Chinese hamster ovary cell mutants with temperature-sensitive defects in endocytosis. I. Loss of function on shifting to the nonpermissive temperature.

Abstract

Abstract. We have isolated three independent Chinese hamster ovary cell mutants (B3853, I223, and M311) with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. Activities affected at 41°C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of ctz-macroglobulin, compartmentalization of newly synthesized acid hydrolases, resistance to ricin, and sensitivity to diphtheria and Pseudomonas toxins and modeccin. The three mutants also displayed decreased sialylation of some secreted glycoproteins at 41°C, reminiscent of the nonconditional mutant DTG1-5-4 that showed both endocytic and Golgi-associated defects (Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman, 1984, J. Cell Biol., 99:1296-1308. Phenotypic changes were detectable within 30 min after transfer of the mutants to 41°C; maximal alteration of most susceptible functions was obtained 4 h after temperature shift. At 39°C, the mutants exhibited many but not all of the changes manifested at 41°C; resistance to diphtheria and Pseudomonas toxins required the higher temperature.Analysis of cell hybrids showed that B3853 and DTG1-5-4 are in one complementation group ("Endl"); M311 and I223 are in another CEnd2"). In the Endl mutants, loss of endocytosis correlated with complete loss of ATP-dependent endosomal acidification in vitro; in the End 2 mutants partial loss of acidification was observed. At the nonpermissive temperature, residual levels of endocytic activity in B3853 and M311 were nearly identical; thus, we conclude that the differences measured in endosomal acidification in vitro reflect the different genetic loci affected, rather than the relative severity of the genetic lesions. The mutations in M311 and I223 appear to have different effects on the same protein; in 1223 (but not in M311) the full spectrum of phenotypic changes could be produced at the permissive temperature by inhibition of protein synthesis. S EVERAL laboratories have described mutant Chinese hamster ovary (CHO) cells that are pleiotropically defective in receptor-mediated endocytosis (17,21,22,28,29). Endosomes isolated from some of these mutants were shown to be defective in ATP-dependent acidification (20,29). Although decreased endosomal acidification has yet to be demonstrated in vivo, many aspects of the mutants' phenotypes are consistent with such a defect; e.g., increased resistance to diphtheria toxin and enveloped RNA viruses (21,22,28,29) and decreased accumulation of Fe from transferrin (13).However, other facets of the mutants' phenotypes do not appear to follow directly from loss of endosomal acidification; decreased release of Sindbis virus and decreased galactosylation of Sindbis glycoproteins (29) suggest a defect in Golgi function. Other phenotypic changes, e.g., oversecretion of newly synthesized acid hydrolases (28,29,36), are ambiguous in that they could result from either Golgiassociated or endosomal defects.Through analysis of revertants and cell-cel...

Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.

“…This allows these ligands to be delivered normally to lysosomes in DTG 1-5-4 and DTF 1-5-1 (27 ; Table 11). 2 Similar results have been found with temperature-sensitive, endocytosis CHO mutants from the same complementation groups (30).…”

A Fluorescent Probe of Polyamine Transport Accumulates into Intracellular Acidic Vesicles via a Two-step Mechanism

“…2A) as expected from the temperature dependence of endocytosis (23). At the permissive temperature (34°C), net uptake of BODIPY FL-transferrin in End1 cells and wild-type cells was similar, but was strongly reduced in End1 cells upon transfer to 40°C, as expected from the known phenotype of these mutants (21). On the other hand, no difference was observed in the uptake of Spd-C 2 -BODIPY between wild-type and End1 cells at either 34 or 40°C (Fig.…”

“…Because a subpopulation of the PSVs could be labeled with Texas Red-transferrin, a marker of recycling endosomes, this suggested that receptor-mediated endocytosis was, at least in part, an integral part of the polyamine uptake mechanism. To address that hypothesis, we used End1 CHO mutants that are strongly defective in receptor-mediated endocytosis and exhibit pleiotropic defects at the non-permissive temperature (40°C) (21). The latter include defective ATP-dependent acidification that mainly affects early endosomes and the TGN, with late endosomes and lysosomes being largely spared (21,22).…”

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

“…The CHO mutants DTF 1-5-1 and DTG 1-5-4 obtained by Robbins and coworkers are defective in the uptake of lysosomal enzymes via the mannose-6-phosphate receptor (21,22) and have a diminished capacity to obtain iron from Tf (7; Robbins, A., personal communication). Genetic complementation has shown that DIG 1-5-4 is in the complementation group "Endl" and DTF 1-5-1 in another group "End2" (23). The characteristics of the CHO mutants suggest that they are defective in delivery of ligands to an appropriate acidic environment.…”