Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p
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Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p. / Schwappach, B; Stobrawa, S; Hechenberger, M; Steinmeyer, K; Jentsch, T J.
In: J BIOL CHEM, Vol. 273, No. 24, 12.06.1998, p. 15110-8.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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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 -