Panaretou, Barry; (1993) The plasma membrane in the tolerance of yeast to stress. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In most organisms a mild sub-lethal stress increases the capacity for survival during a subsequent lethal stress. The mechanisms that implement this acquisition of tolerance have been the subject of numerous studies. Best characterised is the induction of heat shock proteins (hsps) at elevated temperatures. Very little is known about heat shock-associated events occurring at the plasma membrane. This is surprising bearing in mind that the first component of the cell to encounter environmental stress is the cell envelope. Work described in this thesis established action of the plasma-membrane-associated H+-translocating ATPase (H+-ATPase) of S. cerevisiae as an important determinant of tolerance to several types of stress including heat shock, exposure to ethanol and high salt concentrations. Activity of this enzyme also influences the capacity of cells to synthesize hsps. The importance of this enzyme in thermotolerance is probably due to its role in limiting the intracellular acidification that is one of the consequences of heat shock. It is conceivable that cells target proteins with a damage limitation function to the plasma membrane as part of an inducible stress response. Two prominent changes to the protein composition of the S. cerevisiae plasma membrane were seen on heat shock: (I) a marked (55%) decrease in levels of the H+-ATPase; and (II) the acquisition of a previously uncharacterised hsp of Mr 30,000 (hsp30). The ATPase decline must have an important influence over the ability of cells to maintain intracellular pH during extended periods of stress. In vitro assays of ATPase activity revealed that enzyme from heat shocked cells is almost twice as active as that from unstressed cells, indicating the existence of a system that activates ATPase during stress. Hsp30 is an integral membrane protein exclusively located at the plasma membrane which can be phosphorylated in vitro. The strong evolutionary conservation of hsp induction makes it probable that structural (and possibly functional) homologs of hsp30 will be found in organisms other than yeast. Both of these events were also observed when cells entered stationary phase; in addition, hsp30 is induced when cells are starved for nitrogen. Covalent modification of proteins is the most recurrent form of biological control. Any notable differences between phosphoprotein profiles from unstressed and stressed cell fractions may shed light on how events associated with stress are regulated. The in vitro kinase/autophosphorylation activities associated with cytosolic (S100) fractions of unstressed and heat shocked yeast were examined. One polypeptide of Mr 66kDa was heavily phosphorylated in heat shock extracts only. This protein is not an hsp, but its phosphorylation is hsp dependent. The phosphoproteins detected by these experiments are particularly interesting since a large proportion of them involve the poorly-understood (rare) acid labile, N-linked phosphoryl groups. This suggests the existence of a signal transduction system in yeast, based on N-linked phosphorylation, much like the one mediating chemotaxis in bacteria.

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