期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 104, 期 10, 页码 3693-3697出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0611713104
关键词
fluorescent protein; HisZiFiT; live cell labeling; zinc
资金
- NIGMS NIH HHS [P20 GM072033, GM 72033] Funding Source: Medline
- NINDS NIH HHS [NS 27177, R37 NS027177, R01 NS027177] Funding Source: Medline
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 Zn2+ ions binds tightly but reversibly to hexahistidine (Hiss) 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 Ca2+ 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.
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