IDENTIFICATION AND CHARACTERIZATION OF CYTOSOLIC HANSENULA-POLYMORPHAPROTEINS BELONGING TO THE HSP70 PROTEIN FAMILY

We have isolated two members of the Hsp70 protein family from the yeas t Hansenula polymorpha using affinity chromatography. Both proteins we re located in the cytoplasm. One of these, designated Hsp72, was induc ible in nature (e.g. by heat shock). The second protein (designated Hs c74) was constitutively present. Peptides derived from both Hsp72 and Hsc74 showed sequence homology to the cytosolic Saccharomyces cerevisa e Hsp70s, Ssa1p and Ssa2p. The gene encoding Hsp72 (designated HSA1) w as cloned, sequenced and used to construct HSA1 disruption and HSA1 ov erexpression strains. Comparison of the stress tolerances of these str ains with those of wild-type H. polymorpha revealed that HSA1 overexpr ession negatively affected the tolerance of the cells to killing effec ts of temperature or ethanol, but enhanced the tolerance to copper and cadmium. The tolerance for other chemicals (arsenite, arsenate, H2O2) or to high osmolarity was unaffected by either deletion or overexpres sion of HSA1. The nucleotide sequence of HSA1 was submitted to the EMB L data library and given the Accession Number Z29379.