J. Membrane Biol. 56 241–248 (1980) 0022-2631/80/0056-0241 $01.60 © 1980 Springer-Verlag New York Inc. Phosphorylation of Heavy Sarcoplasmic Reticulum Vesicles: Identification and Characterization of Three Phosphorylated Proteins Kevin P. Campbell  and Adil E. Shamoo  Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 Summary. Heavy sarcoplasmic reticulum vesicles de- rived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate pro- teins. Heavy vesicles were phosphorylated at room temperature in 5 m M MgCl 2 , 1 m M EGTA, 10 mM HEPES (pH 7.4) and 10 µM γ- 32 P-ATP. 32 P- phosphoproteins were determined by sodium dodecyi sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (β-aminoethyl ether) N,N,N′ ,N′ -tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase ac- tivity. Phosphorylation of three proteins of 64,000 daltons (El), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1 mM EGTA. Phosphorylation of these proteins was cAMP-inde- pendent, hydroxylamine-resistant, and was seen with- out the addition of protein kinase. In the presence ofHgCl 2 (2.5 mM) or sodium deoxycholate (1%) no protein phosphorylation was observed. Protein E1 was heavily phosphorylated in the presence of 200 mM KCl, while its phosphorylation was inhibited by 20 µM sodium dantrolene, an inhibitor of Ca 2+ release. Phosphoprotein E3 was found in light and heavy sarcoplasmic reticulum vesicles while E1 and E2 were found only in heavy vesicles. The phospho- protein E2 had the properties of an intrinsic membrane protein while the protein E1 behaved as an extrinsic membrane protein. Proteins E2 and E3 corresponded in mobility to minor sarcoplasmic reticulum proteins while E1 had the same mobility as calsequestrin. The presence of high calcium (5 mM) during electrophore-  Present Address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, On- tario M5G1L6, Canada.  Present Address and to whom reprint requests should be made: Department of Biological Chemistry, University of Maryland, 660 West Redwood Street, Baltimore, Maryland 21202. sis caused calsequestrin to run at a lower molecular weight (~56,000 instead of 64,000 daltons), and cor- respondingly the phosphoprotein E1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated. It is generally accepted that depolarization of the transverse tubular system of skeletal muscle initiates the release of calcium from the terminal cisternae of the sarcoplasmic reticulum (Ebashi & Endo, 1968; Endo, 1977; Fuchs, 1974; Sandow, 1970). In recent years there have been several mechanisms proposed for the link between the depolarization of the walls and of the T-tubule and the release of calcium from the sarcoplasmic reticulum (Ebashi & Endo, 1968; Endo, 1977), but it still remains one of the least under- stood processes in muscle contraction. The skeletal muscle membranes directly involved in excitation- contraction coupling are the transverse tubular mem- brane and the junctional sarcoplasmic reticulum membrane (Franzini-Armstrong, 1975). The morpho- logical and chemical differences of the longitudinal sarcoplasmic reticulum and the terminal cisternae are consistent with the hypothesis that the longitudinal SR contains, predominantly, the calcium pump pro- tein and is responsible for calcium uptake resulting in relaxation, and that the terminal cisternae, is re- sponsible for the calcium release to initiate contrac- tion and contains one or more membrane proteins besides the calcium pump protein (Franzini-Arm- strong, 1975; Meissner, 1975, Campbell, Armstrong & Shamoo, 1980). It is now becoming increasingly evident that the phosphorylation of membranes by protein kinases