GENETIC CONTROL OF MEIOTIC DEVELOPMENT IN YEAST

RANDY STRICH, Ph.D., Associate Member; Adjunct Assistant Professor, Department of Biology, University of Pennsylvania

In yeast, the physiological response to changing environmental conditions is controlled by both the cell type (either haploid or diploid) and the nature of the stimuli. For example, both haploid and diploid cells will undergo mitotic cell division in the presence of growth stimuli. However, diploid cells posses multiple options when confronted with nutrient limitation. Unlike haploids, diploid cells can withdraw from the cell cycle and initiate meiotic development when deprived of nitrogen and a fermentable carbon source. Our research is directed toward understanding how yeast coordinate mitotic cell division with meiotic development. Toward this goal, we have isolated several transcription factors (UME1-6) that regulate a subset of genes required for the first meiotic division in yeast. This report will focus on Ume3p, the yeast C-type cyclin.

Previous studies in this laboratory revealed that Ume3p is destroyed in response to oxidative stress. To identify the signal transduction system regulating this response, two candidate pathways were examined. First, Ume3p levels were followed in hog1 mutant strains that are defective in sensing hypertonic conditions. In response to either ethanol shock or oxidative stress, Ume3p is destroyed with similar kinetics in the hog1 mutant compared to wild type (Figure 1A and 1B). These findings indicate that the HOG pathway does not trigger Ume3p destruction in response to these stimuli. Next, Ume3p regulation was examined in mutants lacking a member of the phospholipase C family,

PLC1. Similar to hog1 mutant strains, plc1 mutants are sensitive to hyperosmotic medium. Ume3p was down-regulated normally in the plc1 mutant exposed to ethanol shock (Figure 1A), but not in response to oxidative stress (Figure 1B). The degree of stabilization was nearly complete and similar to that obtained in the presence of the osmostabilizing agent, sorbitol, or in a doa4 mutant. These results indicate that PLC1 is a component of the oxidative stress-induced Ume3p degradation pathway. Moreover, the effects of osmotic perturbations suggest that alterations in membrane integrity may represent the trigger recognized by Plc1p.

The results described above indicate that Plc1p is required for Ume3p degradation in response to oxidative stress. To investigate the physiological significance of this regulation, we first tested the requirement of PLC1 for viability in strains exposed to stress-inducing levels of H₂O₂. A strain deleted for PLC1 and the wild-type control were streaked onto rich plates containing 0.8 mM H₂O₂ and incubated at 30°. The plc1 mutant was viable under these conditions, but grew significantly slower that the control (Figure 2A) indicating a role for PLC1 in the cellular response to oxidative stress. Next, we examined whether this growth defect was due to the inability of the plc1 mutant to down regulate Ume3p. To address this question, the UME3 gene was deleted in the plc1 mutant and again plated on medium containing H₂O₂.

These experiments revealed that the double mutant was able to grow at rates similar to either the wild-type strain or a ume3

single mutant. These results formally demonstrate that the growth defect in plc1 mutants exposed to H₂O₂ is due to the inability to destroy Ume3p.

Loss of Plc1p activity results in a variety of other phenotypes including temperature sensitive growth and aberrantly large cell morphology. To investigate whether aberrant stabilization of Ume3p also contributes to these phenotypes, epistasis studies were again performed. The wild type, both single mutants, and the ume3 plc1 double mutant were streaked onto rich plates and incubated at 30° or 37°C. Following a two-day incubation, the wild-type strain and the ume3 single mutant grew on both plates (Figure 2A). As expected, the plc1 mutant was inviable at 37°. Interestingly, the ume3 plc1 double mutant was able to grow at 37° at a rate similar to the ume3 single mutant. These results indicate that, similar to oxidative stress, failure to destroy Ume3p in response to elevated temperatures may account for the growth defect in plc1 strains. Moreover, cells taken from the 37° plates were examined for the cell morphology abnormality associated with plc1 mutations. Loss of Plc1p activity resulted in large cells with unusual bud development (Figure 2B). Similar to the temperature sensitive growth defect, the ume3 null allele again suppressed this plc1 phenotype. These findings indicate that a central task of Plc1p in response to stress is to trigger the degradation of Ume3p.

IDENTIFICATION OF THE Ume3p HOLOENZYME-ASSOCIATING DOMAIN. COOPER, MALLORY

When tethered to a promoter via the lexA DNA binding domain, lexA-Ume3p is able to activate transcription of the LEU2 gene controlled by lexA operators (lexA_op-LEU2) due to its ability to recruit the RNA polymerase II (RNA Pol II) holoenzyme. This activation allows a leu2 mutant host strain to grow on medium lacking leucine (Figure 3A). However, lexA-Ume3p is unable to promote growth at 37° due to the heat-induced degradation (Figure 3A, right panel). Two mutants (lexA-TAA38 and lexA-TAA53) are able to promote growth on medium lacking leucine at high temperature. Western blot and DNA sequence analyses revealed that lexA-TAA38 and lexA-TAA53 are truncated at amino acid 38 and 53, respectively (Figure 3B). These findings suggested that the first 38 residues of Ume3p can function as a transcriptional activator and, therefore, are sufficient to allow recruitment of the RNA Pol II holoenzyme to the lexA_op-LEU2 promoter.

To test whether the amino terminal region of Ume3p is sufficient for RNA Pol II holoenzyme binding, a series of co-immunoprecipitation experiments were performed. Cultures harboring plasmids expressing either lexA (pEG202) or the lexA-UME3 truncation mutant with a stop codon at amino acid 53 (lexA-TAA53) were grown to mid-log phase and protein extracts prepared. These extracts were immunoprecipitated with either lexA antibodies or antibodies directed against one of two components of the RNA polymerase holoenzyme (TFIIB or Rpb1p). The immunoprecipitates were collected and the presence or absence of lexA or lexA-TAA53 was determined by Western blot analysis. The control immunoprecipitations with antibodies directed against lexA revealed that lexA and lexA-TAA53 were expressed at similar levels in these strains (Figure 3C). Antibodies directed against the general transcription factor TFIIB or against the largest subunit of RNA Pol II (Rpb1p) were able to precipitate lexA-TAA53. Some non-specific co-immunoprecipitation of lexA with the holoenzyme components was also detected in the control reactions, but to a significantly lesser extent than lexA-TAA53. Identical results were obtained in co-immunoprecipitation studies including the lexA-Ume3p protein truncated at amino acid 38 (lexA-TAA38, data not shown). These results demonstrate that the amino terminal region is sufficient to direct the association of Ume3p to the holoenzyme. This domain is now referred to as the RNA Pol II holoenzyme associating domain, or HAD.

PUBLICATIONS

COOPER, K.F., MALLORY, M.J., STRICH, R. Oxidative stress-induced destruction of the yeast C-type cyclin Ume3p requires the Phosphatidylinositol-specific phospholipase C (PLC1) and the 26S proteasome. Mol. Cell Biol. 19:3338-3348, 1999.

COOPER, K.F., STRICH, R. Functional analysis of the yeast C-type cyclin Ume3p and the RNA polymerase II holo-enzyme interaction. Gene Exp. (in press).

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998