Yeast Rpi1 is a putative transcriptional regulator that contributes to preparation for stationary phase

Abstract

The RPI1 gene of Saccharomyces cerevisiae was identified initially as a dosage suppressor of the heat shock sensitivity associated with overexpression of RAS2 (J. Kim and S. Powers, Mol. Cell. Biol. 11:3894-3904, 1991). Based on its failure to suppress mutationally activated RAS2, RPII was proposed to be a negative regulator of the Ras/cyclic AMP (cAMP) pathway that functions at a point upstream of Ras. We isolated RPI1 as a high-copy-number suppressor of the cell lysis defect associated with a null mutation in the MPK1 gene, which encodes the mitogen-activated protein kinase of the cell wall integrity-signaling pathway. Although the sequence of Rpil is not informative about its function, we present evidence that this protein resides in the nucleus, possesses a transcriptional activation domain, and affects the mRNA levels of several cell wall metabolism genes. In contrast to the previous report, we found that RPI1 overexpression suppresses defects associated with mutational hyperactivation of the Ras/cAMP pathway at all points including constitutive mutations in the cAMP-dependent protein kinase. We present additional genetic and biochemical evidence that Rpil functions independently of and in opposition to the Ras/cAMP pathway to promote preparations for the stationary phase. Among these preparations is a fortification of the cell wall that is antagonized by Ras pathway activity. This observation reveals a novel link between the Ras/cAMP pathway and cell wall integrity. Finally, we propose that inappropriate expression of RPI1 during log phase growth drives fortification of the cell wall and that this behavior is responsible for suppression of the mpkl cell lysis defect.

FIG. 1.

Overexpression of RPI1 suppresses the growth defect of an mpk1 mutant. Transformants of yeast strain DL456 (mpk1Δ) harboring episomal vector YEp352 (12), YEp352[RPI1], or centromeric plasmid YCp50[MPK1] were streaked onto a YPD plate and incubated at 37°C for 3 days.

FIG. 2.

Overexpression of MPK1 fails to suppress the heat shock sensitivity of an rpi1 mutant. Transformants of yeast strain DL707 (rpi1Δ) harboring episomal vector YEp352, YEp352[MPK1], or centromeric plasmid pRS316[RPI1] were grown to saturation in SD medium and subjected to heat shock for the indicated times. Cultures were diluted and plated on YPD to determine survival levels. Each value represents the mean and standard deviation from at least three independent experiments.

FIG. 3.

Rpi1 resides in the nucleus. Fluorescence micrographs of wild-type strain 1788 expressing Rpi1-GFP from episomal plasmid pRS425[RPI1-GFP] are shown. Rpi1-GFP was visualized using fluorescein isothiocyanate filter (left). Rpi1-GFP colocalized with nuclear DNA, as judged by DAPI fluorescence (right).

FIG. 4.

The C-terminal domain of Rpi1 possesses a transcriptional activation function. Yeast strain AH109 (GAL2_UAS-GAL2_TATA-ADE2) was transformed with plasmids bearing fusions of RPI1 to the Gal4 DBD in the two-hybrid vector pGBT9. Transformants were patched onto YPD medium for 3 days. Failure to develop a red color is due to GAL2-driven expression of the ADE2 reporter and indicates transcriptional activation by the protein fused to the Gal4 DBD.

FIG. 5.

Overexpression of RPI1 enhances FKS2 mRNA levels independently of the states of the cell wall integrity and Ras/cAMP signaling pathways. Plus signs indicate overexpression of RPI1 from YEp352. Total RNA (10 μg), isolated from log-phase cultures, was probed for FKS2 mRNA. A probe for ACT1 (encoding actin) was used as a negative control.

FIG. 6.

Overexpression of RPI1 suppresses the heat shock sensitivity associated with activating mutations in Ras, adenylate cyclase, and PKA. (A and B) Yeast strain DL2153 (RAS2^Ala18Val19, which expresses constitutive Ras2), was transformed with either episomal vector YEp352 or YEp352[RPI1], and its congenic wild-type (DL2154) was transformed with vector only. Transformants were grown to saturation in SD medium and subjected to heat shock at 48°C (A) or 51°C (B). Cultures were diluted and plated on YPD to determine survival levels. (C) Yeast strain DL2226 (ras1Δ ras2Δ SRA4-6, which expresses constitutive adenylate cyclase) was transformed with either YEp352 or YEp352[RPI1] and tested for heat shock sensitivity as above. (D) Yeast strain DL2156 (bcy1Δ, which expresses constitutive PKA) was transformed with YEp352, YEp352[RPI1], or centromeric plasmid YCp50[BCY1] and tested for heat shock sensitivity as above. Each value represents the mean and standard deviation from at least three experiments.

FIG. 7.

Rpi1 functions outside of the Ras/cAMP pathway. (A) Depression of cAMP levels by overexpression of PDE2 suppresses the heat shock sensitivity of an rpi1 mutant. Yeast strain DL707 (rpi1Δ) was transformed with YEp352, YEp352[PDE2], or centrometric plasmid pRS316[RPI1]. Transformants were grown to saturation in SD medium and subjected to heat shock for the indicated times. Cultures were diluted and plated on YPD to determine survival levels. (B) The heat shock sensitivities associated with rpi1 and ira1 are additive. Wild-type (1783), rpi1Δ (DL2289), ira1Δ (DL2297), and rpi1Δ ira1Δ (DL2329) strains were grown to saturation in YPD and tested for heat shock sensitivity as above. Each value represents the mean and standard deviation from at least three experiments.

FIG. 8.

Overexpression of RPI1 suppresses the inability of an ira1 mutant to survive in stationary phase. Yeast strain DL2297 (ira1Δ) was transformed with either episomal vector YEp352 or YEp352[RPI1]. Wild-type strain 1783 was transformed with YEp352 only. Transformants were inoculated into YPD medium, grown to saturation at 30°C with agitation, and maintained under the same conditions for 6 weeks. Samples were diluted and plated on YPD at the indicated times to test for viability. Experiments were conducted in triplicate. However, loss of two cultures during the course of the experiment prevented calculation of the standard deviation.

FIG. 9.

Overexpression of RPI1 suppresses the deficiency of an ira1 mutant to acquire Zymolyase resistance in saturated cultures. Yeast strain DL2297 (ira1Δ) was transformed with either episomal vector YEp352 or YEp352[RPI1]. Strains 1783 (wild-type) and DL2289 (rpi1Δ) were transformed with YEp352 only. Transformants were grown to saturation in SD medium before being diluted into YPD. Cultures were grown either to mid-log phase (solid symbols) or to saturation (open symbols), washed, and resuspended in water to an initial density of A₆₀₀ ≈ 0.55 prior to treatment with Zymolyase 20T (150 μg/ml). Cell lysis was assessed by A₆₀₀ measurements at the indicated times.

FIG. 10.

An rpi1 mpk1 double mutant loses viability rapidly on exiting log-phase growth. Yeast strains DL456 (mpk1Δ) and DL2291 (mpk1Δ rpi1Δ) were cultured at 30°C in either SD medium (open symbols) or SD medium supplemented with 10% sorbitol (solid symbols), starting with identical inocula of log-phase cells. Samples were diluted and plated on YPD at the indicated times to test for viability. Culture densities were monitored during the course of the experiment to assess the growth phase. Isogenic wild-type and rpi1Δ strains did not display any loss of viability even after 48 h in culture (data not shown). Each value represents the mean and standard deviation from at least three experiments.