A new type of non-Ca2+-buffering Apo(a)-based fluorescent indicator for intraluminal Ca2+ in the endoplasmic reticulum.
Genetically encoded Ca2+ indicators are outstanding tools for the assessment of intracellular/organelle Ca2+ dynamics. Basically, most indicators contain the Ca2+-binding site of a (mutated) cytosolic protein that interacts with its natural (mutated) interaction partner upon binding of Ca2+. Consequently, a change in the structure of the sensor occurs that, in turn, alters the fluorescent properties of the sensor. Herein, we present a new type of genetically encoded Ca2+ indicator for the endoplasmic reticulum (ER) (apoK1-er (W. F. Graier, K. Osibow, R. Malli, and G. M. Kostner, patent application number 05450006.1 at the European patent office)) that is based on a single kringle domain from apolipoprotein(a), which is flanked by yellow and cyan fluorescent protein at the 3'- and 5'-ends, respectively. Notably, apoK1-er does not interact with Ca2+ itself but serves as a substrate for calreticulin, the main constitutive Ca2+-binding protein in the ER. ApoK1-er assembles with calreticulin and the protein disulfide isomerase ERp57 and undergoes a conformational shift in a Ca2+-dependent manner that allows fluorescence resonance energy transfer between the two fluorophores. This construct primarily offers three major advantages compared with the already existing probes: (i) it resolves perfectly the physiological range of the free Ca2+ concentration in the ER, (ii) expression of apoK1-er does not affect the Ca2+ buffering capacity of the ER, and (iii) apoK1-er is not inactivated by binding of constitutive interaction partners that prevent Ca2+-dependent conformational changes. These unique characteristics of apoK1-er make this sensor particularly attractive for studies on ER Ca2+ signaling and dynamics in which alteration of Ca2+ fluctuations by expression of any additional Ca2+ buffer essentially has to be avoided.
Osibow
Karin
K
Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria.
Malli
Roland
R
Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria.
Kostner
Gerhard M
GM
Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria.
Graier
Wolfgang F
WF
Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria.
D001054:Apolipoproteins A / Q000737:chemistry; D001665:Binding Sites; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D037282:Calreticulin / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013997:Time Factors
D001054:Apolipoproteins A; D002021:Buffers; D037282:Calreticulin; D003902:Detergents; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human; D002118:Calcium