HSP70 binds to the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA1a) and prevents thermal inactivation.
This study examined whether HSP70 could bind to and protect against thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. Sarcoplasmic reticulum vesicles prepared from rat gastrocnemius muscle were incubated with purified HSP70 at both 37 and 41 degrees C for either 30, 60, or 120 min. Maximal SERCA1a activity (micromol/g protein/min) in the absence of HSP70 was reduced progressively with time, with greater reductions occurring at 41 degrees C compared with 37 degrees C. HSP70 protected against thermal inactivation of SERCA1a activity at 37 degrees C but not at 41 degrees C and only at 30 and 60 min but not at 120 min. HSP70 also protected against reductions in binding capacity for fluorescein isothiocyanate, a fluorescent probe that binds to Lys515 in the nucleotide binding domain of SERCA, at 30 and 60 min but not at 120 min, an effect that was independent of temperature. HEK-293 cells were co-transfected with cDNAs encoding rabbit SERCA1a and human HSP-EYFP and subjected to 40 degrees C for 1 h. Immunohistochemistry revealed nearly complete co-localization of SERCA1a with HSP70 under these conditions. Co-immunoprecipitation showed physical interaction between HSP70 and SERCA1a under all thermal conditions both in vitro and in HEK-293 cells. Modeling showed that the fluorescein isothiocyanate-binding site of intact SERCA1a in the E2 form lies in its close proximity to a potential interaction site between SERCA1a and HSP70. These results indicate that HSP70 can bind to SERCA1a and, depending on the severity of heat stress, protect SERCA1a function by stabilizing the nucleotide binding domain.
Tupling
A Russell
AR
Department of Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. rtupling@uwaterloo.ca
Gramolini
Anthony O
AO
Duhamel
Todd A
TA
Kondo
Hiroya
H
Asahi
Michio
M
Tsuchiya
Shauna C
SC
Borrelli
Michael J
MJ
Lepock
James R
JR
Otsu
Kinya
K
Hori
Masatsugu
M
MacLennan
David H
DH
Green
Howard J
HJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D046912:Multiprotein Complexes; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology*; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013816:Thermodynamics; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium