Glycosyl phosphatidylinositol-anchored proteins in chemosensory signaling: antisense manipulation of Paramecium tetraurelia PIG-A gene expression.
Glycosyl phosphatidylinositol (GPI)-anchored proteins are peripheral membrane proteins tethered to the cell through a lipid anchor. GPI-anchored proteins serve many functions in cellular physiology and cell signaling. The PIG-A gene codes for one of the enzymes of a complex that catalyzes the first step in anchor synthesis, and we have cloned the Paramecium tetraurelia pPIG-A gene using homology PCR. To understand the function of pPIG-A and the significance of GPI-anchored proteins in Paramecium, we reduced the mRNA for pPIG-A in transformed cells using an expression vector that transcribed antisense mRNA. The amount of transcript is reduced to approximately 0.3% of the mRNA in control-transformed cells. Compared to control cells, cells transformed with the antisense pPIG-A vector show reduced synthesis of GPI anchor intermediates catalyzed in their endoplasmic reticula and a very few GPI-anchored proteins among the peripheral proteins that can be recovered from their surfaces. They also show specific defects in chemoresponse to glutamate and folate. Other cellular functions, such as growth and mating, seem to be normal.
Yano
Junji
J
Department of Biology, University of Vermont, Burlington, Vermont 05405, USA.
Rachochy
Villa
V
Van Houten
Judith L
JL
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002628:Chemoreceptor Cells / Q000187:drug effects / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015870:Gene Expression / Q000187:drug effects; D017125:Genes, Protozoan; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016805:Paramecium tetraurelia / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry; D016372:RNA, Antisense / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D017261:Glycosylphosphatidylinositols; D016376:Oligonucleotides, Antisense; D015800:Protozoan Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger