Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast.

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site.

Rvs167p, the budding yeast homolog of amphiphysin, colocalizes with actin patches.

In this report, we have shown that the yeast amphiphysin-like protein Rvs167p was localized mainly in small cortical patches throughout the cell in unbudding cells. During budding, the patches were polarized at bud emergence site. During mating, Rvs167p was concentrated at the tip of the shmoo. Rvs167p colocalized with actin patches during yeast vegetative growth and mating. Complete disruption of the actin cytoskeleton using Latrunculin-A did not affect Rvs167p localization in patches throughout the cell. In rvs167 mutant cells, actin patches are mislocalized and in rvs161 or abp1 mutant cells, Rvs167p localization is not affected. These observations suggest that Rvs167p may localize the actin cortical complex properly. Finally, the amphiphysin-conserved N-terminal domain of Rvs167p, called the BAR domain, was required but not sufficient for the correct localization of the protein.

Proteome analysis and morphological studies reveal multiple effects of the immunosuppressive drug mycophenolic acid specifically resulting from guanylic nucleotide depletion.

Mycophenolic acid (MPA), one of the most promising immunosuppressive drugs recently developed, is a potent inhibitor of IMP dehydrogenase, the first committed step toward GMP synthesis. We found that all the drug effects on yeast cells were prevented by bypassing GMP synthesis, thus confirming the high specificity of MPA. Although the primary target of MPA is clearly identified, we aimed to further understand how GTP depletion leads to growth arrest and developed a new approach based on proteome analysis combined with overexpression studies. Essential proteins down-expressed in the presence of MPA were identified by protein two-dimensional gel analysis and subsequently overexpressed in yeast. Two such proteins, Cdc37p and Sup45p, when overexpressed allowed partial relief of MPA toxicity, strongly suggesting that their lower amount after MPA treatment significantly contributed to the MPA effect. These conserved proteins involved in cell cycle progression and translation are therefore important secondary targets for MPA. Our data establish that MPA effects occur through inhibition of a unique primary target resulting in guanine nucleotides depletion, thereby affecting multiple cellular processes.