Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol.
Alkaline phosphatase is anchored to the outer leaflet of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol anchor. We have studied the biosynthetic transport and endocytosis of alkaline phosphatase in the choriocarcinoma cell line BeWo, which endogenously expresses this protein. It was demonstrated that the protein was synthesized as a Triton X-100-soluble precursor. During transport to the cell surface the enzyme was converted in a mature form, which was insoluble in Triton X-100 at 0 degrees C. Once at the cell surface 85% of alkaline phosphatase remained in the detergent-insoluble form. Under steady state conditions 15% of alkaline phosphatase was endocytosed. Most interestingly, this fraction of internalized alkaline phosphatase was completely soluble in Triton X-100 at 0 degrees C. After depletion of membrane cholesterol by saponin, alkaline phosphatase became completely soluble in Triton X-100 at 0 degrees C, suggesting that cholesterol plays a critical role in the formation and maintenance of Triton X-100-resistant membrane domains.
Cerneus
D P
DP
Department of Cell Biology, University of Utrecht, Medical School, The Netherlands.
Ueffing
E
E
Posthuma
G
G
Strous
G J
GJ
van der Ende
A
A
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002784:Cholesterol / Q000502:physiology*; D002822:Choriocarcinoma; D002999:Clone Cells; D004705:Endocytosis; D004792:Enzyme Precursors / Q000378:metabolism; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes; D008563:Membrane Lipids / Q000502:physiology*; D016253:Microscopy, Immunoelectron; D008871:Microvilli / Q000201:enzymology / Q000648:ultrastructure; D017830:Octoxynol; D011092:Polyethylene Glycols; D011247:Pregnancy; D012995:Solubility; D013501:Surface-Active Agents; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms
D004792:Enzyme Precursors; D007457:Iodine Radioisotopes; D008563:Membrane Lipids; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D017830:Octoxynol; D002784:Cholesterol; D000469:Alkaline Phosphatase