ABSTRACT
Ca(2+) storage and release in muscle cells are controlled by a complex of junctional sarcoplasmic reticulum (jSR) proteins, that includes the calcium-binding protein calsequestrin (CSQ), the Ca(2+)-release channel (ryanodine receptor or RyR) and two transmembrane proteins that bind to RyR: junctin (JNC) and triadin (Tr). The relationship between CSQ and JNC, and their contributions to the architecture of the jSR vesicle was studied in transgenic mice with combined overexpression of CSQ and JNC. We find that CSQ, on its own, has a diffuse disposition in the sarcoplasmic reticulum (SR) lumen. Overexpression of JNC results in a tighter packing of CSQ in proximity of the SR membrane, presumably due to the binding of CSQ to the membrane by JNC. Quantitative and qualitative analysis of structural changes in the overexpressing as well as in the normally differentiating myocardium illustrate the synthetic pathways and the events in the targeting and delivery of CSQ and JNC to the jSR of the differentiating cardiac myocyte. CSQ is delivered from the Golgi to the SR, where it buds out into precursors of the jSR vesicles. JNC reaches the jSR vesicles directly, but its arrival is delayed relative to CSQ.
Subject(s)
Calcium-Binding Proteins , Calsequestrin/metabolism , Carrier Proteins/metabolism , Membrane Proteins , Mixed Function Oxygenases , Muscle Proteins/metabolism , Myocardium/metabolism , Animals , Blotting, Western , Calsequestrin/biosynthesis , Calsequestrin/ultrastructure , Carrier Proteins/biosynthesis , Carrier Proteins/ultrastructure , Cell Differentiation , Golgi Apparatus/metabolism , Mice , Mice, Transgenic , Muscle Proteins/biosynthesis , Muscle Proteins/ultrastructure , Myocardium/ultrastructure , Myocytes, Cardiac/ultrastructure , Sarcoplasmic Reticulum/chemistry , Sarcoplasmic Reticulum/ultrastructure , Time FactorsABSTRACT
A calsequentrin (CS)-like glycoprotein is present in the sarcoplasmic reticulum (SR) of chicken pectoralis muscle, which displays unusual properties: it binds relatively low amounts of Ca2+, compared to CS in mammalian skeletal muscle (Yap & MacLennan, 1976), it does not exhibit a marked pH-dependent shift in mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and its metachromatic staining properties with Stains All are likewise peculiar (Damiani et al., 1986). We have now definitively localized the same protein to the junctional terminal cisternae (TC) fraction of the SR of chicken pectoralis muscle and have further characterized it, following purification by crystallization with Ca2+ and by Ca2(+)-dependent elution from phenyl-Sepharose columns. The purified protein (apparent Mr: 51 kDa), isoelectrofocuses at pH 4.5, and is readily identified on blots by a 45Ca overlay technique, similar to CS of rabbit skeletal muscle, but it binds half as much Ca2+ (about 20 moles of Ca2+ per mole of protein), as estimated by equilibrium dialysis. However, the chicken protein shares extensive similarities with mammalian CSs, concerning Ca2(+)-induced changes in maximum intrinsic fluorescence and the Ca2(+)-modulated interaction with phenyl-Sepharose, as well as in being protected by Ca2+ from proteolysis by either trypsin or chymotrypsin. We discuss how the presence of a Ca2(+)-regulated hydrophobic site in the CS molecule appears to be the most invariant property of the CS-family of Ca2(+)-binding proteins.
