Abstract
Most ryanodine receptors and their relatives, inositol 1,4,5-trisphosphate receptors, are expressed in the sarcoplasmic or endoplasmic reticulum (ER), where they mediate Ca(2+) release. We expressed fragments of ryanodine receptor type 1 (RyR1) in COS cells alone or fused to intercellular adhesion molecule-1 (ICAM-1), each tagged with yellow fluorescent protein, and used confocal imaging and glycoprotein analysis to identify the determinants of ER targeting and retention. Single transmembrane domains (TMD) of RyR1 taken from the first (TMD1-TMD2) or last (TMD5-TMD6) pair were expressed in the ER membrane. TMD3-TMD4 was expressed in the outer mitochondrial membrane. The TMD outer pairs (TMD1-TMD2 and TMD5-TMD6) retained ICAM-1, a plasma membrane-targeted protein, within the ER membrane. TMD1 alone provided a strong ER retention signal and TMD6 a weaker signal, but the other single TMD were unable to retain ICAM-1 in the ER. We conclude that TMD1 provides the first and sufficient signal for ER targeting of RyR1. The TMD outer pairs include redundant ER retention signals, with TMD1 providing the strongest signal.
Highlights
Ryanodine receptors (RyR)3 compose a family of intracellular Ca2ϩ channels that mediate release of Ca2ϩ from the intracellular stores of excitable and non-excitable cells [1]
The biophysical properties and probably the structure of the pore are similar for RyR and IP3R [14], and a structure similar to the inositol 1,4,5-trisphosphate-binding core is present within a similar part of ryanodine receptor type 1 (RyR1) [15]
Expression of Full-length RyR1 in the endoplasmic reticulum (ER)—When expressed alone, green fluorescent protein (GFP) was diffusively distributed throughout the cytoplasm and nucleoplasm of COS-7 cells and co-localized with neither calreticulin (Fig. 2A) nor cyan fluorescent protein-ER
Summary
Ryanodine receptors (RyR)3 compose a family of intracellular Ca2ϩ channels that mediate release of Ca2ϩ from the intracellular stores of excitable and non-excitable cells [1]. A fragment of RyR1 that includes the six TMD but excludes the N-terminal region (RyTMD1– 6C) (Fig. 1C) was expressed in the ER, co-localized with calreticulin (Fig. 3A) and calnexin (data not shown), and was retained in cells after permeabilization (Fig. 3E).
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