Centro di Studio per la Biologia e la Fisiopatologia Muscolare del Consiglio Nazionale delle Ricerche, Dipartimento di Scienze Biomediche Sperimentali dell'Università di Padova, 35121 Padua, Italy
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ABSTRACT |
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Calsequestrin (CS) is the
Ca2+ binding protein of the
junctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimeric
CS-HA1, obtained by adding the nine-amino-acid viral epitope
hemagglutinin (HA1) to the COOH terminus of CS, was shown to be
correctly segregated to the sarcoplasmic reticulum [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe.
Am. J. Physiol. 272 (Cell Physiol. 41): C1420-C1428,
1997]. A putative targeting mechanism of CS to jSR implies
electrostatic interactions between negative charges on CS and positive
charges on intraluminal domains of jSR integral proteins, such as
triadin and junctin. To test this hypothesis, 2 deletion mutants of
chimeric CS were engineered: CS-HA1Glu-Asp, in which the 14 acidic
residues
[-Glu-(Asp)5-Glu-(Asp)7-] of the COOH-terminal tail were removed, and
CS-HA1
49COOH, in which the
last, mostly acidic, 49 residues of the COOH terminus were removed.
Both mutant cDNAs were transiently transfected in HeLa cells, myoblasts
of rat skeletal muscle primary cultures, or regenerating soleus muscle
fibers of adult rats. The expression and intracellular localization of
CS-HA1 mutants were studied by epifluorescence microscopy with use of
antibodies against CS or HA1. CS-HA1 mutants were shown to be
expressed, sorted, and correctly segregated to jSR. Thus short or long
deletions of the COOH-terminal acidic tail do not influence the
targeting mechanism of CS.
calcium binding protein; protein targeting
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INTRODUCTION |
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THE SARCOPLASMIC RETICULUM (SR) of skeletal muscle, a network of tubules and cisternae devoted to intracellular Ca2+ homeostasis, is composed of two continuous compartments: the nonjunctional SR, enriched in Ca2+ pump molecules, and the junctional SR (jSR), juxtaposed to the transverse tubules (TT) and enriched in Ca2+ release channels [also known as ryanodine receptors (RyR)] and calsequestrin (CS).
The SR is a subcompartment of the endoplasmic reticulum (ER), as indicated by the coexistence of ER general markers, such as BiP, calnexin, and PDI, with specific molecular components of Ca2+ stores, e.g., CS, Ca2+ pump, and RyR (26). The molecular differentiation of SR appears to occur from a wide-mesh ER membrane network, to include at an early stage the concentration of CS within membrane-bound structures also containing general ER markers, and progressively to evolve into the establishment of triad couplings between terminal cisternae (TC) and TT (4, 14, 24).
CS is an acidic (3, 22), low-affinity (dissociation constant ~1 mM), high-capacity (40-50 mol/mol) Ca2+ binding protein, the atomic resolution structure of which has been recently described (27). CS segregates to the jSR lumen, where it has been detected as electron-dense material, aggregates, paracrystalline arrays, or fiberlike network (5, 12), and plays a crucial role in the storage of Ca2+ between uptake and release cycles (16). CS lacks the characteristic COOH-terminal sequence KDEL (3) that ensures luminal retention, without segregation, to several ER proteins (e.g., BiP and PDI) through continuous recycling from pre-Golgi and Golgi compartments. Although a stage through the Golgi complex seems well established for CS (25), subsequent routes of intracellular trafficking are not yet clear. In particular, it is not known whether CS reaches SR via clathrin-coated cis/medial Golgi-derived vesicles (23) or whether it travels back to the ER, diffuses intraluminally to SR via ER/SR continuities (24, 26), and segregates to the jSR (5, 12). The presence of CS in the ER lumen and nonjunctional SR during early postnatal development (24) as well as in ER subdomains of transfected L6 myoblasts (6) would support the latter possibility.
We have developed a cellular and molecular biology approach to investigate the targeting mechanisms (sorting, retention, and segregation) of CS. A first accomplishment was the characterization of a chimeric cDNA, CS-HA1 cDNA (18): a DNA fragment coding for the nine amino acids of the influenza virus hemagglutinin (HA1) was added at the 3' terminus of the whole cDNA of rabbit skeletal muscle CS by PCR cloning. The complete construct was transiently transfected in nonmuscle recipient (HeLa) cells, myoblasts isolated from 0- to 3-day-old rat skeletal muscle, and regenerating skeletal muscles of adult rats. Expression and intracellular localization of CS-HA1 were monitored with anti-CS and anti-HA1 antibodies and revealed that 1) chimeric CS-HA1 is correctly sorted to ER/SR compartments in muscle and nonmuscle systems, 2) the HA1 epitope does not alter the structure and/or the sequence of the signal(s) involved in CS retention, 3) the free COOH terminus is not required for targeting of CS, and 4) CS-HA1 segregates to the jSR of regenerating skeletal muscle fibers of adult rats after in vivo transfection of CS-HA1 cDNA (20). Thus CS-HA1 lends itself as a powerful tool for the identification of targeting sequences of CS by site-directed mutagenesis.
CS segregation to the jSR can be hypothetically accounted for by integral proteins, restricted in their expression to the jSR (8, 11, 13, 29) and able to bind CS [CS-binding protein(s)] with their luminal, mostly basic domain. Triadin (TD) and junctin (JC), both integral membrane proteins, are putative CS-binding proteins and have been recently cloned and sequenced (11, 13); moreover, the RyR can also form complexes with CS (17, 29). CS segregation to the jSR might also be accounted for by specific recognition sites on mostly acidic domains of CS, for interaction with TD and/or JC, and, possibly, with RyR. Given the known primary sequences of CS and of the luminal domains of TD, JC, and RyR, electrostatic interactions are likely. It is not known, however, which and where such putative CS domains might be.
In a first attempt to identify such domains, we have thus engineered
two CS-HA1 deletion mutants, CS-HA1Glu-Asp and
CS-HA1
49COOH. In the first
case, we have removed the COOH-terminal acidic tail (Glu354-Asp367)
made of 14 amino acids
[-Glu-(Asp)5-Glu-(Asp)7-]
and, in the second case, the 49 residues at the COOH terminus, which
are mostly acidic (42% Glu + Asp) (3). The results reported here show that the mostly acidic COOH terminus of CS, irrespective of length of
deletion, appears not to be needed for sorting, retention, and
segregation of CS to the jSR. The results are also interpreted in the
framework of knowledge derived from the crystal structure of CS (27).
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MATERIALS AND METHODS |
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Generation of CS-HA1Glu-Asp cDNA and
CS-HA1
49COOH cDNA
The CS-HA149COOH was developed
using an identical cloning strategy with the same forward primer and a
different reverse primer: 5'-CTA
-
AGTAACATTGACGACTCCTATTTG-3', with underlined nucleotides being the coding sequence of the HA1 tag
(28) and the stop codon being represented by characters in small
capitals. The final construct, devoid of 147 bp at the 3' end,
was called pCS-HA1
49COOH.
Orientation and correct sequence of chimeric mutants were checked by restriction assays, and sequence of the synthetic region was obtained by the dideoxy chain termination method (21) with use of modified T7 DNA polymerase.
Cell Cultures
HeLa cells. HeLa cells were grown in DMEM containing 2 mM glutamine and 10% FCS.
Primary cultures of skeletal muscle rat myoblasts and differentiation into myotubes. Primary myoblasts were isolated from hindlimb skeletal muscles of 0- to 3-day-old rats. After the isolated muscles were washed several times in 125 mM PBS, pH 7.4, and subjected to three 20-min stages of trypsinization (2.5% trypsin in PBS) at 37°C and mixing with a vortex every 4 min, supernatants were collected and trypsin was inhibited by addition of 2% horse serum (HS). Cells were then collected by centrifugation and preplated in 9-cm-diameter plates for 1 h at 37°C. The myoblast-enriched supernatant was centrifuged, and cells were finally resuspended in DMEM supplemented with 20% FCS and 20 mM glucose, counted, and plated. Differentiation was obtained by changing the medium to DMEM with 10-20% HS and subsequently DMEM with 2% HS. A few nonmuscle cells were occasionally transfected (see Fig. 2D).
Bupivacaine-Induced Necrosis and Regeneration of Adult Rat Skeletal Muscle
Male adult Wistar rats (~250 g body wt) were anesthetized with ketamine (1.5 mg/100 g body wt). The right soleus muscles were exposed and injected with 0.4 ml of 0.5% bupivacaine, as described previously (18). Muscles were removed 3 or 10 days later and frozen in liquid nitrogen. In agreement with previous reports (9, 25), the local anesthetic bupivacaine induced almost complete necrosis of the whole soleus by day 3. Regeneration started by day 3 and was completed by day 10.Generation of Transient Transfectants
Twenty-four hours before transfection, HeLa cells or primary myoblasts were seeded onto 25-mm-diameter wells of a 24-well Corning plate containing a 13-mm-diameter round coverslip with a cell density suitable to obtain 50% confluence at the moment of transfection. pCS-HA1Adult rat soleus muscles were exposed 3 days after bupivacaine injection under ketamine anesthesia and injected with 100 µg of plasmid DNA in 20% sucrose. Rats were killed 7 days later, and transfected and mock-transfected, contralateral muscles were excised, frozen, and processed for immunocytochemistry.
Immunofluorescence
Cells were fixed in 4% paraformaldehyde and 0.25% glutaraldehyde in PBS for 15 min and permeabilized with 0.3% Triton X-100, 20 mM phosphate buffer, pH 7.4, 0.45 M NaCl, and 15% goat serum (buffer A) for 30 min. Incubation with polyclonal anti-CS (24, 26) and monoclonal anti-CS (Affinity Bioreagents) or monoclonal and polyclonal anti-HA1 antibodies (BabCO and Santa Cruz Biotechnology, respectively) was performed at room temperature for 1.5 h in buffer A. After they were washed for 1 h, cells were incubated for 30 min with rhodamine isothiocyanate or fluorescein-conjugated anti-mouse or anti-rabbit antibodies (DAKO). Images were obtained with a Zeiss Axioplan microscope or a Leica DMRB microscope. Transverse 9-µm and longitudinal 6-µm sections were obtained for soleus muscles, as described previously (24, 26).Preparation of Homogenates From HeLa Cells and Myotubes and of SR Vesicles From Rabbit Skeletal Muscles
HeLa cells and myotubes were cultured as described above, transiently transfected with pCS-HA1Purified SR vesicles referable to terminal cisternae enriched in CS were prepared as described previously (26) from rabbit skeletal muscles. Protein concentration was determined according to Lowry et al. (15).
SDS-PAGE and Western Blot
SDS-PAGE on 10% acrylamide gels and immunoblot, with anti-CS or anti-HA1 antibodies, were carried out as previously described (19).Materials
DMEM and complements were purchased from Technogenetics (Milan, Italy), Waymouth's MB from ICN, and DNA modification and restriction enzymes from Boehringer Mannheim and New England Biolabs, except T7 DNA polymerase, which was purchased from Pharmacia. All other chemicals were obtained from Sigma Chemical. ![]() |
RESULTS |
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Construction of the CS-HA1Glu-Asp cDNA
Orientation, sequence, and restriction map of the "synthetic"
part of the cDNA were determined by sequencing. Sequence experiments indicate that 1) the synthetic CS
cDNA fragment was correctly fused to the
EcoR I site and the whole cDNA was an
uninterrupted open reading frame, 2)
no additional restriction sites were created by the procedure (e.g.,
due to partial digestion with EcoR I), 3) the sequence of the CS cDNA
corresponded to the original skeletal muscle CS cDNA sequence, and
4) the 3' end of the CS cDNA
had been modified by addition of the tag (HA1) coding sequence and by
deletion of the last 14 amino acid residues. The complete cDNA was
transferred, by directional cloning, downstream from the CMV promoter
of the eucaryotic expression vector pcDNA3, suitable for transient and
stable transfection in eucaryotic cells. The resulting construct
(called pCS-HA1Glu-Asp) included the coding regions for the leader
sequence, the COOH-terminal-deleted CS, the HA1 tag, and the final stop codon.
Thus the construct was useful for transfection and expression of a mutant chimeric CS, immunologically distinguishable from endogenous CS, and suitable to test the role of the COOH-terminal acidic tail in the segregation mechanism of CS.
CS-HA1Glu-Asp Expression in Transiently Transfected
HeLa Cells: Recognition by Anti-CS and Anti-HA1 Antibodies
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The epifluorescence pattern obtained with both antibodies demonstrated
that CS-HA1Glu-Asp was retained into the endomembrane network of
HeLa cells (Fig. 1, A and
B) and did not have a cytoplasmic distribution.
Compartmentalization of CS-HA1Glu-Asp in
SR/ER Membranes of Rat Myotubes in Double-Labeling Experiments
Expression of CS-HA1Glu-Asp was detected in ~20% of rat myotubes
by anti-HA1 antibodies, whereas almost all myotubes are CS positive, as
indicated by reactivity with anti-CS antibodies. The immunofluoresence
pattern was similar with anti-CS antibodies or anti-HA1 antibodies,
i.e., fluorescent strands running parallel to the longitudinal axis of
the myotubes, suggesting the longitudinal arrangement of the ER/SR
membrane network (cf. Ref. 13). Under the
prevailing experimental conditions, discrete CS foci were rarely observed.
Double-labeled transfected myotubes were observed by immunofluoresence:
similar patterns of fluorescence were obtained with anti-CS or anti-HA1
antibodies (cf. Fig. 2,
A and
B). Merge of the two images showed
overlap of the antibody reactivity (Fig. 2C); on the contrary, no red regions
(corresponding to the HA1 epitope) were observed. Thus transfected
myotubes display complete colocalization of recombinant
CS-HA1Glu-Asp with endogenous CS, whereas nontransfected myotubes
express only endogenous CS (green myotubes in Fig. 2,
C and
D).
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Detection of CS-HA1Glu-Asp in Homogenates Derived
From HeLa Cells or Rat Myotubes
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The results also indicate that the epitope(s) recognized by either antibody was within the recombinant protein. Moreover, no proteolytic breakdown products could be detected, and this rules out the possibility that the chimeric protein, as it may happen (6, 20), undergoes accelerated or altered turnover, which, in turn, may result in misleading interpretation of immunofluorescence data.
Targeting of CS-HA1Glu-Asp in Regenerating Skeletal
Muscle Fibers of Adult Rats
Figure 4 shows that, 10 days after
bupivacaine treatment, all skeletal muscle fibers were labeled with
anti-CS antibodies (Fig. 4A),
whereas only a few fibers, as expected, were labeled with anti-HA1
antibodies (Fig. 4B), as judged, in
both cases, by immunofluorescence of transverse sections. Thus a few
fibers express the recombinant CS-HA1Glu-Asp.
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Localization of CS-HA1Glu-Asp was thoroughly investigated by
immunofluorescence of double-labeled longitudinal sections of soleus
muscle fibers. Figure 5,
A (anti-CS antibodies) and
B (anti-HA1 antibodies), shows that
CS-HA1
Glu-Asp was indeed localized at the A-I interface, as
indicated by the typical, regular banding pattern of punctate
fluorescence, i.e., two rows of triads on either side of the Z line.
Merge images (Fig. 5C) clearly
indicate colocalization of endogenous and recombinant CS-HA1
Glu-Asp
at the TC level.
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Targeting of CS-HA149COOH
in Regenerating Skeletal Muscle Fibers of Adult Rats
Localization of CS-HA149COOH
was investigated by immunofluorescence of double-labeled longitudinal
sections of regenerating and transfected soleus muscle fibers. Figure
6, A
(anti-CS antibodies) and B (anti-HA1
antibodies), clearly shows that
CS-HA1
49COOH displayed a
regular pattern of punctate fluorescence, as implied by two contiguous
bands of punctate labeling localized at the A-I interface.
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DISCUSSION |
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Targeting of CS includes sorting, retention to the SR, and segregation to the jSR of skeletal muscle. The rationale of experiments reported here is that by comparing the intracellular routing and subcellular localization of wild, chimeric, or mutant/chimeric CSs that can be secreted, retained but not segregated, or retained and segregated to restricted membrane compartments, i.e., jSR, information is gathered regarding the intrinsic targeting mechanism(s) of CS.
Two deletion mutant cDNA clones, CS-HA1Glu-Asp and
CS-HA1
49COOH, have been
designed and characterized to verify one of the putative targeting
mechanisms of CS to the jSR; such a mechanism would involve
electrostatic interactions between acidic domains of CS and basic
luminal domains of jSR integral proteins, e.g., TD, JC, and possibly
RyR. The two deletion mutants differ in the extent of deletion at the
COOH terminus, 14 vs. 49 amino acid residues.
The first recombinant CS, CS-HA1Glu-Asp, was engineered so that
1) intracellular routing and
subcellular localization could be monitored by specific antibodies
directed to the chimeric tag HA1 and
2) a 42-bp fragment was deleted at
the 3' end of the coding region to investigate the specific role
of the COOH-terminal tail, made of 14 acidic amino acids
[-Glu-(Asp)5-Glu-(Asp)7-],
in the electrostatic interaction(s) with intraluminal basic domains of jSR proteins.
The present results show that the mutant CS-HA1Glu-Asp is
1) sorted to endomembrane
compartments in HeLa cells (Fig. 1), 2) sorted and retained to the SR of
differentiating rat myotubes (Fig. 2), and
3) segregated to the jSR of skeletal
muscle fibers (Fig. 5) after in vivo transfection of the recombinant cDNA.
Clear-cut data are derived from in vivo transfection of
CS-HA1Glu-Asp cDNA into regenerating skeletal muscle fibers of adult rats (Figs. 4 and 5): mutant CS is not only sorted and retained to the
SR but, more importantly, also segregates to TC. Expression of
CS-HA1
Glu-Asp during muscle regeneration, i.e., during SR biogenesis, TC development, and triad formation, indicates that deletion of the COOH-terminal acidic tail does not affect CS
segregation to the jSR. Thus not only is the free COOH terminus of CS
not required for sorting, retention, and segregation to the jSR (18), but also the COOH-terminal acidic tail is not, apparently, involved in
protein-protein electrostatic interactions.
Franzini-Armstrong et al. (5), on the basis of freeze-fracture studies of skeletal muscle triads, described a network of CS aggregates in the jSR lumen as being constituted by long fiber structures, which are fully compatible with the Ca2+-induced linear polymers or multimer of dimers of CS recently described by Wang et al. (27). The present results indicate that deletion of the CS tail does not appear to interfere with dimerization and polymerization of CS, in agreement with crystal structure data (27).
If multiple protein-protein interactions were envisioned to account for
CS targeting and if the CS COOH terminus were indeed involved, one
alternative interpretation of our results would be that
CS-HA1Glu-Asp segregates, despite the "short" CS tail deletion. Because the last 49 amino acid residues of the COOH terminus
are mostly acidic, i.e., 42% Glu-Asp (3), it might be that the
short deletion is not sufficient to abolish the
interaction of CS with the anchoring protein(s). To test the effects of
a longer deletion at the COOH terminus, a second deletion mutant CS
cDNA, CS-HA1
49COOH cDNA, was
developed and transfected in vivo into regenerating skeletal muscle
fibers of adult rats. Because CS-HA1
49COOH is segregated to
TC (Fig. 6), it might be safely concluded that the acid COOH terminus,
as a whole, does not affect CS targeting.
Segregation of CS to jSR may be a complex mechanism and entail docking,
i.e., heterologous protein-protein interactions, and self-aggregation
of CS, i.e., homologous protein-protein interactions. Intra- and/or
intermolecular interactions between the SAH and DBH sites on CS have
been proposed as crucial for
Ca2+-induced folding (10) and
self-aggregation (10, 27). If we assume, despite evidence to the
contrary (present results and Ref. 27), that the COOH-terminal acidic
tail were essential for segregation to the jSR, the presence of
endogenous CS (wtCS) in myotubes and regenerating muscle fibers (Figs.
2, 5, and 6) might lead to erroneous interpretations. Mixed
(CS-HA1Glu-Asp/wtCS or
CS-HA1
49COOH/wtCS) aggregates
could still form, whereas the acidic tails of wtCS might interact with
jSR anchoring proteins. In vitro and in vivo experimental systems are
being developed to address directly the latter issue.
Overall, the present results are in agreement with recent crystal structure data (27): Ca2+-induced CS "dimerization interface forms a large pocket lined with many acidic residues. The disordered C-terminal segments" (residues 352-367) "create a very electronegative enclosure within this pocket." Thus it appears that the mostly acidic COOH terminus, on the basis of not only present data with two different deletion mutants but also distinct structural considerations (27), is not involved in homologous and heterologous protein-protein interaction but in Ca2+ binding.
Finally, we note that the targeting mechanism of CS, based on electrostatic interactions, may still hold true: it is entirely plausible that other CS domains are involved, and among them are to be listed acidic stretches located on the surface of each of the three topological domains (2, 27), the switch points of domain II and III (around residues 228-229), rich in acidic amino acids (27), and the NH2 terminus of CS (8, 27, 29).
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ACKNOWLEDGEMENTS |
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We thank Dr. F. Patiri for help in some immunofluorescence experiments.
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FOOTNOTES |
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This work was supported by Telethon, Italy, Grant 669 and by funds from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (1997, Programma di ricerca di rilevante interesse nazionale on "Biopatologia della fibra muscolare scheletrica").
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Volpe, Dept. di Scienze Biomediche Sperimentali, Università degli Studi di Padova, viale G. Colombo 3, 35121 Padua, Italy (E-mail: volpe{at}civ.bio.unipd.it).
Received 28 December 1998; accepted in final form 16 July 1999.
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