Heat-shock-induced assembly of Hsp30 family members into high molecular weight aggregates in Xenopus laevis cultured cells.
In this study, we have examined whether members of the small heat shock protein family, hsp 30, were capable of forming heat-induced aggregates in Xenopus laevis A6 kidney epithelial cells. Rate-zonal centrifugation coupled with immunoblot analysis demonstrated the presence of hsp30 aggregates with an estimated sedimentation coefficient of 10-16S. Also, pore exclusion limit electrophoretic analysis of labeled protein from heat-shocked A6 cells revealed four heat-induced aggregates, including a prominent 510 kDa aggregate, as well as weaker 350, 290, and 240 kDa aggregates. Immunoblot analysis of the aggregates employing an hsp30C antibody suggested that the 510 and 350 kDa aggregates were comprised of hsp30 protein. One- and two-dimensional SDS-PAGE analysis of the proteins isolated from the 510 kDa region of the pore exclusion limit electrophoretic gel confirmed the presence of 30 kDa heat-induced protein. A total of eight small hsps were present in this aggregate, suggesting that virtually all of the major small hsps in Xenopus A6 cells were involved in aggregate formation. This study also detected the presence of heat-inducible hsp70 in the 510 kDa gel fraction containing the small hsps, but it could not be determined whether it was part of the multimer complex.
Ohan
N W
NW
Department of Biology, University of Waterloo, Ontario, Canada.
Tam
Y
Y
Heikkila
J J
JJ
D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells; D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics*; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000235:genetics*; D008970:Molecular Weight; D005810:Multigene Family; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000235:genetics*
D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D029867:Xenopus Proteins; C494391:hsp30 protein, Xenopus