Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles

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Abstract
We investigated the effects of reduced temperature, the pH elevators NH4 CI, monensin, and HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer, as well as the metabolic poisons NaF/KCN on transport of the fluid phase pinocytic marker, horseradish peroxidase (HRP), to lysosomes in Chinese hamster ovary (CHO) cells. In cell fractionation experiments, these agents appeared to block HRP transit at specific point(s) from “early” to “late” (i.e., low to high density) prelysosomal vesicles and lysosomes. Reduced temperature (17°C) most strongly inhibited HRP transport from low density, early endosomes to lysosomes. In long-term HRP uptakes at 17°C, marked peroxidase accumulation occurred both in early endosomes and in lysosomes. Loss (reversible pinocytosis) of HRP from “very early” endosomes occurred at 17°C. All three pH elevators including the common media supplement HEPES buffer inhibited transit of internalized HRP into lysosomes. For all three pH elevators, inhibition was most pronounced at the “early” endosome stage. The respiratory inhibitors NaF/KCN also inhibited transport most strongly at the early endosome stage. Together these results suggest that “early” steps in the endocytic transport of HRP are the most sensitive and that the conditions tested may exert direct effects on the processing of endocytic vesicles.