Expression in High Yield of Pig α1β1 Na,K-ATPase and Inactive Mutants D369N and D807N in Saccharomyces cerevisiae

Abstract

Studies of structure-function relationships in Na,KATPase require high yield expression of inactive mutations in cells without endogenous Na,K-ATPase activity. In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The ␣1-and ␤1-subunit cDNAs were inserted into a single 2-m-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage. The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 ؎ 3,000

Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

“…The resulting polymerase chain reaction fragments were digested with BamHI and EagI and inserted into similarly digested pPAP1933 (17). The remaining mutations were constructed by oligonucleotide cassette mutagenesis (16).…”

“…Growth of yeast cells expressing wild type Na,K-ATPase at 30°C, and Na,K-pump protein synthesis induced with galactose was performed as before (17). Yeast cells producing all other mutations were grown at 30°C until all glucose was metabolized, transferred to 15°C for 30 min, and then induced with 2% galactose (16).…”

“…Using modified growth media and fermentation protocols with reduced temperatures (15°C) during galactoseinduced Na,K-pump synthesis, these mutations can be ex-pressed in amounts sufficient for assay of high affinity binding of free ATP at equilibrium and interactions with Mg 2ϩ (17). In the present work 14 In the E 2 P form, the two residues are more important for hydrolysis of the acyl phosphate bond at Asp 369 .…”

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

“…Rat acinar cell line AR42J (CRL-1492, ATCC, passages [15][16][17][18][19][20][21][22][23][24][25] was cultured in RPMI media supplemented with 10 % fetal bovine serum (FBS) and penicillin/streptomycin. Cells were cultured in humidified incubators at 37°C, with 5 % CO 2 in air.…”

“…Plasmid pPAP7160 was generated by homologous recombination in yeast strain PAP1500 [19] between a polymerase chain reaction (PCR) fragment encompassing nucleotides 1-2,580 from pEGFP-N1 (Clontech, USA) and pEMBLyex4 [20]. The resulting plasmid can replicate in Escherichia coli and Saccharomyces cerevisiae and carries the elements from pEGFP-N1 required for expression in mammalian cells and EGFP.…”