The transmembrane segment of ryanodine receptor contains an intracellular membrane retention signal for Ca(2+) release channel.
The ryanodine receptor (RyR) is a large homotetrameric protein with a hydrophobic domain at the C-terminal end that resides in the endoplasmic reticulum (ER) or sarcoplasmic reticulum membrane and forms the conduction pore of a Ca(2+) release channel. Our previous studies showed that RyR expressed in heterologous cells localized to the ER membrane. Confocal microscopic imaging indicated that the ER retention signal is likely present within the C-terminal portion of RyR, a region that contains four putative transmembrane segments. To identify the amino acid sequence responsible for ER retention of RyR, we expressed fusion proteins containing intercellular adhesion molecule (ICAM), various fragments of RyR, and green fluorescent protein (GFP) in Chinese hamster ovary and COS-7 cells. ICAM is a plasma membrane-resident glycoprotein and serves as a reporter for protein trafficking to the cell surface membrane. Imaging analyses indicated that ICAM-GFP fusion proteins with RyR sequence preceding the four transmembrane segments, ICAM-RyR-(3661-3993)-GFP, and with RyR sequence corresponding to transmembrane segments 1, 2, and 3, ICAM-RyR-(4558-4671)-GFP and ICAM-RyR-(4830-4919)-GFP, were localized to the plasma membrane; fusion proteins containing the fourth transmembrane segment of RyR, ICAM-RyR-(4913-4943)-GFP, were retained in the ER. Biochemical assay showed that ICAM-RyR-GFP fusion proteins that target to the plasma membrane are fully glycosylated, and those retained in the intracellular membrane are core-glycosylated. Together our data indicate that amino acids 4918-4943 of RyR contain the signal sequence for ER retention of the Ca(2+) release channel.
Bhat
Manjunatha B
MB
The Cleveland Clinic Foundation, Center for Anesthesiology Research/FF40, Cleveland, Ohio 44195, USA.
Ma
Jianjie
J
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017930:Genes, Reporter; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013696:Temperature
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D049452:Green Fluorescent Proteins; D002118:Calcium