COMPLEX-FORMATION BETWEEN JUNCTION, TRIADIN, CALSEQUESTRIN, AND THE RYANODINE RECEPTOR - PROTEINS OF THE CARDIAC JUNCTIONAL SARCOPLASMIC-RETICULUM MEMBRANE

Several key proteins have been localized to junctional sarcoplasmic re ticulum which are important for Ca2+ release. These include the ryanod ine receptor, triadin, and calsequestrin, which may associate into a s table complex at the junctional membrane. We recently purified and clo ned a fourth component of this complex, junctin, which exhibits homolo gy with triadin and is the major I-125-calsequestrin-binding protein d etected in cardiac sarcoplasmic reticulum vesicles (Jones, L. R., Zhan g, L., Sanborn, K., Jorgensen, A. O., and Kelley, J. (1995) J. Biol. C hem. 270, 30787-30796). In the present study, we have examined the bin ding interactions between the cardiac forms of these four proteins wit h emphasis placed on the role of junctin. By a combination of approach es including calsequestrin-affinity chromatography, filter overlay, im munoprecipitation assays, and fusion protein binding analyses, we find that junctin binds directly to calsequestrin, triadin, and the ryanod ine receptor. This binding interaction is localized to the lumenal dom ain of junctin, which is highly enriched in charged amino acids organi zed into ''KEKE'' motifs. KEKE repeats are also found in the common lu menal domain of triadin, which likewise is capable of binding to calse questrin and the ryanodine receptor (Guo, W., and Campbell, K. P. (199 5) J. Biol. Chem. 270, 9027-9030). It appears that junctin and triadin interact directly in the junctional sarcoplasmic reticulum membrane a nd stabilize a complex that anchors calsequestrin to the ryanodine rec eptor. Taken together, these results suggest that junctin, calsequestr in, triadin, and the ryanodine receptor form a quaternary complex that may be required for normal operation of Ca2+ release.