Identification of a cold-sensitive step in the mechanism of modeccin action.

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 ...

Section: Response To Toxinsmentioning

confidence: 99%

Recovery of function in Chinese hamster ovary cell mutants with temperature-sensitive defects in vacuolar acidification.

Roff

Hall

Robbins

1990

“…A third explanation is that the defective lysosomes upset vesicular traffic through late endosomal vesicles. This could impair the action of modeccin which appears to penetrate to the cytoplasm from a late compartment (9,32). Also, diphtheria toxin does not begin arresting protein synthesis in ceils until after a minimum lag period of 15-20 min, consistent with the possibility that this toxin may also need to reach a late endosome before entering the cytoplasm (16,19).…”

Section: Discussionmentioning

confidence: 85%

“…Modeccin is normally internalized by receptor-mediated endocytosis and subsequently penetrates to the cytoplasm from an intracellular vesicle, possibly lysosomes (9,32), where it catalytically inactivates protein synthesis (10,25). We compared the sensitivities of wild-type cells, V.24.1 cells, and a revertant, termed V.24.1R31, to modeccin at a restrictive temperature of 41°C (Table I).…”

Section: Revertants Of V241mentioning

confidence: 99%

Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells.

Colbaugh

Stookey

Draper

1989

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“…Abrin was purchased from Sigma Chemical Corp (St. Louis, MO). Rabbit antimodeccin and anti-DT were prepared as previously described (24,26). Sources for other reagents and for cell culture supplies have been previously identified (17,24,27,28).…”

Section: Methodsmentioning

confidence: 99%

“…Preparation and Use of Radiolabeled Modeccin and DT: ~25I-modeccin was prepared as described by Draper et al (24). The specific activity of recovered modeccin ranged between 3 and 23 x 106 cpm/ ug for different preparations.…”

Section: Growth Curves and Plating Efficiency Experimentsmentioning

confidence: 99%

A Chinese hamster ovary cell mutant with a heat-sensitive, conditional-lethal defect in vacuolar function.

Marnell¹,

Mathis²,

Stookey³

et al. 1984

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