Glycosyl-phosphatidylinositol-anchor addition signals are processed in Nicotiana tabacum.
Recent studies have demonstrated the existence of glycosyl-phosphatidylinositol (GPI)-anchored proteins in higher plants. In this study we tested whether GPI-addition signals from diverse evolutionary sources would function to link a GPI-anchor to a reporter protein in plant cells. Tobacco protoplasts were transiently transfected with a truncated form of the Clostridium thermocellum endoglucanase E reporter gene (celE') fused with a tobacco secretion signal (PR-1a) at the N-terminus and either a yeast (GAS1), mammalian (Thy-1) or putative plant (LeAGP-1) GPI-anchor addition signal at the C-terminus. The yeast and plant C-terminal signals were found to be capable of directing the addition of a GPI-anchor to the endoglucanase protein (EGE') as shown by the sensitivity of the lipid component of GPI to phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. In contrast, the mammalian signal was poorly processed for anchor addition. When EGE' was fused to a truncated form of the LeAGP-1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI-anchor was not linked to the EGE' protein indicating the necessity for the missing amino acids. Our results show the conservation of the properties of GPI-signals in plant cells and that there may be some similar preferences in GPI-addition signal sequences for yeast and plant cells.
Takos
A M
AM
Centre for Plant Membrane Biology, School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia.
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Soole
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism*
D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins