A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure.
Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn(2+) ions binds tightly but reversibly to hexahistidine (His(6)) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca(2+) from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His(6) tags are accessible to the dye or antibodies, demonstrating externalization.
Hauser
Christina T
CT
Department of Pharmacology, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093-0647, USA.
Tsien
Roger Y
RY
D020816:Amino Acid Motifs; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007477:Ions; D008565:Membrane Proteins / Q000737:chemistry*; D008956:Models, Chemical; D009363:Neoplasm Proteins / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D000071737:Stromal Interaction Molecule 1; D020349:Surface Plasmon Resonance; D013499:Surface Properties; D015032:Zinc / Q000737:chemistry*
D005456:Fluorescent Dyes; C471213:His-His-His-His-His-His; D007477:Ions; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009842:Oligopeptides; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D006639:Histidine; D015032:Zinc; D002118:Calcium