The Journal of Cell Biology, Vol 110, 1023-1032, Copyright © 1990 by The Rockefeller University Press
Recovery of function in Chinese hamster ovary cell mutants with temperature-sensitive defects in vacuolar acidification
CF Roff, CW Hall and AR Robbins
Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
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.
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