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

We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Col...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1989
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2115610/
https://www.ncbi.nlm.nih.gov/pubmed/2525560
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collection PubMed
description We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499- 507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes-- beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase-- was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta- glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.
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spelling pubmed-21156102008-05-01 Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells J Cell Biol Articles We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499- 507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes-- beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase-- was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta- glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes. The Rockefeller University Press 1989-06-01 /pmc/articles/PMC2115610/ /pubmed/2525560 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells
title Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells
title_full Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells
title_fullStr Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells
title_full_unstemmed Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells
title_short Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells
title_sort impaired lysosomes in a temperature-sensitive mutant of chinese hamster ovary cells
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2115610/
https://www.ncbi.nlm.nih.gov/pubmed/2525560