Proc. Natd. Acad. Sci. USA Vol. 89, pp. 6142-6146, July 1992 Cell Biology
The endoplasmic reticulum-sarcoplasmic reticulum connection: Distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers POMPEO VOLPE*, ANTONELLO VILLAt, PAOLA PODINIt, ADELINA MARTINI*, ALESSANDRA NOIW*, MARIA CARLA PANZERIt, AND JACOPO MELDOLESIti *Consiglio Nazionale delle Ricerche, Center of Muscle Biology and Physiopathology, Institute of General Pathology, University of Padva, Padva, Italy; and
tConsiglio Nazionale delle Ricerche, Cytopharmacology and B. Ceccareili Centers, Department of Pharmacology and S.Raffaele Institute, University of Milan, Milan, Italy
Communicated by George E. Palade, March 27, 1992
cialization contrasts with the wide spectrum of activities typical of the ER. Recently, a group of ER lumenal resident proteins, which include at their C terminus a tetrapeptide motif, KDEL, and a few variants, has been identified. During their lifespan these proteins are transported to a pre-Golgi compartment, from which, however, they are retrieved to the ER after binding to a specific KDEL receptor (8). Of the SR lumenal proteins, CS (9) and other components-sarcalumenin (10, 11), 53-kDa glycoprotein (10, 11), histidine-rich protein (12)-were found to lack the KDEL terminus. This, however, is not the case with two additional minor proteins, originally described as the high-affinity Ca2+ binding protein and the thyroid hormone binding protein and now recognized as calreticulin and protein disulfide isomerase (PDI), respectively (13, 14). Neither of these proteins is muscle specific; rather, they are both expressed by many (possibly all) nonmuscle cells (15, 16). The latter results appear compatible with the interpretation of the SR as a specialized subcompartment of the ER. The available information is, however, still limited. In fact, we do not know whether the SR contains the entire complement of ER lumenal proteins, whether these proteins are distributed to the entire SR lumen or concentrated within discrete areas, and whether expression of ER markers in the SR concerns also the limiting membrane. These problems have now been investigated by parallel experiments of subcellular fractionation and immunocytochemistry, using antibodies (Abs) against yet another ER lumenal protein, BiP, and against a group of ER membrane proteins. These proteins were found to be present and variously distributed in the skeletal muscle SR. Thus our work not only provides support to the interpretation of the SR as a specialized ER subcompartment but in addition reveals new aspects of the complex organization and regulatory mechanisms in this endomembrane system. MATERIALS AND METHODS The following skeletal muscles were dissected from animals of various species and transferred to ice-cold saline solutions: rabbit, fast-twitch adductor and slow-twitch soleus; rat, extensor digitorum longus; chicken, pectoralis major. Subellular Fractionation. The muscles were homogenized, and the whole SR fraction was isolated by differential centrifugation and processed according to Saito et al. (17) to yield various subfractions. Two of these subfractions are highly
The skeletal muscle sarcoplasmic redculum ABSTRACT (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BIP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fraction revealed the presence of BIP in fast- and slow-twitch muscles of the rabbit as well as in rat and chiken muscles. Analyses of purified SR sub s, together with cryosectlon Immunofluorescence and Immunogold labeling, revealed BIP evenly distributed within the ni SR and the teria cisternae. Within the ter l csternae BiP appeared not to be mixed with calsequestrin but to be distributed around the ggregates of the latter Ca+ binding protein. Of the various membrane markers only cainexin (91 kDa) was found to be distributed within both SR sub ous, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a s i ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The dfferential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system. The sarcoplasmic reticulum (SR) of skeletal muscle has attracted interest as to its biogenesis and cytological nature during the last 35 years (1, 2). On the one hand, extensive membrane continuities, suggestive of a direct biogenetic relationship, between the growing SR and typical roughsurfaced endoplasmic reticulum (ER) cisternae were observed during differentiation (3, 4). On the other hand, protein analyses of isolated subcellular fractions accounting for either the whole system or its two major components, longitudinal SR and terminal cisternae (LSR and TC, respectively), revealed a high degree of specialization (2, 5), quite distinct from the heterogeneous patterns observed with ER fractions. In particular, LSR was found to be massively (=90%) enriched in the Ca2+-ATPase and TC in a peculiar, low-affinity, high-capacity intralumenal Ca2+ binding protein, calsequestrin (CS). Moreover, a subfraction corresponding to the junctional face membrane (JFM), the TC membrane associated with the transverse tubules at the triads (6), was enriched in the SR Ca2+ channel, the so-called ryanodine receptor (2, 6, 7). The identification of these and additional minor SR components, which appear to be also involved in Ca2+ homeostasis (5), documented the key role of the SR in the processes of Ca2+ uptake, storage, and release underlying the relaxation-contraction cycle. This spe-
Abbreviations: Ab, antibody; CS, calsequestrin; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; JFM, junctional face membrane of SR terminal cisternae; JFM-CC, junctional facecompartmental contents subfraction; LSR, longitudinal SR; PDI, protein disulfide isomerase; TC, terminal cisternae of the SR. should be addressed at: Department of flTo whom reprint requests Institute S.Raffaele, Via Olgettina, 60, Pharmacology, Scientific 20132 Milan, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6142
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Biology: Volpe et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
enriched of LSR and TC, respectively (17). The TC subfraction was further processed to separate its various components. A preparation containing JFM with associated compartmental content (JFM-CC) was recovered by high-speed centrifugation from the TC subfraction exposed to 0.7% Triton X-100; the subsequent exposure of JFM-CC to 1 mM EDTA resulted in CS extraction and recovery of JFM (6). Total TC limiting membrane and the lumenal content were separated by treatment with Tris/EDTA (pH 8.3) as described by Duggan and Martonosi (18). Protein concentration of the fractions was estimated by Lowry's method, using bovine serum albumin standards. SDS/PAGE was carried out according to Laemmli (19). In a few experiments the SR fractions were run in parallel with microsomes prepared from either the chicken or the rat cerebellum (20, 21). Electrotransfer of the separated protein bands to nitrocellulose sheets and Western blotting were carried out as described (20), using either alkaline phosphatase (BiP) or 125I-labeled protein A (membrane proteins) for visualization. Immunofluorescence and Immunogold Labeling. For the morphological studies, strips of tissue dissected from the rabbit adductor and soleus muscles were stretched, pinned down over a vax sheet, and then fixed for 2 hr at room temperature with either 4% formaldehyde/0.25% glutaraldehyde in phosphate buffer, followed by 2% OS04 in the same buffer, for conventional thin-section electron microscopy, or with the formaldehyde/glutaraldehyde mixture alone, for immunofluorescence and immunogold labeling. For the latter studies (see ref. 21) the fixed samples were infiltrated with sucrose, frozen in a 3:1 mixture of propane/cyclopentane cooled with liquid nitrogen, and sectioned in a Reichert Ultracut ultramicrotome equipped with a FC4 apparatus. One-micrometer-thick cryosections were immunolabeled with Abs against either BiP, ER, or CS, followed by the appropriate rhodamine-labeled goat Abs (21). Controls were carried out either by using a nonimmune serum or by omitting the first Ab treatment. In the immunogold experiments the cryosections were =50 nm thick. For single labeling these cryosections were exposed to either one of the above Abs, washed, and then decorated with 5-nm gold particles coated with goat IgG against either rabbit or rat IgG. For dual labeling, the rabbit anti-CS and the rat anti-BiP Abs were incubated together, and the same procedure was then followed with appropriately coated 5- and 15-nm gold particles. In contrast, the Abs against CS and ER (both raised in the rabbit) and the corresponding gold particles were applied in A
6143
sequence (20, 21). Extensively washed single- and duallabeled cryosections were finally postfixed, stained, and embedded as recommended by Keller et al. (22). Background labeling was estimated by studying parallel preparations (processed by omitting the exposure to specific Abs) and analyzing organelles and structures (e.g., mitochondria) negative for those Abs in the immunodecorated cryosections. Materials. The primary Abs used in this work have been described elsewhere: anti-BiP, a rat monoclonal Ab (23), was the kind gift of D. G. Bole; anti-ER, a rabbit polyclonal Ab raised against rat liver rough-surfaced ER vesicles stripped of their ribosomes (24, 25), was the kind gift of D. Louvard; anti-CS was a rabbit polyclonal Ab (see ref. 26). Rhodaminelabeled goat anti-rabbit and anti-rat IgGs were purchased from Technogenetics, Milan, Italy; 5- and 15-nm gold particles coated with similar IgGs were from Biocell Laboratories. The chemicals were reagent grade, purchased from Sigma.
RESULTS The Abs herewith employed were extensively characterized in previous studies and found to recognize either a single (antiBiP and anti-CS) or various (anti-ER) proteins (23, 24, 26). These results have been confirmed using microsomal fractions from various cell origins (refs. 20 and 25; unpublished results). Subcellular Fractionation. Fig. 1A compares BiPimmunolabeled Western blots of microsomes from a nonmuscle source, the chicken cerebellum (lane a), and a total SR fraction from the rabbit fast-twitch adductor muscle (lane b). Notice the single band at the expected molecular mass of 78 kDa in both preparations (23). The isolated rabbit skeletal muscle SR was subfractionated according to Saito et al. (17) and Costello et al. (6) to yield well-characterized subfractions containing LSR, TC, and JFM-CC. In separate experiments the TC subfraction was treated with a Tris/EDTA solution (pH 8.3) to release most of the intralumenal SR content (18). In Fig. 1B the distribution of BiP among the various subfractions [immunolabeled in the Western blot (lower panel) and identified in the same blot, stained however with Ponceau red (upper panel) by matching with the Western blot] is compared to that of the SR major proteins, Ca2+-ATPase and CS. As can be seen, distinct, BiP-positive bands were present in the LSR- and TC-enriched subfractions (Fig. 1B, lanes a and b). As expected from previous studies (17), both of these subfiactions were enriched in the Ca2+-ATPase, and the second was also enriched in CS (upper panel). When TC was exposed to low detergent, a C
B ATPase..41,e __
/ ~-
0-
BiPL-uM
w _..
_
~ ~ ~ ~ ~ ~- .4
.AP-CS -.-
O--
BiP k
4ki
BiP_.
'K._
.0-
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b
.............
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--
d
,
e
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_
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c d
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FIG. 1. Distribution of BiP in skeletal muscle SR fractions. (A) SDS/PAGE (5-10%o linear gradient) and Western blotting with anti-BiP Abs were carried out as described in the text. Loading was with 100 ,ug of protein per lane. Lane a, chicken cerebellum microsomes; lane b, rabbit fast-twitch adductor muscle SR. (B) Rabbit adductor SR subfractions. The same blot is shown stained with Ponceau red (upper panel) and immunolabeled with anti-BiP Ab (lower panel). SDS/PAGE was on 10%o gels; loading was with 50 ,ug of protein per lane. Lane a, LSR; lane b, TC; lane c, JFM-CC; lanes d and e, TC membranes and intralumenal content after Tris/EDTA incubation, respectively. The positions of Ca2+-ATPase, CS, and BiP are marked. (C) Protein loading, SDS/PAGE, and Western blotting as A. Lane a, rabbit adductor (fast-twitch muscle) SR; lane b, rabbit soleus (slow-twitch muscle) SR; lane c, rat extensor digitorum longus SR; lane d, chicken pectoralis major SR. Small arrows to the right ofthe blots indicate the positions of molecular mass standards (Bio-Rad; from the top): myosin heavy chain, 200 kDa; ,-galactosidase,
116.25 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa. In A and C, all of the standards are indicated; in B (lower panel), only the three intermediate are indicated.
6144
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Volpe et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
treatment that spares JFM and the segregated content but solubilizes the other membranes of the fraction (6), most BiP remained together with CS in the particulate subfraction (JFM-CC), whereas most Ca2+-ATPase was solubilized (lane c). With the same treatment followed by 1 mM EDTA, which solubilized the CS content, BiP was solubilized to only =50%o and the rest remained in the JFM subfraction (not shown). Likewise, when total TC was exposed to the alkaline EDTA wash (18), =50%o of BiP was released (together with the bulk of CS, lane e), whereas the rest remained with the membranes (lane d). By Western blotting of two-dimensional gels (not shown), the isoelectric point of the SR BiP was found to be around 4.7, as reported for this protein in other cell types (27). Finally, the Western blot of Fig. 1C shows that BiP occurs in the SR fractions obtained not only from the adductor (lane a) but also from another muscle of the rabbit, the slow-twitch soleus (lane b), as well as from muscles of other species-the rat, where the 78-kDa band was accompanied by a smaller band at =82 kDa (lane c); and the chicken (lane d). Thus, SR expression of BiP is widespread and possibly general. Fig. 2 illustrates results obtained with the anti-ER Ab. Three bands were labeled in Western blots of rat cerebellar microsomes: a major band at 91 kDa [recently named calnexin (28)], another band at 64 kDa, and a faint component at 29 kDa (Fig. 2, lane a). In the rabbit muscle SR (fast-twitch adductor, Fig. 2, lane b, and slow-twitch soleus, not shown) the major positive band was again calnexin, which appeared diffuse because of its incomplete separation from the Ca2+ATPase band. Additional ER-positive bands were hardly visible in the blots of the total SR fraction (Fig. 2, lane b). When the SR was subfractionated, calnexin was found to be distributed to LSR and TC and recovered also in JFM-CC (lanes c-e). In the latter subfraction, as well as in TC, additional ER-positive bands were also visible (Fig. 2, lanes d and e). When the TC subfraction was treated with Tris/ EDTA, the markers revealed by the Ab were recovered with the membranes (not shown). Immunofluorescence and Immunogold Labeling. Our studies were carried out on the fast-twitch adductor and the slow-twitch soleus muscles of the rabbit, with consistent results. The data shown here are therefore representative of both muscles. Fluorescence images of 1-,um-thick cryosections immunolabeled with the anti-BiP and anti-ER Abs are compared in Fig. 3 with parallel images obtained with the anti-CS Ab (Fig. 3 A, B, and C, respectively). In agreement with previous results by Jorgensen et al. (29), the CS pattern
was found to include parallel rows of bright spots residing
roughly at the border between the I and A band, where triads are known to be located. The I band also exhibited a distinct, spotty CS positivity, whereas the A band appeared completely negative. With anti-BiP and anti-ER Abs (Fig. 3 A and B) the pattern was different. In fact, the distribution of the fluorescence was not spotty but was almost even, especially with BiP (Fig. 3A). The I band was labeled more than the A band; however, a clear positivity was observed also in the latter, particularly evident in the area including the H line, where the LSR is known to be more developed. As a whole, the A band immunofluorescence with anti-BiP and anti-ER Abs resembled that described for the Ca2+-ATPase (29). In the subplasmalemma region around nuclei, where roughsurfaced ER cisternae are known to be located, the BiP and ER signals were not stronger than in the I band (not shown). These results suggest the distribution of BiP and the ER membrane antigens to include not only TC (as it is the case with CS) but also LSR and the rough-surfaced ER cisternae. Immunofluorescence studies were complemented by highresolution immunogold labeling of ultrathin cryosections (Fig. 4). In some of these experiments labeling with either one of the marker Abs (small gold) was combined with CS labeling (large gold). As shown in Fig. 4 A and B, BiP labeling was not restricted to the CS-positive TC but occurred also over membrane-bound profiles distributed in the depth of the H
AB
I
r-li
IB I
Calnexin-_
a
b
c
d
e
FIG. 2. Distribution of antigens recognized by anti-ER Abs in SR subfractions of rabbit fast-twitch muscle. SDS/PAGE (5-15% linear gradient) and Western blotting with anti-ER Abs were carried out as described in the text. Loading was with 150 pg of protein per lane. Lane a, rat cerebellum microsomes; lane b, total SR; lane c, LSR; lane d, TC; lane e, JFM-CC of the rabbit adductor muscle. The position ofthe 91-kDa band (calnexin) is marked to the left. The small
arrows to the right indicate the positions of molecular mass standards
(Bio-Rad; from the top): phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa; bovine carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa.
FIG. 3. Immunofluorescence of rabbit soleus muscle 1-pm-thick cryosections. All panels are at the same magnification. Decoration
was with anti-BiP Ab (A), with anti-ER Ab (B), and with anti-CS Ab (C). The images in A-C have been aligned. The indications at the top of A refer therefore to all three panels. AB and IB, anisotropic (A) and isotropic (I) bands; H and Z, H and Z lines. (Bar = 8 pn.)
Cell Biology: Volpe et al.
iA.;-fw*s>
I and A bands. In the experimental conditions employed, 25 of the 102 TCs observed were labeled for BiP. Interestingly, the labeling over these structures was distributed not at random but beneath the limiting membrane, at the periphery of (in some cases around) the moderately dense content positive for CS (Fig. 4C). With anti-ER Ab, the immunolabeling distribution (Fig. 4 D and F) was similar to that of BiP; however the fraction of labeled TC (Fig. 4D) was higher (almost 40%6). Additional profiles in the I and A bands were also labeled (Fig. 4 D and E). The gold particles were preferentially localized at the lumenal side of the membrane. This observation confirms in the SR the lumenal distribution of the antigenic determinants previously reported in the ER (24, 25). Under optimal labeling conditions, control sections and the structures negative for the antigens (mitochondria, nuclei, contractile fibrils) exhibited little labeling-i.e., background was low (
The endoplasmic reticulum-sarcoplasmic reticulum connection: Distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers POMPEO VOLPE*, ANTONELLO VILLAt, PAOLA PODINIt, ADELINA MARTINI*, ALESSANDRA NOIW*, MARIA CARLA PANZERIt, AND JACOPO MELDOLESIti *Consiglio Nazionale delle Ricerche, Center of Muscle Biology and Physiopathology, Institute of General Pathology, University of Padva, Padva, Italy; and
tConsiglio Nazionale delle Ricerche, Cytopharmacology and B. Ceccareili Centers, Department of Pharmacology and S.Raffaele Institute, University of Milan, Milan, Italy
Communicated by George E. Palade, March 27, 1992
cialization contrasts with the wide spectrum of activities typical of the ER. Recently, a group of ER lumenal resident proteins, which include at their C terminus a tetrapeptide motif, KDEL, and a few variants, has been identified. During their lifespan these proteins are transported to a pre-Golgi compartment, from which, however, they are retrieved to the ER after binding to a specific KDEL receptor (8). Of the SR lumenal proteins, CS (9) and other components-sarcalumenin (10, 11), 53-kDa glycoprotein (10, 11), histidine-rich protein (12)-were found to lack the KDEL terminus. This, however, is not the case with two additional minor proteins, originally described as the high-affinity Ca2+ binding protein and the thyroid hormone binding protein and now recognized as calreticulin and protein disulfide isomerase (PDI), respectively (13, 14). Neither of these proteins is muscle specific; rather, they are both expressed by many (possibly all) nonmuscle cells (15, 16). The latter results appear compatible with the interpretation of the SR as a specialized subcompartment of the ER. The available information is, however, still limited. In fact, we do not know whether the SR contains the entire complement of ER lumenal proteins, whether these proteins are distributed to the entire SR lumen or concentrated within discrete areas, and whether expression of ER markers in the SR concerns also the limiting membrane. These problems have now been investigated by parallel experiments of subcellular fractionation and immunocytochemistry, using antibodies (Abs) against yet another ER lumenal protein, BiP, and against a group of ER membrane proteins. These proteins were found to be present and variously distributed in the skeletal muscle SR. Thus our work not only provides support to the interpretation of the SR as a specialized ER subcompartment but in addition reveals new aspects of the complex organization and regulatory mechanisms in this endomembrane system. MATERIALS AND METHODS The following skeletal muscles were dissected from animals of various species and transferred to ice-cold saline solutions: rabbit, fast-twitch adductor and slow-twitch soleus; rat, extensor digitorum longus; chicken, pectoralis major. Subellular Fractionation. The muscles were homogenized, and the whole SR fraction was isolated by differential centrifugation and processed according to Saito et al. (17) to yield various subfractions. Two of these subfractions are highly
The skeletal muscle sarcoplasmic redculum ABSTRACT (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BIP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fraction revealed the presence of BIP in fast- and slow-twitch muscles of the rabbit as well as in rat and chiken muscles. Analyses of purified SR sub s, together with cryosectlon Immunofluorescence and Immunogold labeling, revealed BIP evenly distributed within the ni SR and the teria cisternae. Within the ter l csternae BiP appeared not to be mixed with calsequestrin but to be distributed around the ggregates of the latter Ca+ binding protein. Of the various membrane markers only cainexin (91 kDa) was found to be distributed within both SR sub ous, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a s i ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The dfferential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system. The sarcoplasmic reticulum (SR) of skeletal muscle has attracted interest as to its biogenesis and cytological nature during the last 35 years (1, 2). On the one hand, extensive membrane continuities, suggestive of a direct biogenetic relationship, between the growing SR and typical roughsurfaced endoplasmic reticulum (ER) cisternae were observed during differentiation (3, 4). On the other hand, protein analyses of isolated subcellular fractions accounting for either the whole system or its two major components, longitudinal SR and terminal cisternae (LSR and TC, respectively), revealed a high degree of specialization (2, 5), quite distinct from the heterogeneous patterns observed with ER fractions. In particular, LSR was found to be massively (=90%) enriched in the Ca2+-ATPase and TC in a peculiar, low-affinity, high-capacity intralumenal Ca2+ binding protein, calsequestrin (CS). Moreover, a subfraction corresponding to the junctional face membrane (JFM), the TC membrane associated with the transverse tubules at the triads (6), was enriched in the SR Ca2+ channel, the so-called ryanodine receptor (2, 6, 7). The identification of these and additional minor SR components, which appear to be also involved in Ca2+ homeostasis (5), documented the key role of the SR in the processes of Ca2+ uptake, storage, and release underlying the relaxation-contraction cycle. This spe-
Abbreviations: Ab, antibody; CS, calsequestrin; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; JFM, junctional face membrane of SR terminal cisternae; JFM-CC, junctional facecompartmental contents subfraction; LSR, longitudinal SR; PDI, protein disulfide isomerase; TC, terminal cisternae of the SR. should be addressed at: Department of flTo whom reprint requests Institute S.Raffaele, Via Olgettina, 60, Pharmacology, Scientific 20132 Milan, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6142
Cell
Biology: Volpe et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
enriched of LSR and TC, respectively (17). The TC subfraction was further processed to separate its various components. A preparation containing JFM with associated compartmental content (JFM-CC) was recovered by high-speed centrifugation from the TC subfraction exposed to 0.7% Triton X-100; the subsequent exposure of JFM-CC to 1 mM EDTA resulted in CS extraction and recovery of JFM (6). Total TC limiting membrane and the lumenal content were separated by treatment with Tris/EDTA (pH 8.3) as described by Duggan and Martonosi (18). Protein concentration of the fractions was estimated by Lowry's method, using bovine serum albumin standards. SDS/PAGE was carried out according to Laemmli (19). In a few experiments the SR fractions were run in parallel with microsomes prepared from either the chicken or the rat cerebellum (20, 21). Electrotransfer of the separated protein bands to nitrocellulose sheets and Western blotting were carried out as described (20), using either alkaline phosphatase (BiP) or 125I-labeled protein A (membrane proteins) for visualization. Immunofluorescence and Immunogold Labeling. For the morphological studies, strips of tissue dissected from the rabbit adductor and soleus muscles were stretched, pinned down over a vax sheet, and then fixed for 2 hr at room temperature with either 4% formaldehyde/0.25% glutaraldehyde in phosphate buffer, followed by 2% OS04 in the same buffer, for conventional thin-section electron microscopy, or with the formaldehyde/glutaraldehyde mixture alone, for immunofluorescence and immunogold labeling. For the latter studies (see ref. 21) the fixed samples were infiltrated with sucrose, frozen in a 3:1 mixture of propane/cyclopentane cooled with liquid nitrogen, and sectioned in a Reichert Ultracut ultramicrotome equipped with a FC4 apparatus. One-micrometer-thick cryosections were immunolabeled with Abs against either BiP, ER, or CS, followed by the appropriate rhodamine-labeled goat Abs (21). Controls were carried out either by using a nonimmune serum or by omitting the first Ab treatment. In the immunogold experiments the cryosections were =50 nm thick. For single labeling these cryosections were exposed to either one of the above Abs, washed, and then decorated with 5-nm gold particles coated with goat IgG against either rabbit or rat IgG. For dual labeling, the rabbit anti-CS and the rat anti-BiP Abs were incubated together, and the same procedure was then followed with appropriately coated 5- and 15-nm gold particles. In contrast, the Abs against CS and ER (both raised in the rabbit) and the corresponding gold particles were applied in A
6143
sequence (20, 21). Extensively washed single- and duallabeled cryosections were finally postfixed, stained, and embedded as recommended by Keller et al. (22). Background labeling was estimated by studying parallel preparations (processed by omitting the exposure to specific Abs) and analyzing organelles and structures (e.g., mitochondria) negative for those Abs in the immunodecorated cryosections. Materials. The primary Abs used in this work have been described elsewhere: anti-BiP, a rat monoclonal Ab (23), was the kind gift of D. G. Bole; anti-ER, a rabbit polyclonal Ab raised against rat liver rough-surfaced ER vesicles stripped of their ribosomes (24, 25), was the kind gift of D. Louvard; anti-CS was a rabbit polyclonal Ab (see ref. 26). Rhodaminelabeled goat anti-rabbit and anti-rat IgGs were purchased from Technogenetics, Milan, Italy; 5- and 15-nm gold particles coated with similar IgGs were from Biocell Laboratories. The chemicals were reagent grade, purchased from Sigma.
RESULTS The Abs herewith employed were extensively characterized in previous studies and found to recognize either a single (antiBiP and anti-CS) or various (anti-ER) proteins (23, 24, 26). These results have been confirmed using microsomal fractions from various cell origins (refs. 20 and 25; unpublished results). Subcellular Fractionation. Fig. 1A compares BiPimmunolabeled Western blots of microsomes from a nonmuscle source, the chicken cerebellum (lane a), and a total SR fraction from the rabbit fast-twitch adductor muscle (lane b). Notice the single band at the expected molecular mass of 78 kDa in both preparations (23). The isolated rabbit skeletal muscle SR was subfractionated according to Saito et al. (17) and Costello et al. (6) to yield well-characterized subfractions containing LSR, TC, and JFM-CC. In separate experiments the TC subfraction was treated with a Tris/EDTA solution (pH 8.3) to release most of the intralumenal SR content (18). In Fig. 1B the distribution of BiP among the various subfractions [immunolabeled in the Western blot (lower panel) and identified in the same blot, stained however with Ponceau red (upper panel) by matching with the Western blot] is compared to that of the SR major proteins, Ca2+-ATPase and CS. As can be seen, distinct, BiP-positive bands were present in the LSR- and TC-enriched subfractions (Fig. 1B, lanes a and b). As expected from previous studies (17), both of these subfiactions were enriched in the Ca2+-ATPase, and the second was also enriched in CS (upper panel). When TC was exposed to low detergent, a C
B ATPase..41,e __
/ ~-
0-
BiPL-uM
w _..
_
~ ~ ~ ~ ~ ~- .4
.AP-CS -.-
O--
BiP k
4ki
BiP_.
'K._
.0-
a a
b
.............
c
--
d
,
e
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_
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a b
c d
b
FIG. 1. Distribution of BiP in skeletal muscle SR fractions. (A) SDS/PAGE (5-10%o linear gradient) and Western blotting with anti-BiP Abs were carried out as described in the text. Loading was with 100 ,ug of protein per lane. Lane a, chicken cerebellum microsomes; lane b, rabbit fast-twitch adductor muscle SR. (B) Rabbit adductor SR subfractions. The same blot is shown stained with Ponceau red (upper panel) and immunolabeled with anti-BiP Ab (lower panel). SDS/PAGE was on 10%o gels; loading was with 50 ,ug of protein per lane. Lane a, LSR; lane b, TC; lane c, JFM-CC; lanes d and e, TC membranes and intralumenal content after Tris/EDTA incubation, respectively. The positions of Ca2+-ATPase, CS, and BiP are marked. (C) Protein loading, SDS/PAGE, and Western blotting as A. Lane a, rabbit adductor (fast-twitch muscle) SR; lane b, rabbit soleus (slow-twitch muscle) SR; lane c, rat extensor digitorum longus SR; lane d, chicken pectoralis major SR. Small arrows to the right ofthe blots indicate the positions of molecular mass standards (Bio-Rad; from the top): myosin heavy chain, 200 kDa; ,-galactosidase,
116.25 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa. In A and C, all of the standards are indicated; in B (lower panel), only the three intermediate are indicated.
6144
Cell Biology:
Volpe et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
treatment that spares JFM and the segregated content but solubilizes the other membranes of the fraction (6), most BiP remained together with CS in the particulate subfraction (JFM-CC), whereas most Ca2+-ATPase was solubilized (lane c). With the same treatment followed by 1 mM EDTA, which solubilized the CS content, BiP was solubilized to only =50%o and the rest remained in the JFM subfraction (not shown). Likewise, when total TC was exposed to the alkaline EDTA wash (18), =50%o of BiP was released (together with the bulk of CS, lane e), whereas the rest remained with the membranes (lane d). By Western blotting of two-dimensional gels (not shown), the isoelectric point of the SR BiP was found to be around 4.7, as reported for this protein in other cell types (27). Finally, the Western blot of Fig. 1C shows that BiP occurs in the SR fractions obtained not only from the adductor (lane a) but also from another muscle of the rabbit, the slow-twitch soleus (lane b), as well as from muscles of other species-the rat, where the 78-kDa band was accompanied by a smaller band at =82 kDa (lane c); and the chicken (lane d). Thus, SR expression of BiP is widespread and possibly general. Fig. 2 illustrates results obtained with the anti-ER Ab. Three bands were labeled in Western blots of rat cerebellar microsomes: a major band at 91 kDa [recently named calnexin (28)], another band at 64 kDa, and a faint component at 29 kDa (Fig. 2, lane a). In the rabbit muscle SR (fast-twitch adductor, Fig. 2, lane b, and slow-twitch soleus, not shown) the major positive band was again calnexin, which appeared diffuse because of its incomplete separation from the Ca2+ATPase band. Additional ER-positive bands were hardly visible in the blots of the total SR fraction (Fig. 2, lane b). When the SR was subfractionated, calnexin was found to be distributed to LSR and TC and recovered also in JFM-CC (lanes c-e). In the latter subfraction, as well as in TC, additional ER-positive bands were also visible (Fig. 2, lanes d and e). When the TC subfraction was treated with Tris/ EDTA, the markers revealed by the Ab were recovered with the membranes (not shown). Immunofluorescence and Immunogold Labeling. Our studies were carried out on the fast-twitch adductor and the slow-twitch soleus muscles of the rabbit, with consistent results. The data shown here are therefore representative of both muscles. Fluorescence images of 1-,um-thick cryosections immunolabeled with the anti-BiP and anti-ER Abs are compared in Fig. 3 with parallel images obtained with the anti-CS Ab (Fig. 3 A, B, and C, respectively). In agreement with previous results by Jorgensen et al. (29), the CS pattern
was found to include parallel rows of bright spots residing
roughly at the border between the I and A band, where triads are known to be located. The I band also exhibited a distinct, spotty CS positivity, whereas the A band appeared completely negative. With anti-BiP and anti-ER Abs (Fig. 3 A and B) the pattern was different. In fact, the distribution of the fluorescence was not spotty but was almost even, especially with BiP (Fig. 3A). The I band was labeled more than the A band; however, a clear positivity was observed also in the latter, particularly evident in the area including the H line, where the LSR is known to be more developed. As a whole, the A band immunofluorescence with anti-BiP and anti-ER Abs resembled that described for the Ca2+-ATPase (29). In the subplasmalemma region around nuclei, where roughsurfaced ER cisternae are known to be located, the BiP and ER signals were not stronger than in the I band (not shown). These results suggest the distribution of BiP and the ER membrane antigens to include not only TC (as it is the case with CS) but also LSR and the rough-surfaced ER cisternae. Immunofluorescence studies were complemented by highresolution immunogold labeling of ultrathin cryosections (Fig. 4). In some of these experiments labeling with either one of the marker Abs (small gold) was combined with CS labeling (large gold). As shown in Fig. 4 A and B, BiP labeling was not restricted to the CS-positive TC but occurred also over membrane-bound profiles distributed in the depth of the H
AB
I
r-li
IB I
Calnexin-_
a
b
c
d
e
FIG. 2. Distribution of antigens recognized by anti-ER Abs in SR subfractions of rabbit fast-twitch muscle. SDS/PAGE (5-15% linear gradient) and Western blotting with anti-ER Abs were carried out as described in the text. Loading was with 150 pg of protein per lane. Lane a, rat cerebellum microsomes; lane b, total SR; lane c, LSR; lane d, TC; lane e, JFM-CC of the rabbit adductor muscle. The position ofthe 91-kDa band (calnexin) is marked to the left. The small
arrows to the right indicate the positions of molecular mass standards
(Bio-Rad; from the top): phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa; bovine carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa.
FIG. 3. Immunofluorescence of rabbit soleus muscle 1-pm-thick cryosections. All panels are at the same magnification. Decoration
was with anti-BiP Ab (A), with anti-ER Ab (B), and with anti-CS Ab (C). The images in A-C have been aligned. The indications at the top of A refer therefore to all three panels. AB and IB, anisotropic (A) and isotropic (I) bands; H and Z, H and Z lines. (Bar = 8 pn.)
Cell Biology: Volpe et al.
iA.;-fw*s>
I and A bands. In the experimental conditions employed, 25 of the 102 TCs observed were labeled for BiP. Interestingly, the labeling over these structures was distributed not at random but beneath the limiting membrane, at the periphery of (in some cases around) the moderately dense content positive for CS (Fig. 4C). With anti-ER Ab, the immunolabeling distribution (Fig. 4 D and F) was similar to that of BiP; however the fraction of labeled TC (Fig. 4D) was higher (almost 40%6). Additional profiles in the I and A bands were also labeled (Fig. 4 D and E). The gold particles were preferentially localized at the lumenal side of the membrane. This observation confirms in the SR the lumenal distribution of the antigenic determinants previously reported in the ER (24, 25). Under optimal labeling conditions, control sections and the structures negative for the antigens (mitochondria, nuclei, contractile fibrils) exhibited little labeling-i.e., background was low (
