Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts.
A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.
Krishna
P
P
Department of Plant Sciences, The University of Western Ontario, London, Canada.
Reddy
R K
RK
Sacco
M
M
Frappier
J R
JR
Felsheim
R F
RF
D000917:Antibody Formation; D001937:Brassica; D002458:Cell Fractionation; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000737:chemistry*; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis* / Q000276:immunology; D010936:Plant Extracts / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000276:immunology; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018724:Spinacia oleracea; D013237:Stereoisomerism
D003432:Cross-Linking Reagents; D018841:HSP90 Heat-Shock Proteins; D010936:Plant Extracts; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins