Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis.

Abstract

Populations of Chinese hamster ovary cells selected for resistance to diphtheria toxin were found to be highly enriched for mutants deficient in the uptake of lysosomal hydrolases via the mannose 6-phosphate receptor. One doubly defective mutant, DTF 1-5-1, exhibited increased resistance to Sindbis virus, although it was able to bind and internalize virus normally. Normal production of virus was obtained when, subsequent to virus binding, the mutant was exposed for 2 rain to acidic pH. Similarly, a shift to acidic pH increased the sensitivity of DTF 1-5-1 to diphtheria toxin 12-fold. Decreased uptake of lysosomal hydrolases by the mutant correlated with decreased mannose 6-phosphate receptor activity at the cell surface; results of lactoperoxidase-catalyzed iodination indicated that the surface-associated receptor was present but inactive on DTF 1-5-1. Total mannose 6-phosphate receptor activity was also decreased in the mutant and this decrease was reflected by increased secretion of lysosomal hydrolases. The phenotype of DTF 1-5-1 resembles in many ways that of cells treated with ammonia. We suggest that the defect in DTF 1-5-1 stems from an inability to deliver virus, diphtheria toxin, and lysosomal hydrolases to an acidic compartment. Other ligands may be endocytosed through a different pathway since the defect of DTF 1-5-1 did not decrease the endocytosis of ricin, modeccin, or Pseudomonas toxin and had minimal effects on uptake and degradation of low density lipoprotein.

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

“…This is consistent with penetration occurring from large endosomes. The resistance of the mutant cell lines to enveloped viruses (27,28) would appear to be in conflict with our finding that many of the large endosomes in the mutants have a pH <6.2. However, studies of the kinetics of Semliki Forest virus uncoating and penetration have shown that these processes are very similar in the mutants and wild-type cells (Marsh, M., A. Helenius, and I. Mellman, personal communication).…”

“…3). We speculate that the higher pH large endosomes are the sorting endosomes; this would be consistent with the inefficiency of the cells in releasing iron from "IT (13,40) and in delivering endocytosed lysosomal enzymes to lysosomes (27,28). Based on this we assign a pH >6.2 to the sorting endosomes and a pH of 5.2-6.0 to the late prelysosomal endosomes in the mutants.…”

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13,27,28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.…”

“…Acid phosphatase in contrast to other acid hydrolases is not deficient in I-cell fibroblasts, suggesting that this enzyme is not routed to lysosomes via the mannose-6-phosphate (man-6-P) receptor (14). Use of a lysosomal enzyme that is not carried by the man-6-P receptor was necessary since those enzymes are decreased in the CHO mutants (27,28). We confirmed that acid phosphatase is not depleted in the mutant cells by measuring the total cell enzyme levels.…”

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

“…Several groups have obtained CHO mutants pleiotropically defective in endocytosis by selecting cells that were resistant to diphtheria toxin (2,9,14,21). These mutants are also resistant to other toxins and certain viruses that require an acidic pH for toxicity or infectivity.…”