W. Murray, A. Shevchenko, B. L. Bennett, D. B. Young, J. W. Li, G. Pascual, A. Motiwala, H. Zhu, M. Mann, and A. M. Manning. 1999. IκB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex. Mol. Cell. Biol. 19:1526-1538. [PMC free article] [PubMed]
57. Mösch, H. U., E. Kubler, S. Krappmann, G. R. Fink, and G. H. Braus. 1999. Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae. Mol. Biol. Cell 10:1325-1335. [PMC free article] [PubMed]
58. Mösch, H. U., R. L. Roberts, and G. R. Fink. 1996. Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 93:5352-5356. [PMC free article] [PubMed]
59. Murray, L. E., N. Rowley, I. W. Dawes, G. C. Johnston, and R. A. Singer. 1998. A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation. Proc. Natl. Acad. Sci. USA 95:8619-8624. [PMC free article] [PubMed]
60. Nelson, C., S. Goto, K. Lund, W. Hung, and I. Sadowski. 2003. Srb10/Cdk8 regulates yeast filamentous growth by phosphorylating the transcription factor Ste12. Nature 421:187-190. [PubMed]
61. Nemecek, J. C., M. Wuthrich, and B. S. Klein. 2006. Global control of dimorphism and virulence in fungi. Science 312:583-588. [PubMed]
62. Nobile, C. J., and A. P. Mitchell. 2006. Genetics and genomics of Candida albicans biofilm formation. Cell. Microbiol. 8:1382-1391. [PubMed]
63. Novick, P., M. Medkova, G. Dong, A. Hutagalung, K. Reinisch, and B. Grosshans. 2006. Interactions between Rabs, tethers, SNAREs and their regulators in exocytosis. Biochem. Soc. Trans. 34:683-686. [PubMed]
64. O'Rourke, S. M., and I. Herskowitz. 1998. The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev. 12:2874-2886. [PMC free article] [PubMed]
65. O'Rourke, S. M., I. Herskowitz, and E. K. O'Shea. 2002. Yeast go the whole HOG for the hyperosmotic response. Trends Genet. 18:405-412. [PubMed]
66. Pan, X., T. Harashima, and J. Heitman. 2000. Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae. Curr. Opin. Microbiol. 3:567-572. [PubMed]
67. Park, H. O., and E. Bi. 2007. Central roles of small GTPases in the development of cell polarity in yeast and beyond. Microbiol. Mol. Biol. Rev. 71:48-96. [PMC free article] [PubMed]
68. Peter, M., A. M. Neiman, H. O. Park, M. van Lohuizen, and I. Herskowitz. 1996. Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. EMBO J. 15:7046-7059. [PMC free article] [PubMed]
69. Petrakis, T. G., T. M. Sogaard, H. Erdjument-Bromage, P. Tempst, and J. Q. Svejstrup. 2005. Physical and functional interaction between Elongator and the chromatin-associated Kti12 protein. J. Biol. Chem. 280:19454-19460. [PubMed]
70. Petrakis, T. G., B. O. Wittschieben, and J. Q. Svejstrup. 2004. Molecular architecture, structure-function relationship, and importance of the Elp3 subunit for the RNA binding of holo-elongator. J. Biol. Chem. 279:32087-32092. [PubMed]
71. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e45. [PMC free article] [PubMed]
72. Pitoniak, A., B. Birkaya, H. S. Dionne, N. Vadiae, and P. J. Cullen. 2009. The signaling mucins Msb2 and Hkr1 differentially regulate the filamentation mitogen-activated protein kinase pathway and contribute to a multimodal response. Mol. Biol. Cell 20:3101-3114. [PMC free article] [PubMed]
73. Pokholok, D. K., N. M. Hannett, and R. A. Young. 2002. Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. Mol. Cell 9:799-809. [PubMed]
74. Rahl, P. B., C. Z. Chen, and R. N. Collins. 2005. Elp1p, the yeast homolog of the FD disease syndrome protein, negatively regulates exocytosis independently of transcriptional elongation. Mol. Cell 17:841-853. [PubMed]
75. Rahl, P. B., and R. N. Collins. 2008. Analysis of the Elp complex and its role in regulating exocytosis. Methods Enzymol. 439:315-325. [PubMed]
76. Reiser, V., D. C. Raitt, and H. Saito. 2003. Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure. J. Cell Biol. 161:1035-1040. [PMC free article] [PubMed]
77. Reynolds, T. B. 2006. The Opi1p transcription factor affects expression of FLO11, mat formation, and invasive growth in Saccharomyces cerevisiae. Eukaryot. Cell 5:1266-1275. [PMC free article] [PubMed]
78. Reynolds, T. B., and G. R. Fink. 2001. Bakers' yeast, a model for fungal biofilm formation. Science 291:878-881. [PubMed]
79. Reynolds, T. B., A. Jansen, X. Peng, and G. R. Fink. 2008. Mat formation in Saccharomyces cerevisiae requires nutrient and pH gradients. Eukaryot. Cell 7:122-130. [PMC free article] [PubMed]
80. Roberts, C. J., B. Nelson, M. J. Marton, R. Stoughton, M. R. Meyer, H. A. Bennett, Y. D. He, H. Dai, W. L. Walker, T. R. Hughes, M. Tyers, C. Boone, and S. H. Friend. 2000. Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287:873-880. [PubMed]
81. Roberts, R. L., and G. R. Fink. 1994. Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev. 8:2974-2985. [PubMed]
82. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
83. Rupp, S., E. Summers, H. J. Lo, H. Madhani, and G. Fink. 1999. MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene. EMBO J. 18:1257-1269. [PMC free article] [PubMed]
84. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
85. Schlieker, C. D., A. G. Van der Veen, J. R. Damon, E. Spooner, and H. L. Ploegh. 2008. A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway. Proc. Natl. Acad. Sci. USA 105:18255-18260. [PMC free article] [PubMed]
86. Schneider, B. L., W. Seufert, B. Steiner, Q. H. Yang, and A. B. Futcher. 1995. Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast 11:1265-1274. [PubMed]
87. Stevenson, B. J., N. Rhodes, B. Errede, and G. F. Sprague, Jr. 1992. Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 6:1293-1304. [PubMed]
88. Struhl, K., and R. W. Davis. 1977. Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3). Proc. Natl. Acad. Sci. USA 74:5255-5259. [PMC free article] [PubMed]
89. Sun, J., J. Zhang, F. Wu, C. Xu, S. Li, W. Zhao, Z. Wu, J. Wu, C. Z. Zhou, and Y. Shi. 2005. Solution structure of Kti11p from Saccharomyces cerevisiae reveals a novel zinc-binding module. Biochemistry 44:8801-8809. [PubMed]
90. Svejstrup, J. Q. 2007. Elongator complex: how many roles does it play? Curr. Opin. Cell Biol. 19:331-336. [PubMed]
91. Taheri, N., T. Kohler, G. H. Braus, and H. U. Mösch. 2000. Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development. EMBO J. 19:6686-6696. [PMC free article] [PubMed]
92. Tatebayashi, K., M. Takekawa, and H. Saito. 2003. A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway. EMBO J. 22:3624-3634. [PMC free article] [PubMed]
93. Tatebayashi, K., K. Tanaka, H. Y. Yang, K. Yamamoto, Y. Matsushita, T. Tomida, M. Imai, and H. Saito. 2007. Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway. EMBO J. 26:3521-3533. [PMC free article] [PubMed]
94. Tatebayashi, K., K. Yamamoto, K. Tanaka, T. Tomida, T. Maruoka, E. Kasukawa, and H. Saito. 2006. Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway. EMBO J. 25:3033-3044. [PMC free article] [PubMed]
95. TerBush, D. R., T. Maurice, D. Roth, and P. Novick. 1996. The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15:6483-6494. [PMC free article] [PubMed]
96. Vadaie, N., H. Dionne, D. S. Akajagbor, S. R. Nickerson, D. J. Krysan, and P. J. Cullen. 2008. Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast. J. Cell Biol. 181:1073-1081. [PMC free article] [PubMed]
97. Vinod, P. K., N. Sengupta, P. J. Bhat, and K. V. Venkatesh. 2008. Integration of global signaling pathways, cAMP-PKA, MAPK and TOR in the regulation of FLO11. PLoS ONE 3:e1663. [PMC free article] [PubMed]
98. Voynov, V., K. J. Verstrepen, A. Jansen, V. M. Runner, S. Buratowski, and G. R. Fink. 2006. Genes with internal repeats require the THO complex for transcription. Proc. Natl. Acad. Sci. USA 103:14423-14428. [PMC free article] [PubMed]
99. Winkler, G. S., T. G. Petrakis, S. Ethelberg, M. Tokunaga, H. Erdjument-Bromage, P. Tempst, and J. Q. Svejstrup. 2001. RNA polymerase II elongator holoenzyme is composed of two discrete subcomplexes. J. Biol. Chem. 276:32743-32749. [PubMed]
100. Wittschieben, B. O., J. Fellows, W. Du, D. J. Stillman, and J. Q. Svejstrup. 2000. Overlapping roles for the histone acetyltransferase activities of SAGA and elongator in vivo. EMBO J. 19:3060-3068. [PMC free article] [PubMed]
101. Zarrinpar, A., R. P. Bhattacharyya, M. P. Nittler, and W. A. Lim. 2004. Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway. Mol. Cell 14:825-832. [PubMed]
102. Zhang, X., E. Bi, P. Novick, L. Du, K. G. Kozminski, J. H. Lipschutz, and W. Guo. 2001. Cdc42 interacts with the exocyst and regulates polarized secretion. J. Biol. Chem. 276:46745-46750. [PubMed]

Articles from Eukaryotic Cell are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...