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Cardiovascular Research 2007 74(1):114-123; doi:10.1016/j.cardiores.2007.01.006
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Copyright © 2007, European Society of Cardiology

Phospholamban and sarcolipin are maintained in the endoplasmic reticulum by retrieval from the ER-Golgi intermediate compartment

John Butlera,1, Anthony G. Leea,1, David I. Wilsonb,1, Cosma Spallutob,1, Neil A. Hanleyb,1 and J. Malcolm Easta,*

aSchool of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK
bSchool of Medicine, University of Southampton SO16 6YD, UK

* Corresponding author. Tel.: +44 23 80594222; fax: +44 23 80594459. Email address: jme1{at}soton.ac.uk

Objective: Phospholamban and sarcolipin are small transmembrane proteins that modulate cardiac contractility through their interaction with the sarcoplasmic reticulum (SR) calcium pumps (SERCAs). We have examined the hypothesis that phospholamban and sarcolipin are maintained in the SR by a process of retrieval from post-SR compartments and the role of their transmembrane domains in targeting.

Methods: Antibodies directed against phospholamban and protein markers of the endoplasmic reticulum/Golgi intermediate compartment (ERGIC) and the trans-Golgi were used in fluorescence microscopy studies of cultured human fetal cardiac myocytes. In addition, sarcolipin and phospholamban were tagged at the N-terminus with enhanced-green-fluorescent protein (EGFP) and expressed in COS 7 cells. The EGFP-tagged constructs were localised using fluorescence microscopy and cell fractionation. The length of the transmembrane domains of phospholamban and sarcolipin were extended and the effect on cellular location was also examined.

Results: In fetal cardiac myocytes phospholamban was located in the SR and the ERGIC, but did not migrate to the trans-Golgi network. Tagged-phospholamban and sarcolipin were located in the endoplasmic reticulum (ER) of COS 7 cells indicating that their targeting was unaffected by the EGFP tag. Significant proportions of the tagged phospholamban and sarcolipin were also located in the ERGIC but not in the trans-Golgi. Increasing the length of the transmembranous domains of EGFP-tagged phospholamban and sarcolipin resulted in their mis-targeting to the plasma membrane.

Conclusions: Phospholamban and sarcolipin are maintained in the SR/ER by a process that includes their retrieval from the ERGIC following their passage from the SR/ER into the ERGIC. The transmembrane domains of phospholamban and sarcolipin are involved in the retrieval process.

KEYWORDS Phospholamban; Sarcolipin; Targetting; Endoplasmic reticulum; Sarcoplasmic reticulum; Retrieval; ERGIC; Transmembrane domain

1 Tel.: +44 23 80594222; fax: +44 23 80594459.

Time for primary review 23 days


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