A CHINESE-HAMSTER OVARY CELL MUTANT DEFECTIVE IN THE NON-ENDOCYTIC UPTAKE OF FLUORESCENT ANALOGS OF PHOSPHATIDYLSERINE - ISOLATION USING A CYTOSOL ACIDIFICATION PROTOCOL
K. Hanada et Re. Pagano, A CHINESE-HAMSTER OVARY CELL MUTANT DEFECTIVE IN THE NON-ENDOCYTIC UPTAKE OF FLUORESCENT ANALOGS OF PHOSPHATIDYLSERINE - ISOLATION USING A CYTOSOL ACIDIFICATION PROTOCOL, The Journal of cell biology, 128(5), 1995, pp. 793-804
Transmembrane movement of phosphatidylserine (PS) and various PS analo
gs at the plasma membrane is thought to occur by an ATP-dependent, pro
tein-mediated process. To isolate mutant CHO cells defective in this a
ctivity, we first obtained conditions which inhibited the endocytic, b
ut not the non-endocytic pathway of lipid internalization since PS may
enter cells by a combination of these two pathways. We found that aci
dic treatment of cells, which blocks clathrin-dependent endocytosis, e
nhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[{7-nitrobenz-
2-oxa-1,3 azol-4-yl}amino]caproyl-sn-glycero-3-phosphoserine (C-6-NBD-
PS) in CHO cells from donor vesicles (liposomes) by about twofold. Con
trol experiments demonstrated that the enhanced uptake of C-6-NBD-PS a
t acidic pH was not due to: (a) an increase in the capacity of the pla
sma membrane to incorporate C-6-NBD-PS from the donor vesicles; (b) a
decrease in the rate of loss of C-6-NBD-PS from the cells; or (c) fusi
on or engulfment of the donor vesicles. When cytosolic acidification (
to pH 6.3) was imposed without acidification of the extracellular medi
um, C6NBD-PS uptake by intact cells was increased by about 50% compare
d to control values determined in the absence of acidification. These
results suggested that a protein and energy dependent system(s) for tr
ansbilayer movement of the fluorescent PS was stimulated by cytosolic
acidification. A screening method for mutant cells defective in the no
n-endocytic uptake of fluorescent PS analogs with replica cell colonie
s 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 u
ptake of fluorescent PS analogs was reduced to about 25% of the wild t
ype level at either pH 6.0 or 7.4. Control experiments demonstrated th
at the reduced uptake of fluorescent PS analogs in the mutant cells wa
s unrelated to multidrug resistance, and that endocytosis of another p
lasma membrane lipid marker occurred normally in the mutant cells. The
se results suggested that a non-endocytic pathway responsible for upta
ke of fluorescent PS analogs was specifically affected in the mutant c
ells.