HEAT-SHOCK ENHANCES THE AMOUNT OF PRENYLATED DNAJ PROTEIN AT MEMBRANES OF GLYOXYSOMES

Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark-grown cucumber seedling s. Using a cucumber Dnaj protein produced by expression in bacteria, w e raised polyclonal antibodies against the protein and used them for l ocalization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractiona tion of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at 10000 Xg, and in the microsomes. Following subfractionation by sucrose densi ty gradient centrifugation and analysis by immunoblotting, a 53-kDa pr otein was attributed to the glyoxysomal fraction and an 80-kDa protein to the mitochondrial fraction. The glyoxysomal Dnaj protein behaved a s a membrane-bound form. Upon heat shock, a slight increase in the con tent of the glyoxysomal Dnaj protein was found. When glyoxysomes were treated with protease and subsequently isolated by gradient centrifuga tion, virtually all immunologically detectable Dnaj protein was remove d. Administration of radiolabelled mevalonic acid to cotyledons and is olation of glyoxysomes yielded labelled Dnaj protein which remained me mbrane bound during the purification of glyoxysomal membranes by float ation in a density gradient.