Several key proteins have been localized to junctional sarcoplasmic reticulum which are important for Ca 2؉ release. These include the ryanodine receptor, triadin, and calsequestrin, which may associate into a stable complex at the junctional membrane. We recently purified and cloned a fourth component of this complex, junctin, which exhibits homology with triadin and is the major 125 I-calsequestrin-binding protein detected in cardiac sarcoplasmic reticulum vesicles (Jones, L. R., Zhang, L., Sanborn, K., Jorgensen, A. O., and Kelley, J. (1995) J. Biol. Chem. 270, 30787-30796). In the present study, we have examined the binding interactions between the cardiac forms of these four proteins with emphasis placed on the role of junctin. By a combination of approaches including calsequestrin-affinity chromatography, filter overlay, immunoprecipitation assays, and fusion protein binding analyses, we find that junctin binds directly to calsequestrin, triadin, and the ryanodine receptor. This binding interaction is localized to the lumenal domain of junctin, which is highly enriched in charged amino acids organized into "KEKE" motifs. KEKE repeats are also found in the common lumenal domain of triadin, which likewise is capable of binding to calsequestrin and the ryanodine receptor (Guo, W., and Campbell, K. P. (1995) J. Biol. Chem. 270, 9027-9030). It appears that junctin and triadin interact directly in the junctional sarcoplasmic reticulum membrane and stabilize a complex that anchors calsequestrin to the ryanodine receptor. Taken together, these results suggest that junctin, calsequestrin, triadin, and the ryanodine receptor form a quaternary complex that may be required for normal operation of Ca 2؉ release.

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Outer membrane proteins of Vibrio cholerae were purified by sucrose density cenatifugation and Triton X-100 extraction at 10 mM Mg2e. The proteins were separated by polyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.Outer membranes of gram-negative bacilli are composed primarily of proteins, lipids, and lipopolysaccharide. The outer membranes of members of the Enterobacteriaceae and Pseudomonadaceae (8,10,18,19,26,28,34,36) have been characterized, but little has been reported on the outer membrane of Vibrio cholerae (23, 37). V. cholerae differs from members of the Enterobacteriaceae and Pseudomonadaceae by having a lipopolysaccharide which lacks 2-keto-3-deoxyoctonate (29-31).Rough cholera strains are avirulent (16), and recent mutant studies suggest that other cell surface changes may play a role in the virulence of V. cholerae (6). We studied the outer membrane proteins of V. cholerae strain CA401, a virulent wild-type strain.Slightly modified methods, similar to those used for isolation of outer membranes of Enterobacteriaceae, were successful in obtaining outer membrane proteins of V. cholerae. Characterization of these proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed 8 to 10 major proteins, with more than 90% of the outer envelope protein appearing in a band having an Mr of -45,000.In this paper we present a useful method for isolating outer membrane proteins of V. cholerae, assign numbers to outer membrane proteins, and discuss some of the unique features of the V. cholerae outer envelope. MATERIALS AND MErHODS Bacterial strain. V. cholerae CA401 is a classical Inaba strain isolated in 1953 in Calcutta, India (22), which has been extensively characterized (3-6, 35). t Present address: MO 65212.Stock cultures were kept lyophilized or frozen at -70°C in brain heart infusion broth plus 15% glycerol. Working stocks were kept on meat extract agar slants (2) at 49C for no longer than 3 weeks.Cell culture and harvesting. V. cholerae was routinely grown in the semisynthetic medium (Syncase) of R. A. Finkelstein and C. E. Lankford (Bacteriol. Proc., p. 43, 1955) to mid-logarithmic growth phase and was occasionally grown in other media for comparison: complex medium, meat extract broth (2), and defined minimal A medium (27). Cultures were grown aerobically by inoculating approximately 20 ml of medium with 2 ml of an overnight culture in the same medium and incubating it at 35 to 370C with shaking (180 to 200 rpm) in a New Brunswick shaking waterbath until mid-logarithmic phase. Ten milliliters of this culture was used to inoculate 400 ml of the same medium in a 2-liter Erlenmeyer flask and incubated at 37°...