Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p

TY - JOUR

T1 - Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p

AU - Schwappach, B

AU - Stobrawa, S

AU - Hechenberger, M

AU - Steinmeyer, K

AU - Jentsch, T J

PY - 1998/6/12

Y1 - 1998/6/12

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Biological Transport/physiology

KW - Biomarkers/analysis

KW - Chloride Channels/chemistry

KW - Fungal Proteins/chemistry

KW - Golgi Apparatus/physiology

KW - Hydrogen-Ion Concentration

KW - Immunohistochemistry

KW - Iron/metabolism

KW - Mannosyltransferases/analysis

KW - Membrane Proteins/chemistry

KW - Molecular Sequence Data

KW - Mutagenesis/genetics

KW - Oligopeptides

KW - Peptides/immunology

KW - Proprotein Convertases

KW - Quinacrine/metabolism

KW - Saccharomyces cerevisiae/physiology

KW - Saccharomyces cerevisiae Proteins

KW - Sequence Alignment

KW - Sequence Deletion/genetics

KW - Subtilisins/analysis

U2 - 10.1074/jbc.273.24.15110

DO - 10.1074/jbc.273.24.15110

M3 - SCORING: Journal article

C2 - 9614122

VL - 273

SP - 15110

EP - 15118

JO - J BIOL CHEM

JF - J BIOL CHEM

SN - 0021-9258

IS - 24

ER -