K. Hauser et al., 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, MOL MICROB, 37(4), 2000, pp. 773-787
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) ar
e 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 a
mount 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 di
stribution pattern. However, when GFP is inserted in the major cytoplasmic
loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, proba
bly because of the presence of the overt C-terminal KKXX ER-retention signa
l and/or masking of a signal for transfer into ASs. On the overall cell sur
face, new SERCA molecules seem to be permanently delivered from the ER to A
Ss by vesicle transport, whereas in the fission zone of dividing cells ASs
may form anew. In cells overexpressing PtSERCA1 (with C-terminal GFP) in AS
s, [Ca2+](i) regulation during exocytosis is not significantly different fr
om controls, probably because their Ca2+ pump has to mediate only slow reup
take.