Evidence for a glycosylinositolphospholipid-anchored alkaline phosphatase in the aquatic plant Spirodela oligorrhiza.
Glycosylphosphatidylinositol (GPI)-anchored proteins occur widely, perhaps universally, on the surface of animal cells, where they perform a variety of important functions. However, the existence of GPI-anchored proteins on plant cells has never been established. Evidence is presented in this communication for the occurrence of a 50 kDa GPI-anchored alkaline phosphatase (AP) induced in the duckweed Spirodela oligorrhiza by phosphate deprivation. Triton X-114 partitioning of the Spirodela proteins yielded two forms of AP activity. The detergent-associated form was labeled prominently by [3H]ethanolamine, [3H]myristic acid and [3H]palmitic acid. This amphiphilic form of AP, like authentic GPI-anchored AP from mammals, was clearly resolved from the remaining, water-soluble AP activity by two types of incompletely-denaturing polyacrylamide gel electrophoresis. Lipid covalently bound to the solvent-delipidated amphiphilic AP was resistant to cleavage by phosphatidylinositol-specific phospholipase C. Strong acid or alkaline hydrolysis of the 3H-fatty acid-labeled amphiphilic AP yielded radioactive fatty acids and a radioactive lipid tentatively identified as a long chain base. The more abundant water-soluble AP was also radioactive in plants incubated with [3H]ethanolamine and was labeled to a lesser extent by 3H-fatty acids. The water-soluble AP, unlike its amphiphilic counterpart, could be freed of all fatty acid radioactivity by mild alkaline hydrolysis, indicating the continued presence of an ester-linked fatty acid. All evidence supports the conclusion that Spirodela AP is synthesized as an amphiphilic protein with a ceramide-containing GPI anchor.
Morita
N
N
Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
Nakazato
H
H
Okuyama
H
H
Kim
Y
Y
Thompson
G A
GA
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000378:metabolism*; D002240:Carbohydrate Sequence; D003902:Detergents; D004790:Enzyme Induction; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008969:Molecular Sequence Data; D017830:Octoxynol; D010944:Plants / Q000201:enzymology*; D011092:Polyethylene Glycols; D014316:Tritium; D014867:Water
D003902:Detergents; D004983:Ethanolamines; D017261:Glycosylphosphatidylinositols; D014867:Water; D014316:Tritium; D011092:Polyethylene Glycols; D019856:Ethanolamine; D017830:Octoxynol; C010615:Nonidet P-40; D000469:Alkaline Phosphatase