Hsp98 is one of the most prominent proteins synthesized during the heat-shock response of Neurospora crassa. We purified hsp98 and determined the amino acid sequence of two overlapping peptides obtained by cyanogen bromide cleavage. This 28 amino acid sequence from hsp98 has 75% homology with a region of the ClpB protein of Escherichia coli and 86% homology to a 96-kDa protein of Trypanosoma brucei. It also has 71% homology to hsp104 of Saccharomyces cerevisae. Hsp98 was enriched in the microsomal fraction of heat-shocked cells. Sucrose gradient analysis of this cellular fraction showed that the three major high molecular weight heat-shock proteins (hsp98, 83 and 67) were more concentrated in polyribosomes than in monoribosomes. Another newly synthesized protein, p28, was strongly enriched in monoribosomes. After dissociation of the polyribosomes into ribosomal subunits, the three major heat-shock proteins were shown to be localized preferentially in the large subunit. Whereas p28 was also strongly associated with the large ribosomal subunit, a newly synthesized protein of about 22 kDa was exclusively associated with the small subunit.
Bibliographical noteFunding Information:
We thank University of Minnesota colleagues Murray Rosenberg for invaluable help with blotting techniques and Neil Palosaari for assistance with chromatography on the FPLC system. We are also grateful to Ben Madden of the Mayo Clinic for his assistance with peptide sequence analysis. This research was supported by grants from the National Institute of General Medical Sciences (GM-19398) and the USDA Competitive Research Grants Office.
- (N. crassa)
- Amino acid sequence
- Cellular distribution
- Heat-shock protein
- Protein isolation