The product of the geneGEF1ofSaccharomyces cerevisiaetransports Cl^−across the plasma membrane

Abstract

Expression of GEF1 in Xenopus laevis oocytes and HEK-293 cells gave rise to a Cl- channel that remained permanently open and was blocked by nitro-2-(3-phenyl-propylamino) benzoic acid and niflumic acid. NPPB induced petite-like colonies, resembling the GEF1 knock-out. The fluorescent halide indicator SPQ was quenched in a wild-type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB. Immunogold and electron microscopy located Gef1p in the plasma membrane, vacuole, endoplasmic reticulum and Golgi apparatus. Eleven substitutions in five residues forming the ion channel of GEF1 were introduced; some of them (S186A, I188N, Y459D, Y459F, Y459V, I467A, I467N and F468N) did not rescue the pet phenotype, whereas F468A, A558F and A558Y formed normal colonies. All the pet mutants showed reduced O2 consumption, small mitochondria and mostly disrupted organelles. Finally, electron microscopy revealed that the plasma membrane of the mutants develop multiple foldings and highly ordered cylindrical protein-membrane complexes. All the experiments above suggest that Gef1p transports Cl- through the plasma membrane and reveal the importance of critical amino acids for the proper function of the protein as suggested by structural models. However, the mechanism of activation of the channel has yet to be defined.

The metal-coordinated Casiopeína IIIEa induces the petite-like phenotype in Saccharomyces cerevisiae

“…The impaired growth was also associated with the formation of multiple smaller mitochondrial reticules, a probable explanation for the lower respiratory activity. However, we did not find any other clear indication of alterations in the organelles such as those found in petite strains (López-Rodríguez et al 2007).…”

“…This phenotype is derived when the cells are affected in the respiratory chain (Greene et al 1993;López-Rodríguez et al 2007). Figure 2a shows the result of the drop assay after exposing the cells to increasing amounts of Cas IIIEa.…”

“…The sole distinguishable trait observed in the mitochondria was the reduced diameter of the mitochondrial network; but we can not consider it statistically significant because it was not evident in all the analyzed cells and it could be related to the cell-size reduction. In contrast, the plasma membrane of those cells was clearly affected, presenting multiple folds that resemble those found in a petite strain that resulted of the deletion of the gene GEF1 (López- Rodríguez et al 2007). The use of yeast as an experimental model to assess the effect of new antiproliferative molecules offers important advantages with its speed of screening and a deciphering of basic cellular and biochemical processes.…”

“…That was also the case of yeast, where O 2 consumption was severely reduced from 2.62 ± 0.21 Nat of O 2 in cells growing in YPGE to less than 1 Nat of O 2 in those exposed to Cas IIIEa. This experiment revealed that the level of O 2 consumption of yeast growing in Cas IIIEa is even lower than that consumed by a petite strain whose GEF1 gene has been knocked out (López-Rodríguez et al 2007). The reduction of O 2 consumed was consistent with the limited foci of activity indicated by the fluorescent MitoTracker TM .…”

Outwardly Rectifying Anionic Channel from the Plasma Membrane of the Fungus Phycomyces blakesleeanus

“…Thus, homologues of the CLC animal chloride channel were found in the genomes of 15 fungal species (30). So far, the protein product of GEF1, a CLC gene homologue in S. cerevisiae, is the only one that has shown expression and function in yeast endomembranes and plasma membranes when expressed in a heterologous system (Xenopus oocytes) (27).…”