Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes.
Thy-1 is a membrane protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol anchor. Purified rat brain Thy-1 could be reincorporated into the plasma membrane of murine Thy-1- cells directly from aqueous suspension and without the use of detergents. A peripheral staining pattern similar to that observed for endogenous Thy-1 was achieved. Treatment with phosphatidylinositol-specific phospholipase C removed nearly all antibody staining due to either endogenous or inserted Thy-1. Fluorescence recovery after photobleaching (FRAP) was used to compare the lateral mobility of endogenous and inserted Thy-1. Both forms exhibited large lateral diffusion coefficients, but with a substantial immobile fraction (approximately 50%) indicating that the immobile fraction was not due either to chemical differences between inserted and native Thy-1 or to some surface Thy-1 molecules having a protein anchor. However, the inserted Thy-1 failed to activate mouse T lymphocytes upon crosslinking as assayed by [3H]thymidine uptake. Since Thy-1 could be directly labeled with rhodamine, the effect of the size of the labeling ligand on the mobility obtained by the FRAP technique could be explored. Rhodamine-conjugated MRC-OX7 monoclonal antibody or its fragments [R-F(ab)2 or R-Fab] were compared with rhodamine as labels for Thy-1. The measured diffusion coefficients were 1.6 x 10(-9), 2.0 x 10(-9), and 3.2 x 10(-9) cm2/sec for Thy-1 labeled with R-F(ab)2, R-Fab, and rhodamine, respectively; mobile fractions were all in the 40-50% range. Thus, the size of the ligand affects the lateral mobility of this labeled membrane protein to a measurable extent.
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
F
F
Zhang
W G
WG
Schmidt
Y
Y
Hou
A F
AF
Williams
K
K
Jacobson
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D008213:Lymphocyte Activation; D008223:Lymphoma; D008562:Membrane Glycoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008806:Mice, Inbred AKR; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D013154:Spleen / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D018800:Thy-1 Antigens
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D010716:Phosphatidylinositols; D018800:Thy-1 Antigens; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase