Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport.
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 Ca(2+) and Mn(2+) ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca(2+) pumps and screened for loss of Ca(2+) and Mn(2+) transport on the basis of hypersensitivity to 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn(2+) 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 Mn(2+) toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including (45)Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable (45)Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn(774) and Asp(778) 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 Ca(2+) and Mn(2+) 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.
Wei
Y
Y
Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore Maryland 21205, USA.
Chen
J
J
Rosas
G
G
Tompkins
D A
DA
Holt
P A
PA
Rao
R
R
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000633:toxicity; D005656:Fungal Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D016623:Ion Pumps / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D017510:Protein Folding; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic; D015003:Yeasts
D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; D016623:Ion Pumps; D018832:Molecular Chaperones; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D004533:Egtazic Acid; D000251:Adenosine Triphosphatases; C032304:manganese ATPase; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium