A two-dimensional immunoelectrophoretic analysis of the heat-shock response exhibited by Neurospora crassa cells.

Abstract

The heat-shock (HS) response of Neurospora crassa was studied by two-dimensional (2-D) immunoelectrophoresis, in conjunction with in vivo labelling of proteins with [35S]methionine. Antisera against extracts of normally grown and shocked cells were tested with both extracts as antigens. The resolution of normal cell proteins by interaction with homologous antisera yielded at least 35 immunoprecipitates. Using antisera to shocked cells with normal and shocked cell extracts resolved four heat-shock proteins (nhsps): new polypeptides synthesized upon HS and those with an enhanced production. Extracts of partially recovered cells showed proteins typical of normal cells, as well as those specific to the shocked state. Immunogenicity of some proteins appeared altered after heat shock. Following adsorption of HS cell extracts by normal antisera, three unique proteins were recognized in shocked cell extracts, along with a fourth displaying a markedly enhanced level. Stained bands corresponding to two abundant hsps (relative mass 68 000 and 81 000 daltons; referred to as nhsp 70 and nhsp 80) were excised from slab gels and the eluted proteins were employed as immunogens in rabbits. The resulting antibodies were used to identify corresponding proteins in autoradiographs of 2-D immunoelectrophoretograms.