Sarcolipin (SLN) and phospholamban (PLN) are effective inhibitors of the 
sarco(endo)plasmic reticulum Ca(2+)-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 Ca(2+)-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.