The product of the gene GEF1 of Saccharomyces cerevisiae transports Cl- across the plasma membrane.
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.
Instituto de Neurobiología, Departamento de Neurobiología Molecular y Celular, Universidad Nacional Autónoma de México - Campus Juriquilla, Querétaro, México.
Angélica
A
López-Rodríguez
Alfonso Cárabez
AC
Trejo
Leanne
L
Coyne
Robert F
RF
Halliwell
Ricardo
R
Miledi
Ataúlfo
A
Martínez-Torres
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics; D018118:Chloride Channels / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015870:Gene Expression; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009544:Niflumic Acid; D009579:Nitrobenzoates / Q000494:pharmacology; D015388:Organelles / Q000648:ultrastructure; D010101:Oxygen Consumption; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014617:Vacuoles / Q000737:chemistry; D014982:Xenopus laevis
D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; C089713:GEF1 protein, S cerevisiae; D009579:Nitrobenzoates; D029701:Saccharomyces cerevisiae Proteins; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D009544:Niflumic Acid