We describe here the results of complementation studies with six mutant Chinese hamster ovary cells expressing temperature-sensitive lesions affecting the endocytic pathway. The mutants were crossed with representatives of the End1 and End2 complementation groups identified previously by Robbins et al. (J. Cell Biol. 99:1296-1308, 1984). Two mutants, G.8.1 and 31.1, were members of the End1 complementation group. One mutant, 25.2, was a member of the End2 complementation group. The other three mutants each defined new complementation groups, which we have designated End3 (mutant G.7.1), End4 (mutant V.24.1), and End5 (mutant 42.2). Previous work on mutants of the End1, End2, and End3 classes had shown that these mutants were defective in endosomal acidification. We prepared postnuclear supernatants from mutants harvested at the nonpermissive temperature and compared their acidification activities, assessed by ATP-stimulated quenching of acridine orange. Members of the End1, End2, and End2 groups had reduced acidification activity, correlating with the acidification defects known to be expressed by these mutants. Strain V.24.1 (End4) also expressed a 40% reduction in acidification activity, while strain 42.2 (End5) had no reduction of acidification activity.

Abstract. 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

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A new method for isolating mutants defective in fluid-phase endocytosis has been developed based on the observation that endocytosed horseradish peroxidase can be made lethal to cells. The method was used to isolate a mutant from Chinese hamster ovary cells, termed HRP-1, that was temperature-sensitive for viability and had a 70% reduction in the rate of horseradish peroxidase endocytosis at the restrictive temperature. At high temperature, HRP-1 cells were also defective in the secretory path and their Golgi complex disappeared at the resolution of fluorescence microscopy. These properties are similar to two previously described mutants of CHO cells, DS28-6 and V.24.1. In complementation tests, mutants HRP-1, DS28-6, and V.24.1 all appeared to be in the same complementation group.