Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Apr 29;100(9):5040-5.
doi: 10.1073/pnas.0330962100. Epub 2003 Apr 11.

Sarcolipin regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban

Michio Asahi  1 , Yuji Sugita, Kazimierz Kurzydlowski, Stella De Leon, Michihiko Tada, Chikashi Toyoshima, David H MacLennan
Affiliations

Sarcolipin regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban

Michio Asahi et al. Proc Natl Acad Sci U S A. .

Abstract

Phospholamban (PLN), a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs), interacts with both the cytosolic N domain and transmembrane helices M2, M4, M6, and M9 of SERCA. Amino acids in the transmembrane domain of PLN that are predicted to interact with SERCA1a are conserved in sarcolipin (SLN), a functional PLN homologue. Accordingly, the effects of critical mutations in SERCA1a, PLN, and NF-SLN (SLN tagged N-terminally with a FLAG epitope) on NF-SLN/SERCA1a and PLN/NF-SLN/SERCA1a interactions were compared. Critical mutations in SERCA1a and NF-SLN diminished functional interactions between SERCA1a and NF-SLN, indicating that NF-SLN and PLN interact with some of the same amino acids in SERCA1a. Mutations in PLN or NF-SLN affected the amount of SERCA1a that was coimmunoprecipitated in each complex with antibodies against either PLN or SLN, but not the pattern of coimmunoprecipitation. PLN mutations had more dramatic effects on SERCA1a coimmunoprecipitation than SLN mutations, suggesting that PLN dominates in the primary interaction with SERCA1a. Coimmunoprecipitation also confirmed that PLN and NF-SLN form a heterodimer that interacts with SERCA1a in a regulatory fashion to form a very stable PLN/NF-SLN/SERCA1a complex. Modeling showed that the SLN/SERCA1a complex closely resembles the PLN/SERCA1a complex, but with the luminal end of SLN extending to the loop connecting M1 and M2, where Tyr-29 and Tyr-31 interact with aromatic residues in SERCA1a. Modeling of the PLN/SLN/SERCA1a complex predicts that the regulator binding cavity in the E(2) conformation of SERCA1a can accommodate both SLN and PLN helices, but not two PLN helices.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effects of SERCA1a and NF-SLN mutations on formation of the binary NF-SLN/SERCA1a complex. (A and B) SERCA1a mutants were expressed in HEK-293 cells in the presence or absence of NF-SLN. (C) wt SERCA1a was expressed in HEK-293 cells in the presence of wt or mutant NF-SLN. The cells were harvested after 48 h, and microsomal fractions were prepared and dissolved in Tween 20 as described (21, 26). NF-SLN was immunoprecipitated with the FLAG antibody, and the immunoprecipitate was separated by SDS/PAGE and stained with the A52 antibody against SERCA1a to determine the amount of SERCA1a coimmunoprecipitation. Numbers below each lane represent percentage of wt and are the average of three independent experiments.
Figure 2
Figure 2
Effects of NF-SLN and PLN mutations on formation of the ternary PLN/NF-SLN/SERCA1a complex. (A) wt SERCA1a was expressed in HEK-293 cells in the presence of wt PLN and a series of NF-SLN mutants. Microsomal fractions were dissolved and immunoprecipitated with either antibody M2 against NF-SLN (row 1) or antibody 1D11 against PLN (rows 2 and 3). The immunoprecipitates were separated and stained with antibody A52 against SERCA1a (rows 1 and 2), or M2 against NF-SLN (row 3). (B) SERCA1a was expressed in HEK-293 cells in the absence or presence of NF-SLN and in the presence of wt PLN or a PLN mutant. Microsomal fractions were dissolved and immunoprecipitated with either antibody 1D11 against PLN (rows 1 and 3) or antibody M2 against NF-SLN (row 2). Immunoprecipitates were separated and stained with either antibody A52 against SERCA1a (rows 1 and 2) or antibody M2 against NF-SLN (row 3). Numbers below lanes represent percentage of wt and are the average of three independent experiments.
Figure 3
Figure 3
Effects of NF-SLN and the PLN I40A mutation on formation of the ternary PLN/NF-SLN/SERCA1a complex. SERCA1a was expressed in HEK-293 cells in the presence of NF-SLN and either wt or I40A mutant PLN. Microsomal fractions were dissolved and immunoprecipitated with antibody 1D11 against PLN. Immunoprecipitates were separated and stained with antibody A52 against SERCA1a. Numbers below lanes represent percentage of wt and are the average of three independent experiments.
Figure 4
Figure 4
A model for the binary SLN/SERCA1a complex. In this model, based on our previous model for the PLN/SERCA1a complex (10), the water-accessible surface is colored according to lipophilicity (brown, hydrophobic; sky blue, hydrophilic; green, neutral) as calculated with SYBYL6.8 (Tripos Associates, St. Louis). White dots outline SLN. The orange arrow, corresponding to 13 Å, indicates a wide space between SLN and M2 of SERCA1a. Horizontal bars show the boundaries of the hydrophobic core of the lipid bilayer.
Figure 5
Figure 5
A model for the ternary PLN/SLN/SERCA1a complex. (A) A view approximately normal to the membrane from the cytoplasmic side. The water-accessible surface of SERCA1a and van der Waals surfaces of SLN and PLN are shown. Transmembrane helices of SERCA1a (M2, M4, M6, and M9) are identified. (B) A view approximately normal to the membrane from the cytoplasmic side. Details of the interactions in the cytoplasmic half of the transmembrane region are shown. The shaded area in PLN indicates the region used for pentamer formation. Italicized amino acids (I38 and L42) show the two most important residues in hydrophobic contacts between PLN and SERCA, and the corresponding residues in SLN (V15 and V19). Dotted lines show a likely hydrogen bond between N11 (SLN) and C36 (PLN). (C) C-terminal half of SLN and the interaction with M2 of SERCA1a. Cylinders represent α-helices. The model is viewed in a shallow angle to the membrane so that the PLN transmembrane helix becomes approximately upright with the lumen of the sarcoplasmic reticulum at the bottom. M1 and M2 helices of SERCA1a are in green and the PLN transmembrane helix is in red. SLN is shown with a ball-and-stick representation. Note the cluster of aromatic residues near the luminal surface.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. MacLennan D H, Rice W J, Green N M. J Biol Chem. 1997;272:28815–28818. - PubMed
    1. Tada M, Kadoma M. BioEssays. 1989;10:157–163. - PubMed
    1. Simmerman H K, Jones L R. Physiol Rev. 1998;78:921–947. - PubMed
    1. Odermatt A, Taschner P E, Scherer S W, Beatty B, Khanna V K, Cornblath D R, Chaudhry V, Yee W C, Schrank B, Karpati G, et al. Genomics. 1997;45:541–553. - PubMed
    1. Odermatt A, Becker S, Khanna V K, Kurzydlowski K, Leisner E, Pette D, MacLennan D H. J Biol Chem. 1998;273:12360–12369. - PubMed

Publication types

MeSH terms

LinkOut - more resources