Analysis of Prohibitin expression

pWYG2L and pWYGPHBl were used to transform both SUB61 and SUB61AmZ7ï4 to leucine prototrophy. Transformants were obtained and grown to exponential phase on glucose defined media, then transferred to glycerol (time Oh) to induce ADH2 promoter activity and thus production of histagged prohibitin. Cell aliquots were removed at 24, 48 and 72 hours, pelleted and disrupted in SDS extraction buffer. Proteins from the crude extracts were separated by SDS-PAGE and the presence of the Xpress epitope detected by immunodetection with the Xpress monoclonal antibody (Invitrogen). Western blotting detected trace levels of a protein of the correct size (30kDa) in SUB61-pWYGPHBl but not SUB61-pWYG2L samples (data not shown). Prohibitin production was poor, only appearing after 48 hours cultivation and being undetected in 72 hour samples.

In several attempts at transformation it proved impossible to transform SUB61Aw6W cells with pWYGPHBl, although these cells could be transformed with vector minus the P H B l gene (pWYG2L). This failure may be due to toxicity of the prohibitin protein in ubi4 cells. The introduction of pGALUBM (Chen et al. 1994) into SUB61Am^ï4 cells allowed subsequent transformation with pWYGPHBl when cells were plated on galactose. The plasmid pGALUBM contains the yeast polyubiquitin gene, UBI4, under the control of the GALl promoter, together with URA3 for plasmid selection in yeast. This vector was used to transform SUB61Aw6W cells to uracil prototrophy, generating the transformant, SUB61Am^i4+GUB. By maintaining transformants on galactose UBI4 expression was ensured, providing cells with adequate supplies of free ubiquitin. SUB61AmZ?/4 +GUB cells were grown to exponential phase in selective galactose based medium and co-transformed to leucine prototrophy with the prohibitin expression vector pW YGPHBl, generating the transformants SUB61 Aw6W+GUB4-PHB1. Double transformants were selected on YNBGal deficient in both leucine and uracil. When replica plated to glucose containing plates (YNBD) these transformants displayed very weak growth, even

though the ADH2 promoter activity of pWYGPHB was low. Transfer to glucose downregulates GALl promoter activity, preventing UBI4 expression and decreasing the quantity of intracellular ubiquitin available to the cell. Downregulation of ubiquitin expression is therefore responsible for the poor grow th of SUB61AmZ?/^+PHB1 cells.

These results indicated that introduction of the h P H B l episomal vector (pW YGPHBl) is only possible in UBI4 cells, in which high turnover of the overproduced prohibitin protein is possible. Part of the problem could be choice of vector. pWYG2L uses LEU2-d for selection. The weak expression of this gene causes high plasmid copy numbers (Section 1.6.2.1) and therefore high hPHBl gene dosage in transformant cells grown in the absence of leucine. The toxicity that this high hPHBl gene dosage appears to cause to ubi4- cells could be partly overcome with the réintroduction of a UBI4 gene expressed from the GALl promoter (pGALUBM). It is probable therefore that introduction of the prohibitin expression vector requires the complete ubiquitination system of SUB61AmZ?/4+GUB. The prohibitin toxicity apparent in SlJB6lAubi4+G\JB+ PWYGPHBl cells is clearly due to reduction in ubiquitin levels, suggesting that overproduction of the human ageing protein prohibitin is toxic to cells with a reduced capacity for selective ubiquitin-mediated proteolysis. h P H B l expression and prohibitin production in S.cerevisiae cells (SUB61+PHB1) is low, probably due to rapid proteolysis. In retrospect human prohibitin may not have been a good choice of recombinant product for accumulation in yeast cells entering stationary phase.

3.7 Summary

The main conclusions drawn from the work presented in this chapter are:-

1. Depletion of intracellular ubiquitin pools, through the disruption of UBI4, decreases selective ubiquitin-mediated degradation and allows for the accumulation of overproduced homologous GO proteins (such as Adh2 and Hsp82) in stationary phase cells (Figs. 3.2, 3.3 & 3.5). As total protein concentration of the cultures is also increased by this mutation (data not shown) this effect is probably not protein specific. The use of ubi4 strains may therefore assist overproduction of other yeast proteins which are induced by catabolite derepression or starvation.

2. Depletion of vacuolar protease activities hinders both Adh2 enzyme production and cell growth, with both pep4-3, UBI4 and pep4-3, ubi4 cells displaying low Adh activity (Fig. 3.4) and reduced viability as cultures enter stationary phase pep4-3 cells have previously been found to lose viability when nitrogen starved (Jones 1991). 3. ubi4- cells displayed an increased STRE-regulated overproduction of E.coli (3- galactosidase 5h after transfer from glucose to glycerol, but failed to accumulate large quantities of active enzyme in stationary phase (Fig. 3.6), unlike with expression of homologous Adh2 and Hsp90 (Figs. 3.2, 3.3 and 3.5). Possibly, p-galactosidase is a protein of intermediate stability, less stable than native Adh2 or Hsp82, but more stable than recombinant human prohibitin in yeast cells. Low level p-galactosidase production occurred in both UBI4+ (SUB61-LacZ) and ubl4- (SUB63-LacZ) strains throughout the experimental time course (Fig. 3.6.). Poor enzyme yield may be attributed to a number of factors, including low level induction of the STRE-LacZ cassette and, since pep4-3 cells display higher LacZ expression levels (Kirk & Piper 1991), vacuolar proteolysis of the final gene product (STRE-LacZ expression was not studied in pep4-3 cells).

4. Introduction of a high copy number plasmid for production of the human protein, prohibitin, has toxic effects that totally prevent growth in ubi4- cells (SUB61 AwW- PH Bl), but which are less severe in isogenic UBI4+ cells (SUB61-PHB1). The stationary phase induced ubiquitination system of SUB61-PH Bl cells (UBI4) probably ensures that low prohibitin expression levels are maintained. Although introduction of the vector for P H B l expression in ubiquitin-depleted cells (SUB61Aw6W -PH Bl) is apparently lethal, this lethality can be rescued by the introduction of a galactose inducible polyubiquitin cassette (pGALUBM).

C hapter 4; Evidence that the partial proteolysis of H sp82 in