The tRNA modification complex elongator regulates the Cdc42-dependent mitogen-activated protein kinase pathway that controls filamentous growth in yeast

Abstract

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.

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 for 2 days at 30°C. Colonies were washed off the filters, and secreted proteins were examined by immunoblot analysis. Each elp mutant is indicated by an arrow in the lower right corner.

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 growth pathway mutants alongside the elp2Δ mutant (PC2763). (B) Single-cell invasive growth assay. The strains in panel A were used. The black arrows designate the cell elongation phenotype of filamentous cells. The white arrow designates a proximal bud formed in the elp2Δ mutant. Bar, 5 μm. (C) Pgu1p activity in wild-type (PC538) and elp2Δ (PC2763), pgu1Δ (PC1519), and ste12Δ (PC539) mutant cells. (D) FUS1-lacZ expression of the strains described in panel A. β-Galactosidase assays were performed in duplicate, and error bars represent standard deviations. (E) FLO11 expression in wild-type cells, the elp2Δ mutant, and control strains as determined by quantitative real-time RT-PCR analysis.

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 for mat formation (YEPD medium plus 0.3% agar atop a nitrocellulose filter) for 3 days at 30°C. The mats were photographed with transmitted light to reveal the contoured morphology. (B) Close-up of mats in panel A showing Flo11p-dependent and Ste12p-dependent contours. The elp2Δ mutant exhibits a partial defect in mat patterning.

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 elp2Δ (PC2763), GAL-MSB2 (PC1083), and GAL-MSB2 elp2Δ (PC2978) mutants were tested. For the left two panels, cells were spotted onto YEP-GAL medium. The plate was incubated for 2 days, photographed (leftmost panel), washed, and photographed again (second panel from the left). For the right two panels, cells were spotted onto S-GAL plus amino acids (S-GAL+AA) and S-GAL without histidine (S-GAL-HIS) to examine the activity of the growth-dependent FUS1-HIS3 reporter. (B) β-Galactosidase activity of strains in panel A carrying an integrated FUS1-lacZ reporter. Assays were performed in duplicate, and error bars represent standard deviations. (C) Examples of cell morphologies of the strains examined in panel A. Bar, 5 μm. (D to F) Suppression of the elp2Δ mutant defects in cells carrying an activated version of Msb2p, Msb2p^Δ100-818. (D) Equal concentrations of cells were spotted onto YEPD medium. Wild-type (PC538) and elp2Δ (PC2763), MSB2^Δ100-818 (PC1516), and MSB2^Δ100-818 elp2Δ (PC2977) mutant cells were compared. The plate was incubated for 2 days, photographed (leftmost panel), washed, and photographed again (second panel from left). At the right, cells were spotted onto SD medium plus amino acids (SD+AA) and SD medium without histidine (SD-HIS) containing 5 mM 3-amino-1,2,4-triazole. (E) FUS1-lacZ expression of the strains from panel D. Cells were grown for 16 h in SD medium plus amino acids. Assays were performed in duplicate, and error bars represent standard deviations. (F) Examples of cell morphologies of the strains examined in panel D. Bar, 5 μm.

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; FITC, fluorescein isothiocyanate. (B) Immunoblot analysis of Msb2p-HA levels in extracts derived from wild-type (WT; PC999) and elp2Δ mutant (PC2976) cells. Cells (10 ml) were grown in YEPD medium for 16 h at 30°C. Equal numbers of cells, as determined by A₆₀₀, were harvested by centrifugation at 13,000 rpm to separate supernatants (S) from cell pellets (P). Cells were disrupted by addition of 200 μl “Thorner” lysis buffer (8 M urea, 5% sodium dodecyl sulfate, 40 mM Tris-HCl [pH 6.8], 0.1 M EDTA, 0.4 mg/ml bromophenol blue, 1% β-mercaptoethanol) and glass beads, followed by vortexing for 5 min at the highest setting and boiling for 5 min. Supernatants were examined by boiling in 1.5 volumes of lysis buffer for 5 min. Quantitation in parentheses was performed with the ImageJ plug-in Imagequant. (C) MSB2-lacZ expression in wild-type cells and the elp2 mutant. The light band represents MSB2-lacZ expression in mid-log-phase cells (SD-URA). The dark band represents MSB2-lacZ expression in synthetic medium containing 2% galactose and lacking uracil (S-GAL-URA). (D) The Msb2p-HA secretion defect of the elp2Δ and elp4Δ mutants is evident after prolonged incubation. Transformants were pinned onto SD-URA, overlaid with nitrocellulose, and incubated for 1 or 2 days (the top panel shows the 2-day incubation). Colonies were washed off of the filters (middle and lower panels), which were probed by immunoblot analysis. (E) Quantitative mRNA analysis of the MSB2 transcript by real-time RT-PCR analysis. Cells were grown for 8 h in YEP-GAL medium. RNA was harvested and evaluated by real-time RT-PCR as described in Materials and Methods.

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 in YEPD medium containing 30 μM α-factor for 1 or 3 h. Cells were harvested by centrifugation and examined by microscopy at a magnification of ×100. Bar, 5 μm. Right panels, halo assay. Equal amounts of wild-type and elp2Δ mutant cells were spread onto YEPD medium, and 2, 4, or 6 μl of 590 μM α-factor was applied to the plates. Plates were incubated for 48 h at 30°C and photographed. (B) Role of Elp2p in HOG pathway activation. Equal concentrations of wild-type (PC538) and pbs2Δ (PC2053), ssk1Δ ste11Δ (PC2061), and ssk1Δ elp2Δ (PC2991) mutant cells were spotted onto YEPD medium supplemented with the indicated concentrations of KCl. The plates were incubated for 4 days at 30°C and photographed.