FlAsH labelling in living yeast cells.
Christian A. Wurm, Martin Andresen, Stefan Jakobs
Dept. NanoBiophotonics, MPI Biophysical Chemistry, Am Fassberg 11, Goettingen, 37077, Germany
Imaging of protein localizations and dynamics in living cells has developed into an indispensable tool in yeast cell biology. A major driving force has been the introduction of fluorescent proteins as fusion tags. However, fluorescent proteins are at least 220 amino acids in size, so that they are often bigger than the host protein to which they are fused. The biarsenical-tetracysteine labelling system, recently introduced by Roger Tsien and colleagues, provides an opportunity to use tags as small as six amino acids. Such a tag, the TetCys-motif, can bind with high affinity to membrane permeant dyes, like the green fluorescent FlAsH and the red fluorescent ReAsH. In order to complement GFP based tagging approaches, we have created a set of yeast tagging vectors containing various TetCys-motifs. We have developed the staining procedure with FlAsH and ReAsH into a routine method for labelling of (abundant) proteins in live S. cerevisiae. At the example of β -tubulin (Tub2) we demonstrate that large tags like GFP can be detrimental for the functionality of the host protein, whereas a small TetCys tag is tolerable. Stained cells displayed dynamic FlAsH labelled 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.