Abstract
GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.
Highlights
The CLC1 proteins form an evolutionarily old group of membrane proteins with homologues identified in bacteria, archaebacteria, yeast, plants, and animals [1, 2]
This paper is available on line at http://www.jbc.org and COOH termini of the protein were subjected to alanine scanning mutagenesis
A similar pattern was observed with a construct in which the green fluorescent protein was fused to the carboxyl terminus of Gef1p (Fig. 1B)
Summary
The CLC1 proteins form an evolutionarily old group of membrane proteins with homologues identified in bacteria, archaebacteria, yeast, plants, and animals [1, 2]. One possible explanation is that some CLC proteins function as chloride channels in intracellular compartments. The strong homology of the GEF1 gene to CLC chloride channel genes suggests that its gene product might provide an electrical shunt for the Hϩ-ATPase, thereby facilitating the acidification of a common compartment. This paper is available on line at http://www.jbc.org and COOH termini of the protein were subjected to alanine scanning mutagenesis It revealed a 5-amino acid motif in the NH2 terminus that is essential for proper function. Growth assays on titrated media revealed an increased sensitivity of gef cells toward neutral pH These results are compatible with a role for the putative chloride channel Gef1p in compartmental acidification of the Golgi apparatus
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