TY - JOUR
T1 - Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport
AU - Wei, Ying
AU - Chen, Jun
AU - Rosas, Gisele
AU - Tompkins, D. Andrew
AU - Holt, P. Andrew
AU - Rao, Rajini
PY - 2000/8/4
Y1 - 2000/8/4
N2 - Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca2+ and Mn2+ ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca2+ pumps and screened for loss of Ca2+ and Mn2+ transport on the basis of hypersensitivity to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn2+ toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn2+ toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including 45Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable 45Ca transport; of these, a subset also showed defects in traf-ticking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn774 and Asp778 in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca2+ and Mn2+ ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.
AB - Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca2+ and Mn2+ ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca2+ pumps and screened for loss of Ca2+ and Mn2+ transport on the basis of hypersensitivity to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn2+ toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn2+ toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including 45Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable 45Ca transport; of these, a subset also showed defects in traf-ticking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn774 and Asp778 in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca2+ and Mn2+ ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.
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U2 - 10.1074/jbc.M002618200
DO - 10.1074/jbc.M002618200
M3 - Article
C2 - 10801855
AN - SCOPUS:0034604602
SN - 0021-9258
VL - 275
SP - 23927
EP - 23932
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 31
ER -