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...

Abstract. After 4 h at 41°C, B3853 and M311, temperature-sensitive Chinese hamster ovary cell Endl and End2 mutants, respectively, are pleiotropically defective in endocytosis and trans-Golgi network-associated activities (Roff, C. E, R. Fuchs, I. Mellman, and A. R. Robbins. 1986. Z Cell Biol. 103:2283-2297. We have measured recovery of function after return to the permissive temperature. Based on return of normal transferrin-mediated Fe uptake and sensitivity to diphtheria toxin both mutants had restored endosomal function at 10 h; based on delivery of endocytosed lysosomal enzymes to lysosomes and normal sensitivity to modeccin both had functional late endocytic organelles at 10-12 h; and based on retention of newly synthesized lysosomal enzymes and sialylation of secreted glycoproteins both had functional trans-Golgi network at 6 h. At 10 h, M311 had recovered almost all of its ability to endocytose lysosomal enzymes; B3853 required 30 h to recover fully its ability to endocytose lysosomal enzymes. Slow recovery of mannose 6-phosphate-dependent uptake in B3853 reflected altered trafficking of cationindependent mannose 6-phosphate receptors. Although B3853 had normal amounts of receptor at 6-8 h, it had greatly diminished amounts of receptor at the cell surface. Altered trafficking was also suggested by the finding that B3853 rapidly degraded receptor that had been present before the shift to the nonpermissive temperature. F our classes of mutant Chinese hamster ovary (CHO)cells (End1-4) exhibiting temperature-sensitive (is) ~ defects in acidification of organelles have been defined by genetic complementation (7,8,36). Endl-3 mutants were originally characterized as defective in ATP-dependent endosomal acidification (26,27,35,36,44,54,55). Acidification of other organelles is also affected in Endl and End2 mutants; indirect evidence has been presented suggesting altered acidification of the trans-Golgi network (TGN;35,36), and a recent study demonstrates impaired lysosomal acidification in both of these classes (Robbins, A. R., and C. E Roff, manuscript in preparation). To date, evidence that the primary lesion is defective acidification exists only for an End3 mutant: ATP-dependent acidification of a light membrane fraction was ts (49), as were both ATP hydrolysis and ATP-dependent acidification in a coated vesicle preparation (47).We have examined a variety of activities in Endl and 2 mutants (34,35,36) and have shown that both classes of mutants are impaired in receptor-mediated accumulation of lysosomal enzymes, ct2-macroglobulin, and Fe presented as diferric transferrin. In addition, these mutants are resistant to diphtheria and Pseudomonas toxins and modeccin and are hyper-1. Abbreviations used in this paper: M6P, mannose 6-phosphate; CD-M6PR, cation-dependent M6P receptor; CI-M6PR, cation-independent M6P receptor; TGN, trans-Golgi net~rk; ts, temperature-sensitive. sensitive to ricin. They also show defective segregation of newly synthesized lysosomal enzymes out of the secretory pathway and decreased ...