The CHO cell mutant FD 1.3.25 exhibits both increased accumulation and altered distribution of endocytosed fluid phase tracers. Neither the rate of tracer internalization nor the kinetics of recycling from early endosomes was affected, but exocytosis from late endocytic compartments appeared to be decreased in the mutant. Endocytosed tracer moved more rapidly to the cell poles in FD1.3.25 than in wild type cells. An abundant 36-kD polypeptide was found associated with taxol-polymerized microtubules in preparations from wild type and mutant; in the former but not the latter this polypeptide could be dissociated by incubation of the microtubules in ATP or high salt. The 36-kD polypeptide co-electrophoresed in two dimensions with the monomer of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Analysis of cDNA clones showed that the mutant is heterozygous for this enzyme, with approximately 25% of the GAPDH RNA containing a single nucleotide change resulting in substitution of Ser for Pro234, a residue that is conserved throughout evolution. Stable transfectants of wild type cells expressing the mutant monomer at approximately 15% of the total enzyme exhibited the various changes in endocytosis observed in FD1.3.25.

The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events.

After 4 h at 41 degrees C, B3853 and M311, temperature-sensitive Chinese hamster ovary cell End1 and End2 mutants, respectively, are pleiotropically defective in endocytosis and trans-Golgi network-associated activities (Roff, C. F., R. Fuchs, I. Mellman, and A. R. Robbins. 1986. J. 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 cation-independent 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.

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 degrees C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of alpha 2-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 degrees 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 degrees C; maximal alteration of most susceptible functions was obtained 4 h after temperature shift. At 39 degrees C, the mutants exhibited many but not all of the changes manifested at 41 degrees 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 ("End1"); M311 and I223 are in another ("End2"). In the End1 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 I223 (but not in M311) the full spectrum of phenotypic changes could be produced at the permissive temperature by inhibition of protein synthesis.

A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071). DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification. Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 X DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1-5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae. That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.

A Chinese hamster ovary cell mutant defective in the receptor-mediated endocytosis of several unrelated ligands (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071) failed to accumulate iron provided in the form of diferric transferrin. Analysis of the steps of the transferrin cycle indicated that binding and internalization of transferrin proceeded normally in mutant cells. However, the mutant appeared unable to dissociate iron from transferrin, as evidenced by release of diferric transferrin from the mutant versus apotransferrin from the parent. Uptake of ferric ions from the growth medium was enhanced in the mutant.

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 min 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.