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 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 H...