Abstract
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.
Highlights
Functional Interactions between PLN and SERCA1a Molecules Carrying Mutations in Transmembrane Helices M4, M5, M6, and M8 —In earlier studies, we showed that mutations on one face of the PLN transmembrane helix caused loss of functional interaction with SERCA2a [16]
We deduced that mutations in the face of the transmembrane helix in SERCA2a that interacted with PLN would show a complementary loss of functional interaction
Functional Interactions between PLN and SERCA1a—Numerous studies support the view that PLN forms a single transmembrane helix [16, 23,24,25]
Summary
Michio Asahi‡§, Yoshihiro Kimura¶, Kazimierz Kurzydlowski‡, Michihiko Tadaʈ , and David H. Mutation of amino acids on the opposite face of the PLN domain II helix led to loss of the ability of PLN to inhibit SERCA2a, leading us to postulate that it formed a “SERCA interaction face” [16]. We have used SERCA1a instead of SERCA2a because SERCA1a and SERCA2a are inhibited by wild-type and mutant PLN [28], because the sequences of transmembrane helices M4, M5, M6, and M8 are very highly conserved among all three SERCA isoforms [29], because structure/function analysis of SERCA1a is highly advanced, and because a large number of useful mutants derived from earlier alanine-scanning mutagenesis of SERCA1a transmembrane helices M4, M5, M6, and M8 [30] were available for this study. The effects of specific mutations on functional interactions, together with studies of co-immunoprecipitation of wild-type and mutant PLN and SERCA1a, implicate SERCA1a transmembrane helix M6 and Val795 and Leu802, in particular, in the functional interaction between SERCA molecules and PLN
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