Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo.
Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA(i) to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA(i) phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA(i) lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.
Klöppel
Christine
C
University of Kaiserslautern, School of Biology, Erwin-Schrödinger-Strasse, D-67663 Kaiserslautern, Germany.
Müller
Alexandra
A
Marker
Simone
S
Simon
Martin
M
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D020134:Catalytic Domain; D005057:Eukaryotic Cells / Q000201:enzymology / Q000378:metabolism; D019143:Evolution, Molecular; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010247:Paramecium / Q000201:enzymology*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010738:Type C Phospholipases; D016650:Fluorescein-5-isothiocyanate