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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

Received 15 June 2006; revised 5 January 2007; accepted 9 January 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
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. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Phospholamban and sarcolipin are small related membrane proteins (52 and 31 amino-acid residues, respectively) C-terminally anchored in the sarcoplasmic reticulum (SR) of cardiac and skeletal muscle. The role of phospholamban in controlling cardiac output by modulating the activity of the cardiac calcium pump SERCA2a has been well documented [1–5]. Of particular interest is the finding that although the ablation of phospholamban is protective against cardiac hypertrophy in mice models of the disease the situation appears more complex in humans [6,7]. Two human phospholamban mutations that result in dilated cardiomyopathy have been identified. One is essentially a null mutant (L39STOP) [8] and the other (R9C) results in over-activity of this inhibitory non-phosphorylated mutant form of phospholamban [9]. Thus too little or too much phospholamban appear to be pathogenic in humans. Another way in which mutation could alter the influence of phospholamban would be if the phospholamban mutation resulted in incorrect targeting in the muscle cell since phospholamban must be retained in the sarcoplasmic reticulum to interact with SERCA2 (see below).

The role of sarcolipin in the modulation of cardiac contractility is less clear. It has been established that sarcolipin modulates SERCA1, located in fast-twitch muscle [1,10]. However, sarcolipin is also known to be expressed throughout human cardiac muscle [11] although only expressed in the atria of mice [12]. When over-expressed in HEK cells sarcolipin can modulate SERCA activity [1,7,13,14] and over-expression of sarcolipin in rat cardiac myocytes resulted in changes in contractility [15]. The finding that, in mice, atrial sarcolipin mRNA levels are down regulated in cardiac hypertrophy induced by ventricular over-expression of the H-ras gene indicates that sarcolipin may play a part in the pathology of cardiac hypertrophy [12]. Sarcolipin may also be involved in non-shivering thermogenesis by causing slippage of the calcium pump thereby partially uncoupling ATP hydrolysis from calcium transport and generating heat [10]. In addition, in particular muscle types of certain species (e.g., rodent atria [16]) phospholamban and sarcolipin are co-expressed along with SERCA2a. These two regulatory proteins probably act in tandem to effect the modulation of SERCA2a [14].

SERCAs are confined to the endoplasmic/sarcoplasmic reticulum (ER/SR) and in the case of SERCA1 there is evidence to indicate that it is maintained in this cellular compartment by a process of retrieval from the ER-Golgi intermediate compartment (ERGIC) following its passage from the ER [17]. The process of retrieval appears to be mediated through interactions with transmembrane helices M1 and M2 of SERCA1, but the mechanism is unknown [17]. Although phospholamban and sarcolipin are also known to be confined to the ER/SR [1,18,19] the process by which they are confined to this compartment and so are maintained alongside their cognate calcium pumps is unclear. One possibility is that they are maintained in the ER/SR through their interactions with the SERCAs and although one study suggested that Flag-epitope-tagged sarcolipin requires SERCA2 expression for ER localisation this appeared to be important only for truncated variants [19] as full length sarcolipin was localised to the ER in the absence of SERCA. However, the C-terminally anchored cytochrome b5 has been shown to be maintained in the ER mainly by a process of retention [20], although there is a measurable movement of cytochrome b5 to the ERGIC, but not to more distal compartments indicating that retrieval plays a role in maintaining cytochrome b5 in the ER [20]. In contrast, C-terminally anchored Sec61{alpha}, a component of the translocon, is found in significant amounts in the ERGIC and retrieval plays a predominant role in maintaining it in the ER [21]. In this study we have examined the localisation of phospholamban in cultured human fetal cardiac myocytes using immunofluorescence microscopy to determine whether it is prevented from exiting the SR, or whether significant amounts pass from this compartment into the ERGIC. Phospholamban and sarcolipin have also been tagged with EGFP at their N-termini and their targeting has been investigated by immunofluorescence microscopy and cell fractionation in COS 7 cells to determine whether they are maintained in the ER by a process of retention or retrieval. In addition, the role of the transmembrane domains of phospholamban and sarcolipin in the process of retrieval has been evaluated.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Construction of phospholamban and sarcolipin tagged with EGFP
cDNA coding for EGFP was amplified from the pEGFP-N1 vector (Clontech) using the polymerase chain reaction (PCR) with the following primers: GAT TCT AAG CTT ACC ATG GTG AGC AAG GGC GA; ACA GTT GGT ACC CTT GTA CAG CTC GTC CAT GC. The amplified gene was cloned into pcDNA 3.1(+) (Invitrogen) to produce pcDNA3.1EGFP.

The sequence coding for the rabbit sarcolipin gene (Accession number U96091) was synthesised from the oligonucleotides: AAT TCC ATG GAA AGG TCT ACT CG; AGA GCT GTG TCT GAA CTT TAC CGT TGT CCT GAT CAC AGT C; ATC CTT ATT TGG CTA CTA GTG CGG TCT TAC CAG TAC TGA T; GTA AAG TTC AGA CAC AGC TCT CGA GTA GAC CTT TCC ATG G; CAC TAG TAG CCA AAT AAG GAT GAC TGT GAT CAG CAG AAC G; CTA GAT CAG TAC TGG TAA GAC CG.

The canine phospholamban cDNA sequence (Accession number P61012 [GenBank] ) was created by a similar process.

The sarcolipin and phospholamban genes were cloned into pcDNA3.1EGFP downstream of EGFP. EGFP was linked to sarcolipin and phospholamban by the sequence GTELGSTSPVWWNS.

2.2 Construction of an ER-lumenally targeted EGFP
The EGFP sequence from pEGFP-N1 (Clontech) was cloned into the BamHI and NotI sites of pcDNA3.1(+), followed by a sequence coding for the 27 N-terminal residues of human {alpha}1-antitrypsin, amplified from a clone supplied by [22] with primers CTA CCT GCT AGC GAC AGT GAA TCG ACA ATG C and CAG CAT CTC CCT GGG GAT CC using NheI and BamHI sites. A sequence coding for KDEL was then introduced at the 5' end of the coding sequence using BsrGI and NotI sites to clone in the fragment produced by annealing oligonucleotides GTA CAA GAA AGA TGA ACT GTA AAG C and GGC CGC TTT ACA GTT CAT CTT TCT T to generate pAM2.

2.3 Construction of phospholamban and sarcolipin mutants
Primers AAA ATA GAA TTC CAT CGA TAA AGT CCA ATA CCT CAC TAG and AGG ATA TCT AGA TTA GAG GAG GAG GAG GAG AAG CAT CAC AAT AAT ACA G, were used in a PCR with pcDNA3.1(+)EGFP-PLB as template to introduce 4 extra Leu codons at the C-terminus of the phospholamban sequence. The mutated gene was cloned into pcDNA3.1(+)EGFP.

Sarcolipin mutants were produced by QuikChange mutagenesis (Strategene). Four 4 Leu were introduced using the oligonucleotides CAT CCT TAT TTG GCT ACT ACT CCT CCT ACT AGT GCG GTC TTA CC and GGT AAG ACC GCA CTA GTA GGA GGA GTA GTA GCC AAA TAA GGA TG with pcDNA3.1(+)EGFP-SLN as template. To extend the transmembrane domain of this mutant by a further 3 residues oligonucleotides GGC TAC TAC TCC TCC TAC TGC TGC TCC TAG TGC GGT CTT ACC AG and CTG GTA AGA CCG CAC TAG GAG CAG CAG TAG GAG GAG TAG TAG CC were used with the mutated construct as template.

2.4 Transfection of COS 7 cells
COS 7 cells were grown to 80% confluence on 25 mm coverslips for immunofluorescence studies and in tissue-culture dishes for immunoblotting and functional studies before transfecting with plasmid DNA using Fugene 6 (Roche), according to the manufacturer's instructions.

2.5 Subcellular fractionation
Subcellular fractionation was carried out as described previously [17]. Western blotting was performed using antibodies directed against EGFP (Roche), calnexin (Stressgene), β COP (Abcam) and TGN 46 (Serotec). The primary antibodies were detected with the appropriate secondary antibodies conjugated to HRP (Amersham Life Science) and ECL super signal substrate (Pierce).

2.6 Labelling of the plasma membrane with concanavalin A conjugate
COS 7 cells (80% confluent) on glass coverslips were washed with PBS and incubated with 250 µg/ml concanavalin A conjugated with Alexa Fluor 594 in PBS containing 1% BSA for 10 min. The coverslips were then washed with PBS and mounted onto slides in mowiol (CalBiochem) containing 0.1% citifluor (Agar Scientific).

2.7 Determination of transmembrane topology
The orientations of EGFP-phospholamban and EGFP-sarcolipin were determined using the method of [23]. COS 7 cells were grown on 145 cm2 culture plates and transfected with the appropriate construct. Cells were washed with 5 ml of homogenisation buffer (0.25 M sucrose, 50 mM potassium acetate, 5 mM magnesium acetate, 1 mM DTT, 50 mM Tris HCl, pH 7.6) and homogenised in 450 µl of homogenisation buffer. Aliquots of homogenised cells (40 µl) were incubated on ice with 8 µl of homogenisation buffer alone or containing proteinase K (2.4 mg/ml) with or without Triton X-100 (12%) as appropriate. The reactions were stopped using 20 µl of preheated (95 °C) 3xLaemmli sample buffer containing AEBSF (2.4 mg/ml). The samples were then separated by SDS PAGE, transferred to a nitrocellulose membrane and probed with anti-EGFP antibodies (1:1000 dilution) and anti-calnexin antibody (Stressgen SPA-865; 1:2000 dilution).

2.8 Step-wise permeabilisation of the plasma and endoplasmic reticulum membranes
Transfected cells expressing the appropriate constructs were washed in PBS and then fixed in a 24 well plate using 4% formaldehyde PBS for 15 min. The cells were washed once in PBS, containing either Triton X-100 (0.1%) or saponin (0.04 mg/ml) and incubated in buffer P (PBS supplemented with 2% milk powder and either 0.04 mg/ml saponin or 0.1% Triton X-100 as appropriate) for 30 min. The cells were then incubated with mouse anti-EGFP (Roche) (diluted 1:100 in buffer P) for 1 hr at 37 °C followed by washing and anti-mouse Texas Red conjugate (Amersham Life Science) (diluted 1:50). After a final wash in PBS the cells were viewed using a Leica digital epifluorescence microscope (DM IRBE).

2.9 Visualisation of EGFP tagged constructs in COS 7 cells
Transfected cells were viewed under a Leica digital epifluorescence microscope (DM IRBE) fitted with standard FITC and rhodamine filter sets for the excitation of both EGFP and Texas Red fluorophores. Images were also obtained with a Zeiss confocal microscope (LSM 510 META); the EGFP fluorophore was excited at 488 nm with a band pass filter of 505–530 nm and Texas Red and Alexa Fluor 594 at 543 nm with a long pass filter of 560 nm.

2.10 Immunofluorescence microscopy
To detect possible co-localisation of EGFP-phospholamban or EGFP-sarcolipin with SERCA1 COS 7 cells cotransfected with a SERCA1b expression construct were fixed in cold methanol for 10 min and incubated with a monoclonal antibody against SERCA the primary antibody was Y/1F4 [24] diluted 1:10 in PBST followed by anti-mouse Texas Red conjugate antibody (Amersham Pharmacia) diluted 1:50.

To examine the subcellular location of sarcolipin and phospholamban, transfected COS 7 cells were treated with a cold block or brefeldin A to enhance the visualisation of the appropriate compartments (see [17]) before fixation in formaldehyde (4% in PBS) for 15 min. The cells were preincubated for 30 min in PBS Triton X-100 (0.01%) with 2% milk powder. The primary antibodies used were sheep anti-human TGN 46 (Serotech), diluted 1:50 and mouse anti-human ERGIC 53 (a gift from H.P. Hauri) diluted 1:100. The secondary antibody for TGN 46 was donkey anti-sheep IgG conjugated to Texas Red (Amersham Life Sciences) diluted 1:100 and for ERGIC 53 was sheep anti-mouse Texas Red antibody (Amersham Life Sciences) diluted 1:50.

The anti-phospholamban antibody used to investigate targeting in cardiac myocytes was obtained from Abcam.

2.11 Preparation of cardiac myocyte cultures
The collection and use of human and fetal material was carried out following ethical approval from the Southampton and South West Hampshire Local Research Ethics Committee under guidelines issued by the Polkinghorne committee. Cardiac tissues from human embryos were collected with informed consent following surgical termination of pregnancy and staged immediately by stereomicroscopy according to the Carnegie classification [25].

Fetal cardiac myocytes were obtained essentially using a published protocol [26]. The cells were cultured for 15 days before being used for immunofluorescence microscopy.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Localisation of phospholamban in cultured human fetal cardiac myocytes
Fig. 1A and D show that phospholamban is located in the internal membrane system of fetal cardiac myocytes. Co-localisation with the ERGIC marker ERGIC 53 [27] is apparent (Fig. 1B and C) but phospholamban does not co-localise with the trans-Golgi network marker TGN 46 [28] (Fig. 1E and F).


Figure 1
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  		Fig. 1 Phospholamban location in cultured human fetal cardiac myocytes. Cardiac myocytes were seeded onto coverslips and cultured for 15 days. The cells were fixed in cold methanol and labelled with an anti-phospholamban antibody using the appropriate secondary antibody conjugated with fluorescein (panels A and D). Panel B shows the same cells treated with an antibody against the ERGIC marker ERGIC 53 followed by a Texas Red conjugated secondary antibody. Images A and B are merged in panel C. In panel E the trans-Golgi network was identified using an anti-TGN 46 antibody visualised with a Texas Red conjugated secondary antibody. Images D and E are merged in panel F. Cells were viewed with a Zeiss confocal microscope.

 
3.2 ER location of EGFP-tagged phospholamban and sarcolipin
Fig. 2A and D show that, when co-expressed with SERCA1, the cellular locations of EGFP-phospholamban and EGFP-sarcolipin correspond to that expected for ER resident proteins [17,20,21] including native phospholamban and epitope tagged sarcolipin [1,8]. The ER location of these constructs is confirmed in Fig. 2C and F showing that EGFP-phospholamban and EGFP-sarcolipin co-localise with the ER-targeted protein SERCA1 [1]. The cellular locations of EGFP-sarcolipin and EGFP-phospholamban are the same when they are expressed in the absence of heterologous SERCA1 expression (data not shown).


Figure 2
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  		Fig. 2 Co-localisation of EGFP-phospholamban and EGFP-sarcolipin with SERCA1. COS 7 cells were co-transfected with 2 constructs, coding for EGFP-phospholamban and SERCA1 (panels A–C) or EGFP-sarcolipin and SERCA1 (panels D–F). The cells were incubated for 2 days and following fixation in cold methanol SERCA1 was visualised using a primary antibody against SERCA1 and a secondary antibody tagged with Texas Red. The COS 7 cells were viewed by epifluorescence microscopy. Panels A and D show the localisation of EGFP-phospholamban and EGFP-sarcolipin, respectively. Panels B and E show SERCA1 localisation and the fluorescence signals of SERCA1 and EGFP-phospholamban and EGFP-sarcolipin are overlaid in panels C and F, respectively.

 
3.3 EGFP-tagged phospholamban and sarcolipin are inserted into the ER in the correct orientation
Sarcolipin and phospholamban are C-terminally anchored proteins and the data in Figs. 3 and 4Go demonstrate that this is also the case for the tagged variants. The EGFP tags at the N-terminal ends of EGFP-phospholamban and EGFP-sarcolipin are not accessible to EGFP antibody in intact cells (Fig. 3B and D). Fig. 3K and L show that lumenally-targeted EGFP is not detected by EGFP antibodies after saponin treatment to disrupt the plasma membrane [29] consistent with its location in the ER lumen; treatment with Triton X-100, a detergent to disrupt all the cellular membranes, makes the construct accessible to the EGFP antibody (Fig. 3Q, R). In contrast, EGFP antibodies bind to EGFP-phospholamban and EGFP-sarcolipin after treatment of cells with either saponin or Triton X-100 (Fig. 3H–J and N–P, respectively) showing that the EGFP tag on phospholamban and sarcolipin is located in the cytoplasm.


Figure 3
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  		Fig. 3 Selective permeabilisation studies to determine the transmembrane topology of EGFP-phospholamban and EGFP-sarcolipin. COS7 cells were seeded on to 13 mm coverslips at 60–80% confluence and transfected with either constructs coding for EGFP-phospholamban, EGFP-sarcolipin or EGFP targeted to the endoplasmic reticulum. After 2 days the cells were fixed with 4% formaldehyde and then treated with either PBS (A–F), PBS containing saponin (G–L) or PBS containing Triton X-100 (M–R) and probed with antibodies directed against EGFP. Antibody binding was probed with a secondary antibody conjugated with Texas Red. Texas Red and EGFP were visualised by epifluorescence microscopy.

 

Figure 4
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  		Fig. 4 Proteolysis by proteinase K to examine the orientation of EGFP-phospholamban and EGFP-sarcolipin in microsomes. Microsomes made from COS 7 cells expressing EGFP-phospholamban or EGFP-sarcolipin were treated with either buffer (lane 1), proteinase K (lane 2) or Triton X-100 with proteinase K (lane 3). The microsomes were incubated on ice and the component proteins separated by SDS PAGE gels. Proteins were transferred to nitrocellulose and probed with antibodies directed against EGFP or calnexin. Antibody binding was visualized using a secondary antibody conjugated with horseradish peroxidase and a chemiluminescence protocol. The signals were detected using a Biorad Versa Doc detection system.

 
Further evidence for the correct orientation of the EGFP-phospholamban and EGFP-sarcolipin came from proteolysis studies where microsomes containing EGFP-tagged phospholamban or EGFP-tagged sarcolipin were treated with proteinase K (Fig. 4). Treatment with proteinase K of microsomes from COS-7 cells expressing EGFP-phospholamban or EGFP-sarcolipin resulted in the loss of the EGFP tag, as shown by the loss of binding of the EGFP antibody (Fig. 4, lanes 1 and 2). In contrast, a significant amount of the lumenal epitope of the transmembranous ER-resident protein calnexin was protected from the protease, as shown by binding of antibody to calnexin after protease treatment, protection that was lost when the microsomes were treated with Triton X-100 before protease treatment (Fig. 4; lane 3). The reduction in amount of calnexin detected by the antibody following proteinase K treatment may be due to a fraction of leaky microsomes.

3.4 Phospholamban and sarcolipin escape from the ER into the ERGIC
Although EGFP-phospholamban and EGFP-sarcolipin are located in the ER, closer scrutiny reveals that significant amounts of both co-localise with the ERGIC marker ERGIC 53 [27] (Fig. 5A–C and D–F) around the perinuclear region. Note that the use of a "cold block" in these experiments enhances the appearance of the ERGIC compartment [30]. When the technique was repeated using the trans-Golgi marker TGN 46 it is clear that there is negligible overlap between the TGN marker and EGFP-phospholamban (Fig. 5G–I) or EGFP-sarcolipin (Fig. 5J–L). The use of brefeldin A in Fig. 5G–L does alter the distribution of the ER in these cells as anticipated (compare Fig. 5A with G and D with J) (see [17]).


Figure 5
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  		Fig. 5 EGFP-phospholamban and EGFP-sarcolipin expression in COS 7 cells is located in the ERGIC but not the trans-Golgi. COS 7 cells were seeded onto coverslips and transfected with an expression construct coding for EGFP-phospholamban (panels A–C and G–I) or EGFP-sarcolipin (panels D–F and J–L). The cells were then incubated for 2 days before using an anti-ERGIC 53 antibody to reveal the ERGIC compartment (panels B and E). Panel A shows EGFP-phospholamban fluorescence. Panel B shows the same cells treated with an antibody against the ERGIC marker followed by a Texas Red conjugated secondary antibody and panel C shows an overlay of panels A and B. Panel D shows EGFP-sarcolipin fluorescence. Panel E reveals the ERGIC marker as above and panel F shows an overlay of panels D and E. The trans-Golgi network was identified using an anti-TGN 46 antibody visualised with a Texas Red conjugated secondary antibody. Panel G shows EGFP-phospholamban fluorescence. Panel H shows the same cells using the TGN marker. Panel I shows an overlay of panels G and H. Panel J shows EGFP-sarcolipin fluorescence and panel K shows the same cells using the TGN marker. Panel L shows an overlay of panels J and K. Cells were viewed with a Zeiss confocal microscope.

 
Cell fractionation studies were undertaken to validate the finding that EGFP-phospholamban and EGFP-sarcolipin are able to leave the ER. Using antibody markers against proteins located in the ER (calnexin [31]), Golgi (TGN 46 [28]) and ERGIC (β-COP [32]) it can be seen from the analysis of Nycodenz density gradient fractionations (Fig. 6) that the ER separates in the densest fraction, where most of the calnexin resides (Fig. 6A and B fraction 1). The ERGIC marker β-COP is found in the lighter fractions, but is more widely spread (fractions 6–9); the TGN marker is also found in the lighter fractions, mainly between fractions 6–8, with a small amount being located in the densest fraction (fraction 1); the TGN marker in fraction 1 could represent small amounts of newly synthesised material en route to the Golgi. EGFP-phospholamban and EGFP-sarcolipin were found in ER fraction as well as fractions derived from the ERGIC and the trans-Golgi.


Figure 6
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  		Fig. 6 Localisation of EGFP-phospholamban and EGFP-sarcolipin by cell fractionation. COS 7 cells transfected with a construct coding for EGFP-phospholamban (A) or EGFP-sarcolipin (B) were disrupted as described in the method section. After removing the nuclei and undisrupted cells with a preliminary centrifugation step, material was centrifuged on a Nycodenz gradient. Fractions were collected, fraction 1 being the densest. Proteins from each fraction were precipitated with perchloric acid and the pellets obtained by centrifugation were solubilised and the component proteins separated by SDS-PAGE. The proteins were then subjected to western blot analysis using antibodies directed to EGFP, calnexin, β COP and TGN 46. The primary antibodies were detected using secondary antibodies conjugated to horseradish peroxidase and a chemiluminescence protocol. The signal was detected and recorded using a Biorad Versa Doc system.

 
3.5 Increasing the length of the hydrophobic sequence of phospholamban and sarcolipin leads to mis-targeting
Extending the C-terminus of EGFP-phospholamban by the addition of 4 Leu residues lead to relocation of a significant amount of the construct to the plasma membrane (Fig. 7D; compare with Fig. 2A for the normal construct). This was confirmed using Alexa Fluor 594 labelled concanavalin A (Fig. 7E); concanavalin A binds exclusively to the external surface of the plasma membrane in intact cells [33]. Fig. 7F shows that when the images of the location of concanavalin A and the extended EGFP-phospholamban are overlaid these proteins are co-localised at the plasma membrane. Similar studies in which the transmembrane sequence of sarcolipin was extended by 4 Leu residues did not affect the distribution of the extended EGFP-sarcolipin construct (Fig. 7G). However, when the construct was extended to include 7 extra Leu residues the construct was targeted to the plasma membrane (Fig. 7A–C).


Figure 7
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  		Fig. 7 Cellular localisation of mutant forms of EGFP-phospholamban and EGFP-sarcolipin containing extra Leu residues in the transmembrane domain. COS 7 cells were seeded onto coverslips and transfected with an expression construct coding for a mutant form of EGFP-sarcolipin containing 7 extra Leu residues at position 25 (A–C), a mutant form of EGFP-phospholamban containing 4 extra Leu residues at position 52 (D–F), or a mutant form of EGFP-sarcolipin containing 4 extra leucine residues at position 25 (G). The cells were then incubated for 2 days before being treated to reveal the plasma membrane using a concanavalin A conjugated to Alexa Fluor 594. The coverslips were mounted onto microscope slides using mowiol mountant+0.1% citifluor. Panels A and D show EGFP fluorescence. Panels B and E show the same cells tagged with concanavalin A Alexa Fluor 594. Panels C and F show an overlay of panels A and B or D and E, respectively. Panel G shows EGFP fluorescence. The cells were viewed with a Zeiss confocal microscope.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Linkage between β-adrenergic stimulation and increased cardiac output requires the correct targeting of phospholamban [6]. The potential role of sarcolipin in modulating cardiac contraction [15,16] also requires targeting to the same compartment as SERCA2.

Fig. 1A shows that phospholamban is located in the SR of fetal cardiac myocytes, consistent with previous reports [34,35]. Phospholamban in these cells is also found in the ERGIC (Fig. 1A–C) indicating that retention of phospholamban in SR is not absolute and that significant amounts of phospholamban pass from the ER into the ERGIC. However, phospholamban does not proceed further down the secretion pathway since it fails to enter the trans-Golgi network (Fig. 1D–F). These data indicate that phospholamban is retained in SR by a process of retrieval from the ERGIC as has been demonstrated previously for ER resident proteins [17,21,27].

Sarcolipin targeting cannot be investigated by immunofluorescence in cardiac myocytes because, although there are antibodies that recognise sarcolipin in blots [16], there are no suitable antibodies for immunofluorescence applications. To circumvent this problem and the difficulties associated with transfecting primary cells we have expressed phospholamban and sarcolipin tagged at their N-termini with EGFP in COS 7 cells to investigate ER targeting.

SERCA1 when expressed in non-muscle cells locates to the ER [1,17]. We have used this property of SERCA1 to demonstrate that EGFP-phospholamban and EGFP-sarcolipin are located in the same compartment in COS 7 cells (Fig. 2), consistent with immunolocalisation studies of phospholamban and epitope-tagged sarcolipin [1,18,19]. Importantly the EGFP tags on EGFP-phospholamban and EGFP-sarcolipin are located on the cytoplasmic side of the ER, as shown by antibody binding studies in intact and permeabilised cells and by examining the accessibility of the EGFP tag to proteinase K in intact and solubilised microsomes (Figs. 3 and 4Go). These data show that, like their wild type counterparts, EGFP tagged phospholamban and sarcolipin are C-terminally anchored into the ER membrane [1,2].

There are two possible mechanisms by which proteins can be maintained within the ER. Proteins such as calnexin and cytochrome b5 are maintained in the ER largely by a process of retention [20,36]. However, other proteins such as Sec61{alpha} and the calcium pump SERCA1 are maintained in the ER by retrieval of ER proteins that pass into the ERGIC; these proteins do not proceed further down the secretion pathway as they are not found in the trans-Golgi [17,21]. ER lumenal proteins such as protein disulphide isomerase contain a C-terminal KDEL sequence that is recognised in the ERGIC leading to return to the ER [37]. Similarly some transmembrane proteins contain retrieval signals at the C- or N-termini [38,39]. Notably however Sec61{alpha}, SERCA1 and the pump regulators phospholamban and sarcolipin do not contain any of the characterised retrieval signal sequences.

The experiments shown in Fig. 5A–F demonstrate that EGFP-phospholamban and EGFP-sarcolipin co-localise with ERGIC markers, but not with markers from the trans-Golgi (Fig. 5G–L). The cell fractionation studies (Fig. 6) also show that significant amounts of EGFP-phospholamban and EGFP-sarcolipin are located in fractions that contain COP1 proteins; COP1 is located in post ER compartments and is involved in the process of retrieving ER resident proteins from post ER compartments [40]. We therefore conclude that phospholamban and sarcolipin are maintained in the ER by a process of retrieval from the ERGIC.

The mechanism or mechanisms by which membrane-bound ER-resident proteins that do not contain any of the characterised retrieval signals is unclear. Retention of sarcolipin and phospholamban in the ER by interaction with their cognate partners, the SERCAs, seems not to be important since when phospholamban and sarcolipin are over-expressed in COS 7 cells, the proteins are not mis-targeted to the trans-Golgi or plasma membrane (Fig. 3A and C).

Sarcolipin contains just 31 amino acid residues of which about 19 will form the transmembrane {alpha}-helix leaving 8 and 5 polar residues on the cytoplasmic and lumenal sides of the ER, respectively. It is therefore difficult to avoid the hypothesis that the transmembrane domain or its flanking residues must play the major role in directing this protein back to the ER. In one study of a truncated form of sarcolipin in which the lumenal residues (RSYQY) were removed, the truncated sarcolipin escaped from the ER to reach the plasma membrane [19]. However it can be argued that the sequence RSYQY is unlikely to represent a specific retrieval signal for this type of protein since no equivalent sequence exists in phospholamban.

Studies of the C-terminally anchored cytochrome b5 and the type III membrane protein Sec71p have shown that extending the transmembrane domain leads to relocation of the protein from the ER to the plasma membrane [20,41]. It has been suggested that the length of the transmembranous domain is important because of the requirement for hydrophobic matching between the protein and the surrounding lipid bilayer; the hydrophobic thickness of the plasma membrane is greater than that of the ER or Golgi and so membrane proteins containing long transmembrane helices favour the plasma membrane [42]. Hydrophobic matching between a long transmembrane helix and a thin bilayer could require tilting of the helix in the membrane [43], which would affect the interaction between the helix and the proteins involved in retention/retrieval. Addition of 4 extra Leu residues to the transmembrane domain of EGFP-phospholamban redirected the protein to the plasma membrane (Fig. 7A–C). In contrast, sarcolipin was only mis-targeted when the transmembrane domain was extended by 7 Leu (Fig. 7D–G). This is consistent with a role for the length of the transmembrane domain in the targeting process since the transmembrane domain of sarcolipin is about 4 residues shorter than that of phospholamban.

At least two mechanisms could account for the mis-targeting of the extended forms of phospholamban and sarcolipin. Mis-targeting could result from the loss of an ER retention signal, but this seems unlikely because significant amounts of native phospholamban and sarcolipin leave the ER normally to be returned from the ERGIC. The alternative mechanism, and the one favoured here, is that the retrieval mechanism is disrupted allowing phospholamban and sarcolipin to escape from the ERGIC into the endomembrane system en route for the plasma membrane.

The most likely candidate for involvement in the retrieval of phospholamban and sarcolipin from the ERGIC is RER1p [44] that appears to recognise ER targeting information contained within and flanking the transmembrane domains of ER resident proteins, returning them from post-ER compartments [41].


   Acknowledgements
 
We thank the BBSRC for the studentship awarded to JB at the outset of this work and the British Heart Foundation for providing financial support. Thanks are also due to Matthew Cuttle for assistance with fluorescence microscopy, Richard Foreman for providing the {alpha}1-antitrypsin cDNA clone and H-P Hauri for providing the anti-ERGIC53 antibody.


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

Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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