Green fluorescent protein-tagged sarco(endo)plasmic reticulum Ca2+-ATPase overexpression in Paramecium cells: isoforms, subcellular localization, biogenesis of cortical calcium stores and functional aspects.
We have followed the time-dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP) attached to the C-terminus of the PtSERCA1 gene. The outlines of alveolar sacs (ASs) are labelled, as is the endoplasmic reticulum (ER) throughout the cell. When GFP fluorescence is compared with previous anti-PtSERCA1 antibody labelling, the much wider distribution of GFP (ER+ASs) indicates that only a small amount of SERCA molecules is normally retained in the ER. A second isoform, PtSERCA2, also occurs and its C-terminal GFP-tagging results in the same distribution pattern. However, when GFP is inserted in the major cytoplasmic loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, probably because of the presence of the overt C-terminal KKXX ER-retention signal and/or masking of a signal for transfer into ASs. On the overall cell surface, new SERCA molecules seem to be permanently delivered from the ER to ASs by vesicle transport, whereas in the fission zone of dividing cells ASs may form anew. In cells overexpressing PtSERCA1 (with C-terminal GFP) in ASs, [Ca2+]i regulation during exocytosis is not significantly different from controls, probably because their Ca2+ pump has to mediate only slow reuptake.
Hauser
K
K
Department of Biology, University of Konstanz, Germany.
Pavlovic
N
N
Klauke
N
N
Geissinger
D
D
Plattner
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D010247:Paramecium / Q000235:genetics*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000201:enzymology*
D017931:DNA Primers; D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000252:Calcium-Transporting ATPases; D002118:Calcium