Single amino acid mutations in transmembrane domain 5 confer to the plasma membrane Ca2+ pump properties typical of the Ca2+ pump of endo(sarco)plasmic reticulum.
Conserved residues in some of the transmembrane domains are proposed to mediate ion translocation by P-type pumps. The plasma membrane Ca(2+) pump (PMCA) lacks 2 of these residues in transmembrane domains (TM) 5 and 8. In particular, a glutamic acid (Glu-771) residue in TM5, which is proposed to be involved in the binding and transport of Ca(2+) by the sarcoplasmic reticulum Ca(2+) pump (SERCA), is replaced by an alanine (Ala-854) in the PMCA pump. Ala-854 has been mutated to Glu, Asp, or Gln; Glu-975 in TM8, which is an Ala in the SERCA pump, has been mutated to Gln, Asp, or Ala. The mutants have been expressed in three cell systems, with or without the help of viruses. When expressed in large amounts in Sf9 cells, the mutated pumps were isolated and analyzed in the purified state. Two of the three TM8 mutants were correctly delivered to the plasma membrane and were active. All the TM5 mutants were retained in the endoplasmic reticulum; two of them (A854Q and A854E) retained activity. Their properties (La(3+) sensitivity and decay of the phosphorylated intermediate, higher cooperativity of Ca(2+) binding with a Hill's coefficient approaching 2) differed from those of the expressed wild type PMCA pump, and resembled those of the SERCA pump.
Guerini
D
D
Institute of Biochemistry, Swiss Federal Institute of Technology, 8092 Zürich, Switzerland.
Zecca-Mazza
A
A
Carafoli
E
E
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D027682:Cation Transport Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007313:Insecta; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases; D017434:Protein Structure, Tertiary; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013997:Time Factors
D027682:Cation Transport Proteins; D018076:DNA, Complementary; D018698:Glutamic Acid; D007811:Lanthanum; D053499:Plasma Membrane Calcium-Transporting ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium