Co-isolation of heat stress and cytoskeletal proteins with plasma membrane proteins.
Previous reports have suggested the possible existence of a plasma-cell-membrane function associated with some heat stress proteins (HSPs). To investigate the effect of hyperthermia on plasma membrane proteins, rat mammary tumour clone C (MTC) cells were heated at 42 degrees C for 1 h. Their surface proteins were (1) labelled with [3H]leucine, (2) biotinylated, (3) affinity isolated with streptavidin-agarose beads under denaturing or non-denaturing conditions, and (4) analysed by one- and two-dimensional polyacrylamide-gel electrophoresis and protein blotting under denaturing conditions. Affinity isolation of biotinylated proteins enriched for a protein subfraction believed to be membrane-associated. Several proteins analogous to HSP or their heat-stress cognates (HSC) were present with these biotinylated protein subfractions in control or heated cells. The major and most consistent feature of affinity isolates from heated cells was the presence of a small fraction of the induced 68-kD HSP. The 112-, 90-, 70- and 22-kD HSC/HSP were also present in small amounts in affinity isolates of control cells, and the fraction increased in heated cells. Several structural proteins, including actin and the tubulins were present in the same affinity isolates. Protein blotting experiments indicated that none were exposed on the exterior of the plasma-cell membrane or biotinylated and thus none were exposed on the exterior of the plasma-cell membrane or biotinylated intracellularly through membrane damage. These results suggest that small fractions of several HSC are located at or near the cytoplasmic face of the plasma membrane along with cytoskeletal proteins, and that additional submembranous localization of HSP occurs after heat stress and may be part of the processes associated with membrane damage or cellular responses to heat. Further studies will be directed at establishing the relationships between these proteins and the role, if any, of the changes associated with heat stress.
Tomasovic
S P
SP
Department of Tumor Biology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Simonette
R A
RA
Wolf
D A
DA
Kelley
K L
KL
Updyke
T V
TV
D000230:Adenocarcinoma / Q000032:analysis; D000818:Animals; D003598:Cytoskeletal Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006360:Heat-Shock Proteins / Q000302:isolation & purification*; D006979:Hyperthermia, Induced; D008325:Mammary Neoplasms, Experimental / Q000032:analysis; D008565:Membrane Proteins / Q000302:isolation & purification*; D051381:Rats; D014407:Tumor Cells, Cultured / Q000032:analysis*
D003598:Cytoskeletal Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins