Association of Clathrin with Microsomes Isolated from Canine Myocardium

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1 120 Assocaton of Clathrn wth Mcrosomes Isolated from Canne Myocardum Davd R. Caprette, Mark L. Entman, and W. Barry Van Wnkle From the Secton of Cardovascular Scences, Department of Medcne, Baylor College of Medcne, Houston, Texas SUMMARY. We and others have observed specalzed regons of sarcoplasmc retculum membranes that resemble coated vescles, n the I-band regon of myocardal cells. These structures have been named "corbular' sarcoplasmc retculum, and are dstnct n appearance from Golgassocated coated vescles, n that they are larger and contan a flocculent materal that has been dentfed as calsequestrn. Whereas t has been suggested that these structures have a role n cardac calcum metabolsm, ther functon(s) and the molecular dentty of the characterstc 'brstle* coat reman unknown. Mcrosomes enrched n sarcoplasmc retculum were prepared from canne ventrcular muscle by Polytron homogenzaton n ph 6.5 buffer, followed by dfferental centrfugaton. Proten was released by ncubaton n 50 rtm Trs/HCl, ph 8, followed by centrfugaton. We found these extracts to be enrched n a proten that was dentcal to bran clathrn n moblty on a Sepharose 4B gel fltraton column, fnal poston of the natve proten followng nondenaturng electrophoress, relatve moblty n denaturng (sodum dodecyl sulfate) electrophoress on 6% and 7.5% gels, and antgencty to ant-clathrn IgG. These fndngs confrmed the presence of clathrn trskelons n the cardac mcrosome extract. On ths bass, we suggest that clathrn may be a component of the electron dense "coat* of corbular sarcoplasmc retculum. (Crc Res 58: , 1986) COATED vescles (CV) are ubqutous organelles found n varyng amounts n most eukaryotc cells (Pearse, 1980). Despte ther nvolvement n dverse cellular functons, e.g., receptor medated endocytoss (Anderson et al., 1978; Goldsten et al., 1979), membrane recyclng (Heuser and Reese, 1973; Pearse and Bretscher, 1981) and secreton (Kartenbeck, 1980), CV possess a common feature, the characterstc brstle coat seen n electron mcrographs. Ths coat has been shown to be a closed lattce made up of proten subunts called trskelons (Ungewckell and Branton, 1981), several assocated protens of 100, ,000 daltons (Unanue et al., 1982; Zaremba and Keen, 1983), and a trplet of 50,000-55,000 daltons that have been dentfed as a- and /3-tubuln and an 'assembly proten* (Kelly et al., 1983; Pfeffer et al., 1983). The trskelon s a unque three-armed macromolecule, dentfable by rotary shadowng n the electron mcroscope. It conssts of three dentcal 175,000 dalton "heavy* polypeptde chans called clathrn (Pearse, 1975) and three heterogeneous "lght* chans of 32,000-35,000 daltons, termed clathrn-assorated protens, or CAP (Lsant et al., 1982). We and others (Forbes and Sperelaks, 1980) have noted the presence of CV-lke structures n cardac cells, assocated wth sarcoplasmc retculum (SR) membranes n the I-band regon of mammalan ventrcular and atral muscle (Fg. 1) and n the pernuclear area n assocaton wth Golg membranes. CV have been observed "free" n the cytoplasm of neonatal skeletal muscle cells (Bursztajn and lbby, 1981), but have not been seen n assocaton wth SR membrane or transverse tubules n adult skeletal muscle. The CV-lke structures assocated wth cardac SR have also been called 'corbular SR" (Sommer and Waugh, 1976), and are dstnct n appearance compared wth Golg-assocated CV. They contan flocculent materal n the lumen that has been dentfed as calsequestrn (Jorgensen and Campbell, 1984). A role for these structures n cardac calcum metabolsm has been suggested, based on ther membrane contnuty wth the termnal portons of the SR membrane (Forbes and Sperelaks, 1980). However, the presence of clathrn n the brstle coat of these SR-assocated, calsequestrn-contanng vescles has not been confrmed. As part of our studes to determne the role of corbular SR n myocardal cell functon, we report the dentfcaton of clathrn trskelons n extracts of an SR-enrched mcrosomal fracton of canne myocardum. On the bass of these results, we suggest that t s hghly lkely that clathrn s a component of the electron-dense coat of corbular SR. Methods Bran Clathrn Preparaton Crude CV were prepared from trmmed cortces by the method of Pearse (1975), and were further purfed by sucrose gradent centrfugaton. Brans were removed from Nembutal-anesthetzed dogs, placed n ce-cold sotonc salne soluton, and strpped of ther mennges. Subse-

2 Caprette et al. /Assocaton of Clathrn wth Cardac Mcrosomes 121 FIGURE 1. Panel a: thn secton electron mcrograph of canne left ventrcular papllary muscle. Coated vescles (arrows) assocated wth membrane of the sarcoplasmc retculum (SR) occur only n the regon of the Z-band and I-band (bar = 0.5 nm). panel b: thn secton of canne papllary llustratng assocaton of two coated vescles (arrows) wth the transverse CD tubule, and smlarty of ther flocculent contents (bar = 0.5 xm). quent steps were carred out at 0-4 C. The trmmed cortces were placed n 1 ml/g buffer A, consstng of 100 rrtm 2[N-morpholno]ethanesulfonc acd (MES), 1 rrtm EGTA, 0.5 rrm MgCl^ and 5 HIM NaN3/ ph 6.5, and homogenzed 3 x 10 seconds at hgh speed n a Warng Blendor. The homogenate was centrfuged 30 mnutes at 20,000 g, and the supernatant was fltered and recentrfuged, then centrfuged for 60 mnutes at 85,000 g. The pellets (crude mcrosomes) were washed by resuspenson n 50 ml buffer A, wth several strokes of a Dounce homogenzer wth a loose-fttng pestle, followed by centrfugaton for 30 mnutes at 85,000 g. CV were further purfed by sucrose gradent centrfugaton, as descrbed by Keen et al. (1979). The coat proten was solated from ether coated vescles or crude mcrosomes by suspenson n 50 mm Trs/HCl, ph 8, by Dounce homogenzaton, followed by strrng for 30 mnutes. Vescular materal was separated from soluble proten by centrfugaton for 60 mnutes at 165,000 g. Myocardal Clathrn Preparaton The followng s a modfcaton of our preparaton for SR-enrched fractons from canne myocardum (Van Wn- kle et al., 1978). Hearts were removed from Nembutalanesthetzed dogs and placed n sotonc salne soluton. Subsequent steps were carred out at 0-4 C. The rght ventrcular free wall, extramyocardal tssues, and epcardal and endocardal surfaces were removed, and the remanng left ventrcle and septum were blotted and weghed, mnced wth scssors, and homogenzed (Polytron, 3 x 25 seconds at 3/4 maxmum speed n 3 ml/g buffer A). Ths homogenate was centrfuged 20 mnutes at 4,000 g, the supernatant (SN) was fltered through two layers of cheesecloth and set asde, and the pellet was rehomogenzed (Polytron, 25 seconds n 3 ml/g buffer A) and recentrfuged. Fltered SN were combned and centrfuged 20 mnutes at 8,000 g, followed by 30 mnutes at 100,000 g. The pellets were washed n buffer A, then were resuspended n 50 mm Trs/HCl, ph 8, and extracted as descrbed for bran mcrosomes. Column Chromatography Extracts from bran and heart mcrosomes were further fractonated on a column of Sepharose 4B, 1.6 X 90 cm (Pharmaca), equlbrated wth buffer B (25 mm Trs base, 192 mm glycne, ph 8.8). The column was calbrated wth

3 122 thyroglobuln (669,000 daltons), apoferrtn (443,000 daltons), /3-amylase (200,000 daltons), and alcohol dehydrogenase (150,000 daltons), obtaned from Sgma. Vod volume was determned wth blue dextran 2000, and K. v = (V e V 0 )/(V, V o ) was determned for each proten standard, where V e = eluton volume, V o = vod volume, and V, = total volume. Proten n each extract was desalted on a column of Sephadex G-25 equbrlated wth 20 ITIM Nr,HCO3 and lyophlzed. Samples were prepared for chromatography by resuspenson n buffer B wth 40 ITLM dthothretol (DTT), and were degassed before loadng 2 to 4 ml of sample per run. Eluton was performed at ml/hr wth buffer B, and 2-ml fractons were collected. Fractons were dentfed by absorbance at 280 nm. Proten was recovered by desaltng and lyophlzaton, as descrbed above, and pellets were resuspended n dstlled water. Electrophoress Electrophoress was performed on slab gels prepared from a stock soluton of 30% acrylamde/0.8% bs-acrylamde. Proten bands were vsualzed by stanng wth Coomasse-Weber stan, followed by destanng wth 7.5% methanol, 10% acetc acd. Relatve amounts of clathrn trskelons or heavy chan polypeptde n dfferent fractons were estmated by scannng Coomasse-staned bands n an LKB laser denstometer. Molecular weghts of polypeptdes were estmated from standard curves prepared from proten standards (Sgma). Sodum dodecyl sulfate-polyacrylamde gel electrophoress (SDS-PAGE) was performed on fractons of bran and heart mcrosomal preparatons, after proten estmaton by the buret method. Samples were dluted to 2-4 mg/ml wth dstlled water, then mxed 1:1 wth buffer C, consstng of 125 ITIM Trs base, 80 ITIM DTT, 20% glycerol, 10% SDS, and 0.002% bromophenol blue dye (BPB). After 5 mnutes at room temperature, the solublzed samples were centrfuged 5 mnutes n a Beckman mcrofuge (10,000 g) and loaded n amounts of Mg buret proten nto sample wells of 1.5-mm-thck slab gels. Acrylamde concentraton was from 6-15% (resolvng gel), wth 3% polyacrylamde stackng gels. Stackng was performed at 120 V and unstackng at 200 V, wth a dscontnuous SDS/Trs/glycne buffer system (Hames, 1981). Nondenaturng PAGE on 3-mm-thck lnear gradent slab gels of 3-15% polyacrylamde, prepared as descrbed by Hames (1981), was used to assay for the presence of ntact clathrn trskelons. Electrophoress was performed wth buffer B (nondenaturng hgh-ph contnuous-buffer system). Samples were prepared by mxng undluted sample 1:1 wth buffer D, consstng of 40 parts buffer B, 40 parts glycerol, and 20 parts 0.08% BPB. For nondenaturng PAGE under reducng condtons, 80 ITIM DTT was ncluded n buffer D. From j/g buret proten were loaded n each sample well, and electrophoress was performed at 100 Vat room temperature overnght (16 hours). Gel peces contanng trskelon proten were obtaned from nondenaturng gels by stanng a vertcal strp from the preparatve gel, then excsng peces from dentcal but unstaned lanes, at the approprate locaton. Gel peces were soaked 2 hours n buffer C, and placed n sample wells of 6% SDS gels wth 3% stackng gel, whle preservng ther orgnal orentaton. SDS-PAGE was performed as descrbed above. Immunoblottng Immune serum and affnty-purfed antbodes to clathrn were a gft from Saul Puszkn and coworkers, Dvson Crculaton Research/Vo/. 58, No. 1, January 1986 of Molecular Pathology, Pathology Department, Mt. Sna School of Medcne of the Cty Unversty of New York. Immune serum was obtaned from New Zealand Whte male rabbts njected subcutaneously on a weekly bass wth purfed bovne bran clathrn, assembled as baskets, and emulsfed wth Freund's complete adjuvant. Antclathrn mmunoglobuln G (IgG) was affnty purfed by cyanogen bromde-actvated Sepharose 4B affnty chromatography. Antbodes were characterzed by the method descrbed by Bloom et al. (1980). Reactvty of clathrn-enrched fractons wth ant-clathrn IgG was assessed by the "Western" blottng technque wth radoodnated proten A (Towbn et al., 1979; Burnette, 1981). Electron Mcroscopy Samples contanng crude mcrosomes or coated vescles were examned by negatve stan electron mcroscopy employng 1% uranyl acetate (ph 4.5) on Formvar-coated, carbon-stablzed grds. Papllary muscles from canne left ventrcle were processed for electron mcroscopy by the method descrbed by Van Wnkle and Schwartz (1978). Thn sectons were examned n a Semens 1A electron mcroscope or n a JEOL 200 CX electron mcroscope. The volume of SR-assocated coated vescles relatve to myocardal cell volume was determned by the stereologcal methods descrbed by Van Wnkle and Schwartz (1978). Electron Mcroscopy Results Coated vescles occurred n precse locatons n canne ventrcular muscle cells n agreement wth prevous descrptons. Most often, they occurred as specalzatons n the I-band regon of each sarcomere, wth ther membranes contnuous wth those of sarcoplasmk retculum (Fg. la). We also observed them attached to the transverse (T) tubule membrane (Fg. lb), suggestng that ths speces of coated vescle represents a mechansm of transfer between the SR lumen and the extracellular space. Stereologcal analyses of electron mcrographs of canne myocardum ndcated that the coated vescles assocated wth SR or T-tubule membranes occupy 0.3% of the volume of cardac ventrcular cytoplasm. In addton to the flocculent matrx, SRcoated vescles were dstngushed from Golg-assocated coated vescles by ther sze, the former beng 75.1 ± 2.5 nm (mean ± SD) n dameter, compared to 51.3 ± 1.6 nm for the latter. Both possessed a 20-nm-thck brstle coat, as observed prevously (Forbes and Sperelaks, 1980). Preparatve Coated vescles solated from canne bran exhbted the same morphology as those solated from other tssues. Yelds of bran and heart mcrosomes, followng the wash step, were 2.13 ± 0.2 and 1.23 ± 0.2 mg buret proten/g wet weght startng tssue, respectvely. SDS-PAGE of bran mcrosomes revealed a major hgh molecular weght band shown by others to be clathrn heavy chan (Pearse, 1976; Garbern and Wu, 1981). The molecular weght of canne bran clathrn heavy chan, estmated from a

4 Caprette et al. /Assocaton of Clathrn wth Cardac Mcrosomes standard curve of molecular weght vs. relatve moblty (Rf), was 165,000 daltons. A fant band (band 1) wth the same Rf as bran clathrn was revealed by electrophoress of crude cardac mcrosomes (Fg. 2). Extracton of the membranes wth 50 HIM Trs/ HC1, ph 8, resulted n enhancement of ths band n the soluble fracton. Yelds of the crude mcrosome extracts were 77 ± 19 and 88 ± 28 Mg/g startng tssue for bran and heart, respectvely. Band 1 represented 15% of heart mcrosome extract proten. Calbraton of the Sepharose 4B column yelded Kav values of 0.44, 0.47, 0.60, and 0.81 for thyroglobuln, apoferrtn, catalase, and aldolase, respectvely. Chromatography of bran extract yelded three major proten peaks. Fractons contanng the second peak (Kav = 0.29) were pooled. Analyss of ths proten peak by SDS-PAGE revealed a sngle major polypeptde band wth a molecular weght of 165,000 daltons (lane E, Fg. 2; lane A, Fg. 5). The eluton patterns for both extracts are shown n Fgure 3. The most promnent proten peaks from heart mcrosome extract eluted ether n the vod fracton or wth a Kav of A mnor peak wth a Kav dentcal to that of bran trskelons was dentfed, and fractons were pooled as ndcated n Fgure 3. Subsequent gradent SDS-PAGE showed ths fracton to be enrched wth band 1 proten. Nondenaturng PAGE of CV extract revealed a sngle major band ("natve" clathrn) mgratng to a poston of 4.1% acrylamde monomer concentraton (%T) on the gradent gel (Fg. 4). Extracts from heart mcrosomes also revealed ths band (band 2), but 123 other protens of lower apparent molecular weght were also evdent. The fnal %T was the same for band 2 and bran clathrn. As shown n Fgure 4, treatment wth DTT dd not affect the fnal %T for ether band 2 or bran clathrn. Nondenaturng electrophoress was carred out on DTT-treated heart and bran extracts wth 3-15% lnear gradent gels, and the proten bands were transferred to ntrocellulose paper (Fg. 4). Followng mmunoblottng of dentcal transfers wth antclathrn antbody, bran trskelons and band 2 of heart extract were labeled. Irrevelant proten bands and control transfers were not labeled. To ensure the presence and predomnance of clathrn n band 2 compared wth trskelon proten from bran mcrosomes, a form of two-dmensonal E c o CO CM». CO > > l A / / /^ / / // // / V / Y L c ID O >, g 5 a O s IHIIm 1 1!A' /V / S 100 Fracton Number B A B C D FIGURE 2. Analyss of clathrn-erched fractous from bran and heart tssue by sodum dodecyl sulfate-polyacrylamde gel electrophoress (SDS-PAGE) on a 7.5% slab gel wth 3% stackng gel. Lanes A and B: bran mcrosomes (50 fg) and hgh ph extract (20 fg), respectvely. Lanes C and D: heart mcrosomes and extract (50 fg each). Lane E: clathrn trskelons, purfed from bran extract on Sepharose 4B (10 fg). Standard protens are myosn (200,000 daltons), f-galactosdase (116,250 daltons), phosphorylase B (9Z500 daltons), bovne serum albumn (66,200 daltons), and ovalbumn (45,000 daltons). FIGURE 3. Top: eluton patterns of bran (sold lne) and heart (dashed lne) mcrosome extracts on Sepharose 4B. Fractons between arrows (Br, He) were pooled, desalted, and lyophlzed. Bottom: analyss of labeled proten peaks by SDS-PACE on lnear gradent gels of 7.515% polyacrylamde. Lane A: 50 fg bran mcrosome extract. Lane B: 50 fg peak Br. Lane G 50 fg heart mcrosome extract. Lane D: 25 fg peak He. Standard protens are myosn (200,000 daltons), P-galactosdase (116,250 daltons), phosphorylase B (92,500 daltons), bovne serum albumj (66,000daltons), ovalbumn (45,000 daltons), carbonc anhydrase (29,000 daltons), and cytochrome c (12,000 daltons).

5 124 Crculaton Research/Vo/. 58, No. 1, January 1986 C F A B C 3X T-1 2 T ~-15 /. fc. FIGURE 4. Analyss of coated vescle and heart mcrosome extracts by nondenaturng PACE on lnear gradent gels of 3-15% polyacrylamde wth a contnuous buffer system. T: bran trskelon band. 2: band 2 from heart mcrosome extract. Lanes A and C 100 fg coated vescle extract. Lanes B and D: heart mcrosome extract, 100 and 270 ng, respectvely. Dthothretol (40 m was ncluded n the sample buffer n lanes C and D. Lanes E and F: X-ray flm exposed 16 hours to a "Western' transfer of samples dentcal to those shown n lanes C and D. Transfers were exposed to ant-clathrn gg and radoodnated proten A. The bands correspond to the postons of bran clathrn trskelons (E) and band 2 of heart extract (F). electrophoress was performed. Gel peces contanng band 2 and the clathrn band from bran mcrosome extract were removed from preparatve nondenaturng gels, denatured, and SDS-PAGE was carred out (Fg. 5). Electrophoress on 6% polyacrylamde gels resolved a sngle band wth Rf of 0.37, correspondng to 165,000 daltons. Dscusson The prncpal queston addressed by ths study s whether or not the proten clathrn s assocated, n sgnfcant amounts, wth cardac SR. Cardac mcrosomes, prepared by Polytron homogenzaton of ventrcular tssue followed by dfferental centrfugaton, are enrched n SR (Van Wnkle et al., 1978). Whereas other membrane vescles are present n the mcrosomal fracton, the only membranes lkely to contrbute clathrn, as determned by morphologcal examnaton of ntact tssue, are corbular SR and the Golg-assocated coated vescles. The Golg-assocated structures are smaller n dameter than corbular SR, and are much fewer n number, located prmarly n the pernuclear regon of the myocardal cell. We therefore sought to determne f the proten clathrn could be solated from suspensons of cardac mcrosomes. Clathrn lattces are dssocated nto subunts by ncubaton n buffers of low onc strength and alkalne ph,.e., >7.5, and by buffers contanng monoamnes such as Trs base (Schook and Puszkn, 1983). To preserve the structure of clathrn that may FIGURE 5. Analyss of band 2 and trskelon proten from heart and bran mcrosomes, respectvely, by two-dmensonal PACE. Lane A: clathrn trskelons, purfed from bran mcrosome extract on Sepharose 4B (10 g). Lane B: band 2 from 80 pg heart extract, cut from a nondenaturng 3-15% gradent gel. Lane C: clathrn trskelons from 16 tg bran extract, cut from a nondenaturng gel. Proten standards are as descrbed for Fgure 3. have been assocated wth SR membranes, we prepared the mcrosomes n buffer contanng MES (pka = 5.96) at ph 6.5. Clathrn lattces were subsequently dssocated nto subunts by ncubaton n Trs buffer, ph 8. To dentfy cardac clathrn, we compared analyses of extracts of cardac mcrosomes wth extracts known to contan clathrn. These extracts were prepared from coated vescles and crude mcrosomes from canne bran, and clathrn was purfed further by gel fltraton on a column of Sepharose 4B. Coated vescles solated from canne bran by the method of Pearse (1975) exhbted the same morphology n negatvely staned electron mcrographs as that reported for coated vescles solated from porcne or bovne brans (Pearse, 1975, 1976). Examnaton of crude mcrosome preparatons by both electron mcroscopy and SDS-PAGE revealed canne bran to be an especally rch source of coated vescles and clathrn (Fg. 2). Structures resemblng bran-coated vescles were not seen n negatvely staned preparatons of cardac mcrosomes, although obscure vescles of the sze and shape of coated vescles occasonally were seen. We offer three possble explanatons for ths observaton. Frst, corbular SR vescles would represent a very small mnorty of the populaton of vescles n a crude mcrosome suspenson. Second, the degree of contrast obtaned n negatve stanng s based on the prncple that protenaceous structures repel the

6 Caprette et al. /Assocaton of Clathrn wth Cardac Mcrosomes 125 heavy metal stan, whereas spaces surroundng the protens stan darkly. The hgh proten:lpd rato of SR vescles relatve to bran vescles may obscure the postulated clathrn lattces n negatvely staned preparatons. Thrd, a characterstc of corbular SR and other forms of junctonal SR s the presence of hgh molecular weght "feet' protens (Dolber and Sommer, 1984; Seler et al., 1984). In addton to the repulson of stan by SR feet protens, an nteracton of clathrn wth these extrnsc protens may affect the confguraton of the lattce structure tself. These structures may have to be dstngushed through the use of mmunocytochemcal labelng usng antbodes to clathrn and feet protens, or, perhaps, through the use of transmsson electron mcroscopc gonometry (specmen tltng). The dfference between our estmate of 165,000 daltons for the molecular weght of bran clathrn heavy chan and the estmate by others (Pearse, 1975; Pfeffer et al., 1983) of 175, ,000 daltons may reflect our choce of proten standards and the gel electrophoress system. We dd not attempt to estmate the molecular weght of 'natve* clathrn trskelons by gel fltraton, snce the three-armed structure of the trskelon gves an effectve molecular radus greater than that expected for a globular proten of the same molecular weght. Thus, the eluton volume for trskelons (approxmate molecular weght, 640,000 daltons) was less than that of thyroglobuln (669,000 daltons). SDS-PAGE of cardac mcrosomes prepared at ph 6.5 revealed a fant band wth the same R f as bran clathrn heavy chan. Ths suggests the presence of clathrn n cardac SR. Enhancement of ths band n the soluble fracton by extracton at ph 8 of cardac mcrosomes suggests that the alkalne ph released the clathrn coat from corbular SR. Proten bands n ths molecular weght range must be greater than 2,000 daltons apart n order to be resolved, assumng a mnmum resoluton dstance of 0.5 mm between bands. To test the hypothess that the band from the cardac source was a dfferent proten wth an dentcal apparent molecular weght, we analyzed hgh ph extracts by electrophoress under nondenaturng condtons. Analyss of coated vescle or bran mcrosome extract revealed a sngle hgh molecular weght proten band, representng 'natve' clathrn trskelons. A band n heart extract mgrated to the same percent acrylamde concentraton (%T) on the gradent gel. Snce the subunt structure of the trskelon has been shown to be ndependent of dsulfde bondng (Gargern and Wu, 1981), electrophoress was also performed under reducng condtons. Ths reduced the number of proten bands n the cardac mcrosome extract, but the moblty of band 2 was unaffected, and remaned the same as that of bran clathrn trskelons. An addtonal pece of evdence (Fg. 4) for molecular dentty between band 2 of heart extract and bran trskelons s ther cross-reactvty wth affnty-purfed rabbt IgG antbody to bovne bran clathrn. The mmunologcal smlarty among clathrns solated from canne bran and heart tssue and from bovne bran reflects the absence of sgnfcant varaton n structure of clathrn heavy chan among tssues of dverse speces. A form of two-dmensonal PAGE, employng nondenaturng followed by SDS-PAGE on 6% polyacrylamde slab gels, confrmed that both the bran trskelon band and the correspondng band n heart extract conssted prmarly of subunts wth the same Rf, correspondng to 165,000 daltons. We chose a 6% resolvng gel n order to acheve good resoluton of bands at the molecular weght of clathrn heavy chan,.e., wth R f between 0.3 and 0.7. We consder the dentcal mobltes of band 2 proten and bran clathrn n both nondenaturng and SDS-electrophoress systems to be strong evdence that band 2 represents natve clathrn trskelons. In transmsson electron mcrographs, corbular SR vescles resemble clathrn-coated vescles that have been dentfed n mcrographs of dverse tssues. Most roles postulated for coated vescles n other tssues have been dynamc, n whch assembly of clathrn lattces leads to vescle formaton, followed by transport of substances nto or out of the cell, or between ntracellular structures. An assocaton of clathrn wth the electron-dense coat of corbular SR mght suggest that such 'coated SR' s nvolved n a dynamc transport process. The assocaton of corbular SR vescles wth the termnal portons of the SR membrane, and the presence of calsequestrn n the electron-dense matrx, suggest a role for these structures n the regulaton of calcum levels n the myocardal cell. One possble functon could be the transport of excess calcum from the lumen of the SR membrane to the transverse (T)-tubule for delvery to the T-tubuIe lumen. A proposed alternatve role for clathrn s the formaton of stable clathrncoated 'pts," n whch a specalzed regon of the membrane s segregated for a purpose such as the approxmaton of smlar receptor complexes. The clathrn lattce, f assocated wth corbular SR, mght serve to segregate and stablze portons of SR contanng of hgh concentraton of calsequestrn, and/ or to bnd the 'feet" protens to the membrane surface. The exact functon of corbular SR, and the complete bochemcal makeup of the electron-dense coat, reman unknown. We wsh to thank Ellot L Hertzbcrg and Robert Skbbens, Department of Bochemstry, Baylor College of Medcne, for ther assstance n the Western mmunoblottng experments. Ths work was supported by a grant-n-ad from the Amercan Heart Assocaton, wth funds contrbuted n part by the Texas afflate, and by NIH Tranng Grant T31-HL Address for reprnts: Davd R. Caprette, Secton of Cardovascular Scences, Department of Medcne, Baylor College of Medcne, One Baylor Plaza, Houston, Texas Receved July 22, 1985; accepted for publcaton October 30, 1985.

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