Molecular characterization of the chaperone and heat shock protein HSP90 in the steroid responsive eukaryotic microbe Achlya ambisexualis

The eukaryotic microbe Achlya ambisexualis is a member of the Oomycetes, a group of microbes that is less well characterized and has little taxonomic similarity to the 'true' fungi. A. ambisexualis is one of the few organisms outside the animal kingdom in which well characterized steroid hormones mediate sexual differentiation. Proteins, the expression of which was altered during antheridiol (steroid)-mediated development, included those with molecular masses of 85 kDa and several in the 68 to 78 kDa range. The 85 kDa proteins and 68 to 78 kDa proteins were identified as members of the Hsp90 and Hsp70 heat shock protein families, respectively.; In vegetatively grown A. ambisexualis, heteromeric protein complexes containing Hsp9O and up to eight different proteins were identified. The 74 kDa protein was identified as Hsp70, and the 23 kDa and 56 kDa proteins were shown to be related to vertebrate protein p23 and immunophilin FKBP51, respectively.; In studies presented herein, a 38 kDa secreted protein that accumulates in the culture medium of vegetatively growing mycelia and antheridiol-treated mycelia, which is antigenically related to the C-terminal region of A. ambisexualis Hsp90, was identified. The secretion of a 38 kDa Hsp90-related protein into the extracellular medium may suggest a role for Hsp90-related proteins in extracellular protein trafficking.; Two incomplete genomic clones and two cDNA clones, representing distinct, and most likely allelic A. ambisexualis hsp90 genes, called hsp90-1 and hsp90-2, were isolated and characterized. Two distinct hsp90 transcript populations are encoded by these cDNAs. One mRNA population contained 2.8 kb transcripts, was constitutively expressed, and exhibited enhanced expression in both heat-shocked mycelia and mycelia undergoing antheridiol-induced differentiation. The second hsp90 transcript population contained transcripts of 2.9 kb and was strictly heat shock-inducible. The level of a number of constitutively expressed hsp70 transcripts was also increased during both treatments.; The increased level of hsp90 and hsp70 transcripts, observed during steroid-mediated differentiation or heat shock, was due to increased transcription of their respective genes. Nucleotide sequence analysis of the 5' flanking region of each hsp90 gene revealed a number of possible trariscription factor binding sites. Among these were motifs with some similarity to the steroid receptor-binding elements found in steroid responsive genes of animals. Knowledge gained from this study increases our basic understanding of biology, and specifically indicates a role for Hsp90 and Hsp70 proteins during steroid-mediated hyphal branching. These molecular studies will be useful to studies on other Oomycetes, many of which are plant and animal pathogens that are poorly characterized.