Proc. Natd. Acad. Sci. USA Vol. 89, pp. 6142-6146, July 1992 Cell Biology The - 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

ABSTRACT The skeletal muscle sarcoplasmic redculum cialization contrasts with the wide spectrum of activities (SR) was investigated for the presence of well-known endo- typical of the ER. plasmic reticulum (ER) markers: the lumenal BIP and Recently, a group of ER lumenal resident , which a group of membrane proteins recognized by an antibody include at their C terminus a tetrapeptide motif, KDEL, and raised against ER membrane vesicles. Western blots of SR afew variants, has been identified. During their lifespan these fraction revealed the presence of BIP in fast- and slow-twitch proteins are transported to a pre-Golgi compartment, from muscles of the rabbit as well as in rat and chiken muscles. which, however, they are retrieved to the ER after binding to Analyses ofpurified SR sub s, together with cryosectlon a specific KDEL receptor (8). Ofthe SR lumenal proteins, CS Immunofluorescence and Immunogold labeling, revealed BIP (9) and other components- (10, 11), 53-kDa evenly distributed within the ni SR and the teria (10, 11), histidine-rich protein (12)-were found cisternae. Within the ter l csternae BiP appeared not to be to lack the KDEL terminus. This, however, is not the case mixed with but to be distributed around the with two additional minor proteins, originally described as ggregates of the latter Ca+ binding protein. Of the various the high-affinity Ca2+ binding protein and the thyroid hor- membrane markers only cainexin (91 kDa) was found to be mone binding protein and now recognized as and distributed within both SR sub ous, whereas the other protein disulfide isomerase (PDI), respectively (13, 14). Nei- markers (apparent molecular masses of64 kDa and 58 kDa and ther ofthese proteins is muscle specific; rather, they are both a doublet around 28 kDa) were concentrated in the terminal expressed by many (possibly all) nonmuscle cells (15, 16). that the SR is a s i ER The latter results appear compatible with the interpretation of cisternae. These results suggest the SR as a specialized subcompartment of the ER. The subcompartment in which general markers, such as the ones we available information is, however, still limited. In fact, we do have investigated, coexist with the major SR proteins specifi- not know whether the SR contains the entire complement of cally responsible for Ca2+ uptake, storage, and release. The ER lumenal proteins, whether these proteins are distributed dfferential distribution of the ER markers reveals new aspects to the entire SR lumen or concentrated within discrete areas, of the SR molecular structure that might be of importance for and whether expression of ER markers in the SR concerns the functioning of the endomembrane system. also the limiting membrane. These problems have now been investigated by parallel The sarcoplasmic reticulum (SR) of skeletal muscle has at- experiments of subcellular fractionation and immunocyto- tracted interest as to its biogenesis and cytological nature chemistry, using antibodies (Abs) against yet another ER during the last 35 years (1, 2). On the one hand, extensive lumenal protein, BiP, and against a group of ER membrane membrane continuities, suggestive of a direct biogenetic re- proteins. These proteins were found to be present and lationship, between the growing SR and typical rough- variously distributed in the skeletal muscle SR. Thus our surfaced endoplasmic reticulum (ER) cisternae were observed work not only provides support to the interpretation ofthe SR during differentiation (3, 4). On the other hand, protein anal- as a specialized ER subcompartment but in addition reveals yses of isolated subcellular fractions accounting for either the new aspects of the complex organization and regulatory whole system or its two major components, longitudinal SR mechanisms in this endomembrane system. and terminal cisternae (LSR and TC, respectively), revealed a high degree of specialization (2, 5), quite distinct from the MATERIALS AND METHODS heterogeneous patterns observed with ER fractions. In par- The following skeletal muscles were dissected from animals ticular, LSR was found to be massively (=90%) enriched in the ofvarious species and transferred to ice-cold saline solutions: Ca2+-ATPase and TC in a peculiar, low-affinity, high-capacity rabbit, fast-twitch adductor and slow-twitch soleus; rat, intralumenal Ca2+ binding protein, calsequestrin (CS). More- extensor digitorum longus; chicken, pectoralis major. over, a subfraction corresponding to thejunctional face mem- Subellular Fractionation. The muscles were homogenized, brane (JFM), the TC membrane associated with the transverse and the whole SR fraction was isolated by differential cen- tubules at the triads (6), was enriched in the SR Ca2+ channel, trifugation and processed according to Saito et al. (17) to yield the so-called (2, 6, 7). The identification of various subfractions. Two of these subfractions are highly these and additional minor SR components, which appear to be also involved in Ca2+ homeostasis (5), documented the key Abbreviations: Ab, antibody; CS, calsequestrin; ER, endoplasmic role of the SR in the processes of Ca2+ uptake, storage, and reticulum; SR, sarcoplasmic reticulum; JFM, junctional face mem- release underlying the relaxation-contraction cycle. This spe- brane of SR terminal cisternae; JFM-CC, junctional face- compartmental contents subfraction; LSR, longitudinal SR; PDI, protein disulfide isomerase; TC, terminal cisternae of the SR. The publication costs ofthis article were defrayed in part by page charge flTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Pharmacology, Scientific Institute S.Raffaele, Via Olgettina, 60, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 20132 Milan, Italy. 6142 Downloaded by guest on September 27, 2021 Cell Biology: Volpe et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6143 enriched of LSR and TC, respectively (17). The TC subfrac- sequence (20, 21). Extensively washed single- and dual- tion was further processed to separate its various compo- labeled cryosections were finally postfixed, stained, and nents. A preparation containing JFM with associated com- embedded as recommended by Keller et al. (22). Background partmental content (JFM-CC) was recovered by high-speed labeling was estimated by studying parallel preparations centrifugation from the TC subfraction exposed to 0.7% (processed by omitting the exposure to specific Abs) and Triton X-100; the subsequent exposure of JFM-CC to 1 mM analyzing organelles and structures (e.g., mitochondria) neg- EDTA resulted in CS extraction and recovery of JFM (6). ative for those Abs in the immunodecorated cryosections. Total TC limiting membrane and the lumenal content were Materials. The primary Abs used in this work have been separated by treatment with Tris/EDTA (pH 8.3) as de- described elsewhere: anti-BiP, a rat monoclonal Ab (23), was scribed by Duggan and Martonosi (18). Protein concentration the kind gift of D. G. Bole; anti-ER, a rabbit polyclonal Ab of the fractions was estimated by Lowry's method, using raised against rat liver rough-surfaced ER vesicles stripped of bovine serum albumin standards. SDS/PAGE was carried their ribosomes (24, 25), was the kind gift of D. Louvard; out according to Laemmli (19). In a few experiments the SR anti-CS was a rabbit polyclonal Ab (see ref. 26). Rhodamine- fractions were run in parallel with microsomes prepared from labeled goat anti-rabbit and anti-rat IgGs were purchased either the chicken or the rat cerebellum (20, 21). Electro- from Technogenetics, Milan, Italy; 5- and 15-nm gold parti- transfer of the separated protein bands to nitrocellulose cles coated with similar IgGs were from Biocell Laboratories. sheets and Western blotting were carried out as described The chemicals were reagent grade, purchased from Sigma. (20), using either alkaline phosphatase (BiP) or 125I-labeled protein A (membrane proteins) for visualization. RESULTS Immunofluorescence and Immunogold Labeling. For the The Abs herewith employed were extensively characterized in morphological studies, strips of tissue dissected from the previous studies and found to recognize either a single (anti- rabbit adductor and soleus muscles were stretched, pinned BiP and anti-CS) or various (anti-ER) proteins (23, 24, 26). down over a vax sheet, and then fixed for 2 hr at room These results have been confirmed using microsomal fractions temperature with either 4% formaldehyde/0.25% glutaralde- from various cell origins (refs. 20 and 25; unpublished results). hyde in phosphate buffer, followed by 2% OS04 in the same Subcellular Fractionation. Fig. 1A compares BiP- buffer, for conventional thin-section electron microscopy, or immunolabeled Western blots of microsomes from a non- with the formaldehyde/glutaraldehyde mixture alone, for muscle source, the chicken cerebellum (lane a), and a total immunofluorescence and immunogold labeling. For the latter SR fraction from the rabbit fast-twitch adductor muscle (lane studies (see ref. 21) the fixed samples were infiltrated with b). Notice the single band at the expected molecular mass of sucrose, frozen in a 3:1 mixture of propane/cyclopentane 78 kDa in both preparations (23). cooled with liquid nitrogen, and sectioned in a Reichert The isolated rabbit skeletal muscle SR was subfractionated Ultracut ultramicrotome equipped with a FC4 apparatus. according to Saito et al. (17) and Costello et al. (6) to yield One-micrometer-thick cryosections were immunolabeled well-characterized subfractions containing LSR, TC, and with Abs against either BiP, ER, or CS, followed by the JFM-CC. In separate experiments the TC subfraction was appropriate rhodamine-labeled goat Abs (21). Controls were treated with a Tris/EDTA solution (pH 8.3) to release most of carried out either by using a nonimmune serum or by omitting the intralumenal SR content (18). In Fig. 1B the distribution of the first Ab treatment. In the immunogold experiments the BiP among the various subfractions [immunolabeled in the cryosections were =50 nm thick. For single labeling these Western blot (lower panel) and identified in the same blot, cryosections were exposed to either one of the above Abs, stained however with Ponceau red (upper panel) by matching washed, and then decorated with 5-nm gold particles coated with the Western blot] is compared to that of the SR major with goat IgG against either rabbit or rat IgG. For dual proteins, Ca2+-ATPase and CS. As can be seen, distinct, labeling, the rabbit anti-CS and the rat anti-BiP Abs were BiP-positive bands were present in the LSR- and TC-enriched incubated together, and the same procedure was then fol- subfractions (Fig. 1B, lanes a and b). As expected from lowed with appropriately coated 5- and 15-nm gold particles. previous studies (17), both ofthese subfiactions were enriched In contrast, the Abs against CS and ER (both raised in the in the Ca2+-ATPase, and the second was also enriched in CS rabbit) and the corresponding gold particles were applied in (upper panel). When TC was exposed to low detergent, a A B C

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a c d a b c d e b a 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 ofmolecular 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. Downloaded by guest on September 27, 2021 6144 Cell Biology: Volpe et al. Proc. Natl. Acad. Sci. USA 89 (1992) treatment that spares JFM and the segregated content but was found to include parallel rows of bright spots residing solubilizes the other membranes of the fraction (6), most BiP roughly at the border between the I and A band, where triads remained together with CS in the particulate subfraction are known to be located. The I band also exhibited a distinct, (JFM-CC), whereas most Ca2+-ATPase was solubilized (lane spotty CS positivity, whereas the A band appeared com- c). With the same treatment followed by 1 mM EDTA, which pletely negative. With anti-BiP and anti-ER Abs (Fig. 3 A and solubilized the CS content, BiP was solubilized to only =50%o B) the pattern was different. In fact, the distribution of the and the rest remained in the JFM subfraction (not shown). fluorescence was not spotty but was almost even, especially Likewise, when total TC was exposed to the alkaline EDTA with BiP (Fig. 3A). The I band was labeled more than the A wash (18), =50%o of BiP was released (together with the bulk band; however, a clear positivity was observed also in the ofCS, lane e), whereas the rest remained with the membranes latter, particularly evident in the area including the H line, (lane d). By Western blotting of two-dimensional gels (not where the LSR is known to be more developed. As a whole, shown), the isoelectric point of the SR BiP was found to be the A band immunofluorescence with anti-BiP and anti-ER around 4.7, as reported for this protein in other cell types (27). Abs resembled that described for the Ca2+-ATPase (29). In Finally, the Western blot of Fig. 1C shows that BiP occurs in the subplasmalemma region around nuclei, where rough- the SR fractions obtained not only from the adductor (lane a) surfaced ER cisternae are known to be located, the BiP and but also from another muscle of the rabbit, the slow-twitch ER signals were not stronger than in the I band (not shown). soleus (lane b), as well as from muscles of other species-the These results suggest the distribution of BiP and the ER rat, where the 78-kDa band was accompanied by a smaller membrane antigens to include not only TC (as it is the case band at =82 kDa (lane c); and the chicken (lane d). Thus, SR with CS) but also LSR and the rough-surfaced ER cisternae. expression of BiP is widespread and possibly general. Immunofluorescence studies were complemented by high- Fig. 2 illustrates results obtained with the anti-ER Ab. resolution immunogold labeling of ultrathin cryosections Three bands were labeled in Western blots of rat cerebellar (Fig. 4). In some ofthese experiments labeling with eitherone microsomes: a major band at 91 kDa [recently named cal- of the marker Abs (small gold) was combined with CS nexin (28)], another band at 64 kDa, and a faint component As shown in 4 A and BiP at 29 kDa (Fig. 2, lane a). In the rabbit muscle SR (fast-twitch labeling (large gold). Fig. B, labeling adductor, Fig. 2, lane b, and slow-twitch soleus, not shown) was not restricted to the CS-positive TC but occurred also the major positive band was again , which appeared over membrane-bound profiles distributed in the depth ofthe diffuse because of its incomplete separation from the Ca2+- H I ATPase band. Additional ER-positive bands were hardly AB IB visible in the blots of the total SR fraction (Fig. 2, lane b). r-li I 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 stud- ies 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 cryosec- tions 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

Calnexin-_

a b c d e FIG. 2. Distribution ofantigens 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 FIG. 3. Immunofluorescence of rabbit soleus muscle 1-pm-thick position ofthe 91-kDa band (calnexin) is marked to the left. The small cryosections. All panels are at the same magnification. Decoration arrows to the right indicate the positions of molecular mass standards was with anti-BiP Ab (A), with anti-ER Ab (B), and with anti-CS Ab (Bio-Rad; from the top): phosphorylase b, 97.4 kDa; bovine serum (C). The images in A-C have been aligned. The indications at the top albumin, 66.2 kDa; ovalbumin, 45 kDa; bovine carbonic anhydrase, of A refer therefore to all three panels. AB and IB, anisotropic (A) 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa. and isotropic (I) bands; H and Z, H and Z lines. (Bar = 8 pn.) Downloaded by guest on September 27, 2021 Cell Biology: Volpe et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6145

I and A bands. In the experimental conditions employed, 25 tively, but also in molecular terms. Indeed, comparison ofour of the 102 TCs observed were labeled for BiP. Interestingly, present SR blots with those ofmicrosomes from other sources the labeling over these structures was distributed not at revealed identical migration only for the major band (most random but beneath the limiting membrane, at the periphery probably calnexin) and slight differences for the others. It of (in some cases around) the moderately dense content should be emphasized, however, that in all cells so far inves- positive for CS (Fig. 4C). With anti-ER Ab, the immunola- tigated labeling with this and the anti-BiP Abs was always beling distribution (Fig. 4 D and F) was similar to that of BiP; found to be restricted to the ER (21, 23-25, 27, 28, 30, 31, however the fraction of labeled TC (Fig. 4D) was higher 33-35). The present demonstration of the corresponding an- (almost 40%6). Additional profiles in the I and A bands were tigens in the skeletal muscle SR therefore represents a direct, also labeled (Fig. 4 D and E). The gold particles were strong argument in favor ofthe ER subcompartment nature of preferentially localized at the lumenal side of the membrane. the latter system, a possibility proposed already 35 years ago This observation confirms in the SR the lumenal distribution (1), which, however, was still supported by limited experi- of the antigenic determinants previously reported in the ER mental evidence. (24, 25). Under optimal labeling conditions, control sections Taken together, our ER markers appear to be bona fide and the structures negative for the antigens (mitochondria, components of the SR. In fact, the concentration of BiP and nuclei, contractile fibrils) exhibited little labeling-i.e., back- calnexin, revealed by Western blotting, was in the same order ground was low (<2 particles perjum). ofmagnitude as that ofthe cerebellar microsomes. Moreover, the immunofluorescence signal over the muscle fiber I band, DISCUSSION which is rich in SR, was not weaker than that over the Of the Abs employed, one (anti-BiP) is known to be highly perinuclear area, where rough-surfaced ER cisternae are specific for its antigen, which appears to be expressed by all concentrated. However, the distribution ofthe various mark- cells (8, 23, 25, 27, 30-32). In contrast, the anti-ER Ab, raised ers within the SR was not uniform. In fact, only BiP and against rat liver ER membranes, is known to recognize various calnexin were found in the LSR and TC, whereas the minor antigens that might differ from cell to cell, not only quantita- membrane markers were concentrated in the TC and, par-

AB t B.l lB .~~~~~~~~~~~~4

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FIG. 4. Immunogold labeling of rabbit soleus muscle ultrathin cryosections. A-C are dually labeled for CS (large gold) and BiP (small gold). (A) I band (IB) with part of an adjacent A band (AB) at the top. The boundary between the two bands is marked by a broken line. CS immunolabeling marks the TC pairs arranged at both sides ofcross-sectioned plasmalemma transverse tubule (T) to form the triads. BiP labeling is marked by small arrows. It occurs within or in the proximity of CS-positive TCs and within other membrane-bound vesicles and tubules that may be part of the LSR. Z, Z line; M, mitochondria. (B) Transverse tubule (T) sectioned longitudinally. The TCs, identified by the labeling for CS (large gold), are also positive for BiP (small arrows): a single small gold particle is visible to the right and a row of three particles is visible to the left, at the tip of a grazingly sectioned TC extension. (C) Cross-sectioned TC heavily positive for CS in its dense content and showing BiP labeling (small arrow) at the periphery, presumably below the limiting membrane. (D and E) Single labeling with anti-ER Ab (small gold, marked by small arrows). An oblique section of a triad is shown in D. Labeling is evident over the lower TC and the small structures to the right, which might correspond to expansions of the same TC grazingly sectioned. Labeling of a LSR cisterna running obliquely in the A band is shown in E. (Bar = 0.1 ,um.) .. i *A.... ;- f *w*s > Downloaded by guest on September 27, 2021 6146 Cell Biology: Volpe et al. Proc. Natl. Acad. Sci. USA 89 (1992) ticularly, in its junctional membrane, recovered in the 4. Schiaffino, S. & Margreth, A. (1969) J. Cell Biol. 41, 855-875. JFM-CC subfraction. The distribution of BiP resembled that 5. Campbell, K. P. (1986) in Sarcoplasmic Reticulum in Muscle Phys- of some minor lumenal proteins specific for the SR, sarcalu- iology, eds. Entman, M. L. & van Winckle, W. B. (CRC, Boca Raton, FL), Vol. 1, pp. 65-99. menin and the 53-kDa glycoprotein (36), and might be shared 6. Costello, B., Chadwick, C., Saito, A., Chu, A., Maurer, A. & also by the other ER lumenal marker, PDI (14). In other cell Fleischer, S. (1986) J. Cell Biol. 103, 741-753. types, BiP and PDI have been shown in fact to be intermixed 7. Ma, J., Fill, M., Knudson, C. M., Campbell, K. P. & Coronado, R. within the ER lumen (31, 35). In contrast, within TC BiP was (1988) Science 242, 99-102. not mixed together with CS but was concentrated beneath the 8. Pelham, H. R. B. (1988) EMBO J. 7, 913-918. 9. Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reith- limiting membrane, around the latter protein (37). These meier, R. A. F. & MacLennan, D. H. (1987) Proc. NatI. Acad. Sci. results, which resemble those reported with the intracisternal USA 84, 1167-1171. (ER) granules of pancreatic acinar cells (35), confirm that 10. Leberer, E., Charuk, J. H. M., Clarke, D., Green, N. M., Zu- within the TC lumena CS is not free to diffuse but rather is brzycka-Gaarn, E. & MacLennan, D. H. (1989) J. Biol. Chem. 264, arranged into aggregates, anchored to the limiting membrane 3484-3493. by discrete strands (38). 11. Leberer, E., Charuk, J. H. M., Green, N. M. & MacLennan, D. H. (1989) Proc. Natd. Acad. Sci. USA 86, 6047-6051. An important question raised by our results concerns the 12. Hofmann, S. L., Goldstein, J. L., Orth, K., Moorman, C. R., pathways by which the ER markers and the other components Slaughter, C. A. & Brown, M. S. (1989) J. Biol. Chem. 264, reach theirfinal destination in the SR. So far, two SR membrane 18083-18090. proteins, Ca2+-ATPase and the ryanodine receptor, have been 13. Fliegel, L., Bums, K., MacLennan, D. H., Reithmeier, R. A. F. & investigated. Ca2+-ATPase appears to reach the SR rapidly Michalak, M. (1989) J. Biol. Chem. 264, 21522-21528. 14. Fliegel, L., Newton, E., Burns, K. & Michalak, M. (1990) J. Biol. after synthesis by membrane-bound ribosomes, probably by Chem. 265, 15496-15502. simple diffusion along ER-SR continuities (39). In contrast, 15. Fliegel, L., Bums, K., Opas, M. & Michalak, M. (1989) Biochim. developmental studies have suggested that the ryanodine re- Biophys. Acta 982, 1-8. ceptor first concentrates in specific vesicles that then fuse with 16. Freedman, R. B. (1984) Trends Biochem. Sci. 9, 438-441. the SR and adhere to the sarcolemma transverse tubules to yield 17. Saito, A., Seiler, S., Chu, A. & Fleischer, S. (1984) J. Cell Biol. 99, the triads (40). This alternative pathway could be followed by 875-885. 18. Duggan, P. F. & Martonosi, A. (1970) J. Gen. Physiol. 56, 147-167. the membrane proteins concentrated in the TC, in particular the 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 64-, 58-, and 28-kDa proteins that seem to coexist with the 20. Volpe, P., Villa, A., Damiani, E., Sharp, A. H., Podini, P., Snyder, ryanodine receptor in the JFM. Also for lumenal proteins, S. H. & Meldolesi, J. (1991) EMBO J. 10, 3183-3189. pathways seem to be multiple. The direct ER-SR continuities 21. Villa, A., Podini, P., Clegg, D. O., Pozzan, T. & Meldolesi, J. (1991) could support transport ofBiPand PDI but not ofCS. The latter J. Cell Biol. 113, 779-791. in fact to travel the cis- 22. Keller, G. A., Tokuyasu, K. T., Dutton, A. H. & Singer, S. J. Ca2+ binding protein appears along (1984) Proc. Natl. Acad. Sci. USA 81, 5744-5747. medium Golgi complex, as documented by its 23. Bole, D. G., Hendershot, L. M. & Kearney, J. F. (1986) J. Cell chain (41, 42), its phosphorylation by casein II (42), and Biol. 102, 1558-1566. its recovery during pulse-chase experiments in a coated vesicle 24. Louvard, D., Reggio, H. & Warren, G. (1982) J. Cell Biol. 92, fraction (43). These results document the existence of a way 92-106. back, from the Golgi complex to the ER-a possibility widely 25. Villa, A., Sharp, A. H., Racchetti, G., Podini, P., Bole, D. G., which has not Dunn, W. A., Pozzan, T., Snyder, S. H. & Meldolesi, J. (1992) accepted, however, only in muscle (2, 42, 43), Neuroscience, in press. been seriously investigated in nonmuscle cells. 26. Volpe, P., Alderson Lang, B. H., Madeddu, L., Damiani, E., The expression of ER markers in the SR might also have Collins, J. H. & Margreth, A. (1990) Neuron 5, 713-721. functional consequences. In particular, BiP is an ATPase 27. Macer, D. R. J. & Koch, G. L. E. (1988) J. Cell Sci. 91, 61-70. specifically devoted to assist the correct folding of proteins 28. Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J. J., and peptide loops exposed to the ER lumen (32), whereas II, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y. & Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610. calnexin has been shown to bind Ca2+ and proposed to play 29. Jorgensen, A. O., Kalnins, V. & MacLennan, D. H. (1979) J. Cell a role in the docking of specific lumenal proteins to the ER Biol. 80, 372-384. membranes (27). In the SR this putative function could 30. Bole, D. G., Dowin, R., Doriaux, M. & Jamieson, J. D. (1989) J. concern sarcalumenin and the 53-kDa glycoprotein, two Histochem. Cytochem. 37, 1817-1823. proteins that fail to express the KDEL sequence (10, 11) and 31. Tooze, J., Hollinshead, M., Fuller, S. D., Tooze, S. A. & Huttner, that need an alternative mechanism to be retained. W. B. (1989) Eur. J. Cell Biol. 49, 259-273. therefore 32. Flynn, G. C., Pohl, J., Flocco, M. T. & Rothman, J. E. (1991) Of potentially even greater interest are the membrane pro- Nature (London) 353, 726-731. teins revealed by the anti-ER Ab in TC and JFM, especially 33. Tougard, C., Louvard, D., Picart, R. & Tixier-Vidal, A. (1984) J. the 28-kDa protein. The latter seems in fact to correspond to Cell Biol. 96, 1197-1207. the doublet described by Costello et al. (6) and shown to bind 34. 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