Subcellular targeting of the endoplasmic reticulum and plasma membrane Ca2+ pumps: a study using recombinant chimeras.
ATP-powered Ca2+ pumps are located in the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). The two pump types share numerous structural and functional features; nevertheless, they are strictly targeted to different cell compartments. Chimeric SERCA/PMCA pumps were constructed to investigate the structural determinants responsible for their specific cellular location. The level of expression of the chimeric constructs and of the wild-type pumps in COS-7 and Sf9 cells was the same and so was their stability. One exception was chimera D, which showed special propensity to degradation. The chimeric constructs had no Ca(2+)-dependent ATPase activity, although in one trace amounts of the Ca(2+)-dependent phosphoenzyme intermediate were detected. Thus, the exchange of the regions encompassing the (NH2-terminal) transmembrane domains apparently is incompatible with the activity of the pump. The immunofluorescence experiments showed that the 85 NH2-terminal residues of the SERCA pump, encompassing the first transmembrane domain, contain a signal-promoting retention of chimeric constructs otherwise consisting of the PMCA pump structure in the endoplasmic reticulum. Additional structural determinants most likely also contribute to the retention of the SERCA pump in the endoplasmic reticulum: one chimeric construct (E) composed of the first two transmembrane domains of the PMCA pump, followed by the remainder of the SERCA pump structure, was still retained, even if not completely, in the endoplasmic reticulum.
Foletti
D
D
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich.
Guerini
D
D
Carafoli
E
E
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004247:DNA; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera
D011993:Recombinant Fusion Proteins; D004247:DNA; D000252:Calcium-Transporting ATPases; D002118:Calcium