Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca(2+)-ATPase forms a functional interaction site with phospholamban. Evidence for physical interactions at other sites.
In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), mutation of amino acids on one face of the phospholamban (PLN) transmembrane helix led to loss of PLN inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) molecules. This helical face was proposed to form a site of PLN interaction with a transmembrane helix in SERCA molecules. To determine whether predicted transmembrane helices M4, M5, M6, or M8 in SERCA1a interact with PLN, SERCA1a mutants were co-expressed with wild-type PLN and effects on Ca(2+) dependence of Ca(2+) transport were measured. Wild-type inhibitory interactions shifted apparent Ca(2+) affinity of SERCA1a by an average of -0.34 pCa units, but four of the seven mutations in M4 led to a more inhibitory shift in apparent Ca(2+) affinity, averaging -0.53 pCa units. Seven mutations in M5 led to an average shift of -0.32 pCa units and seven mutations in M8 led to an average shift of -0.30 pCa units. Among 11 mutations in M6, 1, Q791A, increased the inhibitory shift (-0.59 pCa units) and 5, V795A (-0.11), L802A (-0.07), L802V (-0.04), T805A (-0.11), and F809A (-0.12), reduced the inhibitory shift, consistent with the view that Val(795), Leu(802), Thr(805), and Phe(809), located on one face of a predicted M6 helix, form a site in SERCA1a for interaction with PLN. Those mutations in M4, M6, or M8 of SERCA1a that enhanced PLN inhibitory function did not enhance PLN physical association with SERCA1a, but mutants V795A and L802A in M6, which decreased PLN inhibitory function, decreased physical association, as measured by co-immunoprecipitation. In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCA1a and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites. Thus, both functional and physical data confirm that PLN interacts with M6 SERCA1a.
Asahi
M
M
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Kimura
Y
Y
Kurzydlowski
K
K
Tada
M
M
MacLennan
D H
DH
D001692:Biological Transport / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008565:Membrane Proteins; D000099211:Phospholamban