Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum.
Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of the sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs were expressed in COS-1 cells, and mutant Ca2+-ATPases were assayed for Ca2+ transport function and phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, and 3 resulted in partial loss of Ca2+ transport function. In most cases, subsequent mutation of individual amino acids in the cluster had no effect on Ca2+ transport activity. In one cluster, however, it was possible to assign the reduction in Ca2+ transport activity to alterations of Asn111 and Asn114. The mutant Asn114 to alanine retained about 50% activity, whereas the change Asn111 to alanine retained only 10% activity. None of the mutations affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+. The combined experiments suggest that the reduced Ca2+ uptake observed in the Asn111 and Asn114 mutants was not due to a defect in enzyme activation by Ca2+ or in formation of the phosphorylated enzyme intermediate but rather to incompetent handling of the bound Ca2+ following ATP utilization. These results demonstrate that the acidic and amidated residues within the stalk region do not constitute the high affinity Ca2+-binding sites whose occupancy is required for enzyme activation. They may, however, act to sequester cytoplasmic Ca2+ and to channel it to domains that are involved in enzyme activation and cation translocation. Simultaneous mutation of 4 glutamate residues to alanine in the lumenal loop between transmembrane sequences M1 and M2 did not affect Ca2+ transport activity, indicating that acidic residues in this lumenal loop do not play an essential role in Ca2+ transport. Similarly, mutation of Glu192 and Asp196 in the beta-strand domain between stalk helices 2 and 3 did not affect Ca2+ transport activity, although mutation of Asp196 did diminish expression of the protein.
Clarke
D M
DM
Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Ontario, Canada.
Maruyama
K
K
Loo
T W
TW
Leberer
E
E
Inesi
G
G
MacLennan
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D005973:Glutamine / Q000235:genetics*; D015151:Immunoblotting; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D010766:Phosphorylation; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection
D005971:Glutamates; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D000252:Calcium-Transporting ATPases; D002118:Calcium