Localization and characterization of the 86- and 84-kDa heat shock proteins in Hepa 1c1c7 cells.
The 90-kDa heat shock protein (hsp90) is present in cells at high levels in the cytoplasm and is composed of two separate gene products, hsp86 and hsp84. Rabbit polyclonal antibodies to the murine N-terminal sequences of the 86- and 84-kDa heat shock proteins were isolated from serum by peptide affinity chromatography. Antibodies against each form of hsp90 are capable of immunoprecipitating hsp90. Each antibody preparation is specific against either hsp86 or hsp84 when tested on a protein blot of Hepa 1c1c7 cytosol. The over-all ratio of hsp84/hsp86 in Hepa 1 cytosol was estimated to be 2 to 1. Each antibody preparation was used to immunoprecipitate hsp84 or hsp86 from Hepa 1 cytosol to test whether hsp86/84 exists as a homo- and/or heterodimer. After electrophoresis, silver staining revealed that anti-hsp86 antibodies immunoprecipitated both hsp86 and hsp84. This result would suggest that hsp86 forms heterodimers with hsp84. In contrast, the anti-hsp84 antibodies immunoprecipitated almost entirely hsp84, suggesting that hsp84 exists largely as homodimers. Both anti-hsp86 and hsp84 antibodies were able to immunoprecipitate the 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dixoin-labeled Ah receptor from Hepa 1 cytosol, indicating that these antibodies are able to bind to hsp90 when it is complexed with other proteins. Both antibody preparations recognize hsp90 in mouse, rat, and human cell lines. Immunofluorescence and confocal microscopy were performed using both antibody preparations, and the results indicated that both hsp86 and hsp84 were located in the cytoplasm and nucleus of Hepa 1 cells. Hsp86 was found to localize unevenly in the cytoplasm, while hsp84 was found evenly dispersed throughout the cytoplasm. Hsp86 also appeared to be localized to a greater degree than hsp84 in the vicinity of the nuclear envelope.
Perdew
G H
GH
Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907.
Hord
N
N
Hollenback
C E
CE
Welsh
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006360:Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D011233:Precipitin Tests; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism
D006360:Heat-Shock Proteins; D010455:Peptides; D018336:Receptors, Aryl Hydrocarbon