A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol.
Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6-NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non-endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.
Hanada
K
K
Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210.
Pagano
R E
RE
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D009154:Mutation; D010718:Phosphatidylserines / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D012641:Selection, Genetic
C092351:1-palmitoyl-2-(6-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)caproyl)glycero-3-phosphoserine; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008081:Liposomes; D010718:Phosphatidylserines; D010743:Phospholipids; D009327:4-Chloro-7-nitrobenzofurazan