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CELL BIOLOGY
Sarcolipin retention in the endoplasmic reticulum depends on its C-terminal RSYQY sequence and its interaction with sarco(endo)plasmic Ca²+-ATPases

Anthony O. Gramolini ^*, Thomas Kislinger ^*, , Michio Asahi ^* , Wenping Li ^*, Andrew Emili ^*, , and David H. MacLennan ^*, 

^*Banting and Best Department of Medical Research and Program in Proteomics and Bioinformatics, University of Toronto, Charles H. Best Institute, 112 College Street, Toronto, ON, Canada M5G 1L6

Contributed by David H. MacLennan, October 20, 2004

Sarcolipin (SLN) and phospholamban (PLN) are effective inhibitors of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). These homologous proteins differ at their N and C termini: the C-terminal Met-Leu-Leu in PLN is replaced by Arg-Ser-Tyr-Gln-Tyr in SLN. The role of the C-terminal sequence of SLN tagged N-terminally with the FLAG epitope (NF-SLN) in endoplasmic reticulum (ER) retention was investigated by transfecting human embryonic kidney-293 cells with cDNAs encoding NF-SLN or a series of NF-SLN mutants in which C-terminal amino acids were deleted progressively. Immunofluorescence and immunoblotting of transfected cells by using anti-FLAG antibodies indicated that NF-SLN and PLN tagged at its N terminus with the FLAG epitope, even when overexpressed, were restricted to the ER. However, C-terminal truncation deletions of SLN, which lacked RSYQY, were not localized to ER and did not inhibit Ca²⁺-dependent Ca²⁺ uptake by SERCA. The shortest deletion constructs, NF-SLN 1-22 and NF-SLN 1-23, did not express stable protein products. However, all NF-SLN cDNA constructs, including NF-SLN 1-22 and NF-SLN 1-23, were expressed stably and localized to the ER when they were coexpressed with SERCA2a. These results show that NF-SLN subcellular distribution depends on SERCA coexpression and on its luminal, C-terminal RSYQY sequence. By using immunoprecipitation and MS, glucose-regulated protein 78/BiP and glucose-regulated protein 94 were identified as proteins that interact with NF-SLN through the RSYQY sequence. Thus, in the absence of SERCA, retention of NF-SLN in the ER is mediated through its association with other components through the C-terminal RSYQY sequence.

Abbreviations: SERCA, sarco(endo)plasmic reticulum Ca²⁺-ATPase; PLN, phospholamban; SLN, sarcolipin; NF-SLN, SLN tagged N-terminally with a FLAG epitope; NF-PLN, PLN tagged N-terminally with a FLAG epitope; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; HEK, human embryonic kidney; IP, immunoprecipitation.

Present address: Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan.

To whom correspondence should be addressed. E-mail: david.maclennan{at}utoronto.ca.

© 2004 by The National Academy of Sciences of the USA

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