The new tagging modules allow the generation of transcriptional fusions in their normal chromosomal loci under the endogenous promoter. By using integrative epitope tagging in haploid cells, the native ORF is replaced by its TetCys-tagged form. As a consequence, the potential functional artifacts possibly introduced by the tag to the host protein are not masked by additionally expressed native protein.
Heterozygous diploid cells readily incorporated tagged Tub2 into microtubules, irrespective of the size of the C-terminal tag. Similarly, GFP-tagged α-tubulin coexpressed with native α-tubulin has previously been used to study the dynamic behavior of yeast microtubules, giving important insight into microtubule turnover and dynamics (Carminati and Stearns, 1997
; Straight et al., 1997
; Maddox et al., 2000
). However, although the GFP-tagged Tub2 is localized to microtubules when coexpressed with Tub2, we found Tub2-GFP to be functionally restricted. Haploid cells expressing Tub2-GFP from the endogenous promoter were nonviable. This agrees with the finding of Huh et al
) who failed to generate a haploid strain expressing Tub2-GFP. Tub2 tagged with the 3×TetCys motif also did not complement the tub2
null mutant, although the 8 times smaller Tub2-3×TetCys was less disturbing to sporulation than Tub2-GFP in heterozygous cells. Hence, various cellular processes show different reactions, depending on the size of the tag to Tub2. We found that haploid cells expressing Tub2-2×Tet-Cys were viable. Therefore, the 9-aa size difference between the 2×TetCys-tag and the 3×TetCys-tag had a crucial impact on the functionality of the host protein. The C-terminal region of Tub2 seems to be located on the outside of the microtubule (Nogales et al., 1999
). This region has been shown to be important for the binding of motor proteins to microtubules (Okada and Hirokawa, 2000
). This prominent localization of the C terminus is a reasonable explanation for the observation that small C-terminal tags to Tub2 are better tolerated by the cells than large tags. Still, even the small 1×TetCys-tag apparently interferes with the full functionality of microtubules, because Tub2-1×TetCys-expressing cells have a reduced growth rate, which also might be reflected in the seemingly shorter microtubules in cells expressing Tub2-1×TetCys. It will require further analysis to determine to what extend the dynamical behavior of the microtubules is affected by the 1×TetCys-tag to Tub2.
In general, as in the case of microtubules, a large FP-tag may be especially interfering if the host protein is part of a highly ordered macromolecular complex that does not allow any extra space for a bulky tag. It is reasonable that small tags are also beneficial, for example, for mitochondrial proteins that must be in an unfolded linear conformation to pass through both the outer and inner membrane translocation channels (Truscott et al., 2003
). Bulky FPs, which are known to be relatively resistant against unfolding, are likely to cause problems in this process. We assume therefore that a substantial number of proteins could be identified where a small tag would be functionally superior to a large and disturbing FP tag.
With the FlAsH staining procedure presented here, unspecific background fluorescence was almost negligible in haploid cells. This is a surprising finding because high unspecific background has been reported to be the most serious drawback of the method in mammalian cell lines (Griffin et al., 2000
; Stroffekova et al., 2001
). FlAsH seems to have the highest affinity for the sequence Cys-Cys-Pro-Gly-Cys-Cys (Adams et al., 2002
). In the sequenced S. cerevisiae
genome only one ORF (YKL131W) can be identified that encodes a protein containing this sequence. However, YKL131W is currently annotated as a hypothetical ORF so the expression level of the corresponding protein is unclear but likely to be low, or the ORF is even not transcribed at all (Dolinski et al., 2004
). FlAsH also has been reported to bind, albeit with lower affinities, to other amino acid sequences, namely, to Cys-Cys-Xaa-Xaa-Cys-Cys or Cys-Cys-Xaa-Cys-Cys, where Xaa is a noncysteine amino acid (Adams et al., 2002
). The S. cerevisiae
genome contains another four ORFs coding for proteins with these motifs. All of them are low expressed proteins. Hence, yeast apparently contains only few endogenous proteins that potentially bind FlAsH. Together with the optimized staining procedure, this is the likely explanation for the low cellular background upon labeling with FlAsH-EDT2
Albeit its clear advantages, the biarsenical-tetracysteine system has several limitations. Using the tagging modules presented here, it is straightforward to omit the current spacer between the host protein and the 1×TetCys-tag, resulting in the minimal tag size of six aa. Still, even such a minimal tag may interfere with the full functionality of the host protein. Because the addition of EDT and FlAsH-EDT2
reduced the growth rate of the cells by only ~25%, toxicity of these compounds is not likely a major caveat of the system in S. cerevisiae
. The quantum efficiencies and extinction coefficients of FlAsH and GFP are in the same range, whereas FlAsH is more prone to photobleaching (Adams et al., 2002
). Therefore, it is not surprising that a direct comparison of cells expressing either Atp1-GFP or Atp1 tagged with 1×TetCys, 2×TetCys, or 3×TetCys resulted, under identical imaging conditions, in a higher fluorescence intensity in case of the GFP-tagged protein. Remarkably, concatenating multiple copies of the TetCys motif did not increase the fluorescence intensity, but the fluorescence seemed to be more resistant against photobleaching with increasing numbers of TetCys-tags. Similar observations have been made previously with multiple GFP-tags to the microtubule binding domain of the microtubule-associated protein E-MAP-115 (Faire et al., 1999
). In that study, it was shown that concatenating of GFPs did not result in an increase in fluorescence intensity but lead to a reduction of photobleaching. It is a well know phenomenon that concatenation of fluorophores frequently leads to mutual quenching of their fluorescence emission (Eggeling et al., 1999
; Lakowicz, 1999
). This may prevent an increase in fluorescence intensity with increasing numbers of attached fluorophores and also might account for the observed photostabilization effect. It can be anticipated that it will be a challenging task to use the biarsenical-tetracysteine system in combination with weakly expressed proteins or for extended time-lapse studies. We expect that other fluorophores with higher quantum efficiencies and less sensitivity to photobleaching would help to overcome this potential restriction of the biarsenical-tetracysteine system.
ReAsH, another TetCys-motif binding fluorophore with a red shifted emission wavelength, has been shown to be useful for correlating live-cell fluorescence microscopy with electron microscopy in mammalian cell lines (Gaietta et al., 2002
). Furthermore, it has been recently shown that ReAsH- or FlAsH-mediated chromophore-assisted light inactivation can be used to inactivate selected proteins within a cell (Marek and Davis, 2002
; Tour et al., 2003
). With the modules for epitope tagging presented here and the described methodology for live cell labeling, it can be anticipated that these concepts can be readily transferred to S. cerevisiae
on a genome-wide scale to complement and extend GFP-based approaches.
In summary, we have shown that small TetCys-tags can be highly advantageous for the functionality of the host protein as compared with large fluorescent protein tags. FlAsH-labeled proteins can be used to investigate protein dynamics in time and space. We have substantially enlarged the scope of the biarsenical-tetracysteine system by creating the tools to tag and visualize proteins expressed in S. cerevisiae from their endogenous promoters on a genome-wide scale.