Multimerization of Hsp42p, a Novel Heat Shock Protein of Saccharomyces cerevisiae, Is Dependent on a Conserved Carboxyl-terminal Sequence

D. Wotton, K. Freeman, D. Shore
1996 Journal of Biological Chemistry  
Rap1p is a transcriptional regulator of Saccharomyces cerevisiae, which plays roles in both transcriptional activation and silencing. To identify proteins involved in Rap1p-dependent regulation of transcription, we used the two-hybrid system to screen for Rap1p-interacting proteins. Two of the clones isolated from this screen encode a truncated protein with homology to small heat shock proteins (HSPs). Here we present an analysis of this novel S. cerevisiae HSP, which we name Hsp42p. Expression
more » ... of HSP42 is regulated by a range of stress conditions similar to S. cerevisiae HSP26, with which Hsp42p shares most homology. However, HSP42 expression is more sensitive to increased salt concentration and to starvation and, in contrast to HSP26 is expressed in unstressed cells. Hsp42p interacts with itself in the two-hybrid assay. This interaction is dependent on a hydrophobic region which is conserved among small HSPs. Using bacterially expressed Hsp42p fusion proteins, we demonstrate that this is a direct interaction. Fractionation of yeast protein extracts by size demonstrates that all of the Hsp42p in these extracts is present in complexes with a molecular mass of greater than 200 kDa, suggesting that Hsp42p exists in high molecular mass complexes. The repressor/activator protein (Rap1p) of Saccharomyces cerevisiae plays an important role in transcriptional silencing at both HM loci and telomeres (1-3). Rap1p is also able to activate gene expression and is essential for viability (4), presumably because of its role in the activation of glycolytic and ribosomal protein genes (5-7). We were interested in identifying other proteins involved in these processes, in an attempt to gain further insight into the different functions of Rap1p. We have previously used the two-hybrid system to identify proteins which play a role in the silencing functions of Rap1p. Sir3p and Sir4p interact with the carboxyl terminus of Rap1p in a twohybrid assay (8), and the silencing protein, Rif1p, was identified in a similar way (9). We decided, therefore, to extend this search for Rap1p-interacting proteins. Of the clones identified by this screen, two encoded the same truncated protein, with homology to small heat shock proteins. When eukaryotic cells are exposed to conditions of stress, such as increased temperature, the expression of proteins known as heat shock proteins (HSPs) 1 is induced. HSPs can be divided into four classes; the hsp90 and hsp70 families, the GroEL-related HSPs, and the small HSPs, which are typically up to 40 kDa in size (10). Some of these HSPs, such as hsp70, are highly conserved between organisms as divergent as mammals, yeast and bacteria (11, 12). However, the small HSPs, share far less sequence similarity between species, with the main region of homology being a hydrophobic stretch of about 35 amino acids, located near the carboxyl terminus of the protein (13, 14). The number of small HSPs identified in different species varies greatly. For example, in many species of plants such as the soybean, more than 20 small HSPs have been identified (14) , whereas in Drosophila six small HSPs are known (15) and in humans only one has been identified (16). The function of small heat shock proteins remains unclear. However, in Dictyostelium a mutation that abolishes induction of small HSP gene expression causes reduced stress tolerance, suggesting that these proteins do indeed play a role in stress resistance (17). In S. cerevisiae, the major small HSP is Hsp26p, the expression of which is rapidly induced when cells are transferred to higher temperatures. Hsp26p is one of the major polypeptides produced on heat shock (18 -21). To date, one other small HSP has been identified from S. cerevisiae, a 12-kDa protein with no homology to Hsp26p (22). In addition to heat shock, HSP26 expression is induced under other conditions of stress, such as increased salt concentration and starvation (20, 23). However, no phenotype has been observed on disruption of HSP26 (18, 19) , suggesting that the function of Hsp26p in stress tolerance may overlap with the functions of other HSPs. The identification of other S. cerevisiae HSPs may, therefore, provide a greater insight into the function of the small HSPs in yeast. Here we present the identification and analysis of a novel small HSP of S. cerevisiae. This HSP (Hsp42p) is most similar to S. cerevisiae Hsp26p. HSP42 expression is up-regulated by all stress conditions tested. In contrast to HSP26, HSP42 is expressed at a relatively high level in cells growing exponentially at 25°C. By sucrose gradient fractionation, it has been demonstrated that Hsp26p is present in large complexes (21). We show that Hsp42p is present in high molecular mass complexes, which are heterogeneous in size. Our results also demonstrate that Hsp42p interacts with itself and that this interaction is direct. The interaction of Hsp42p with itself is dependent on the carboxyl-terminal region of the protein including the conserved hydrophobic region.
doi:10.1074/jbc.271.5.2717 pmid:8576246 fatcat:hnw3uu6aunbfho6w2xemp7ebkq