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MBC in Press, published online ahead of print October 6, 2004
Mol. Biol. Cell 10.1091/mbc.E04-06-0454

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Submitted on June 6, 2004
Revised on September 13, 2004
Accepted on September 23, 2004

Short Tetracysteine Tags to {beta}-Tubulin Demonstrate the Significance of Small Labels for Live Cell Imaging

Martin Andresen, Rita Schmitz-Salue, and Stefan Jakobs*

Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany

Monitoring Editor: Ted Salmon

Genetically encoded tags are of fundamental importance for live cell imaging. We show that small tetracysteine (TetCys) tags can be highly advantageous for the functionality of the host protein as compared with large fluorescent protein tags. One to three concatenated small TetCys tags as well as the large green fluorescent protein (GFP) were fused by integrative epitope tagging to the C-terminus of {beta}-tubulin (Tub2) in the budding yeast S. cerevisiae. The increasing tag size correlated with functional interference to the host protein. Tub2 tagged with either 1 x TetCys (10 amino acids (aa)) or 2 x TetCys (20 aa) was able to substitute Tub2 in haploid cells. In contrast, C-terminal tagging of Tub2 with 3 x TetCys (29 aa) or with GFP (244 aa) resulted in nonviable haploid cells. Cells expressing Tub2-1 x TetCys or Tub2-2 x TetCys were stained with FlAsH-EDT2, which selectively binds to the TetCys tag. The stained cells displayed dynamic FlAsH labeled microtubules and low cellular background fluorescence. The presented approach to tag ORFs at their native loci with very small TetCys tags and the subsequent visualization of the tagged proteins in vivo can be extended in principle to any ORF in S. cerevisiae.

*Corresponding author. E-mail: sjakobs{at}gwdg.de




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