Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

Abstract

Abstract. During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes" constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoreticaUy distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH ~6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH <5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH <6.2 in early endosomes with a t~/2 of 5 min. Although a fraction of the virus reached a pH of <5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t~/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41°C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH -,<5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H + pump to fluctuations in the endosomal membrane potential. WE have previously shown, using cell fractionation by free flow electrophoresis (FFE) ~ that internalized Semliki Forest virus (SFV) passes sequentially through two physically distinct subpopulations of endosomes en route to lysosomes in Chinese hamster ovary (CHO) cells (Schmid et al., 1988). Internalized SFV first appears (tja = 3-5 min) in an anodally deflected endosomal compartment of relatively low electrophoretic mobility, defined as "early" endosomes. After an •2 min lag, SFV next appears (t,i2 = Dr. S. Schmid's current address is Research

Endocytosis of influenza viruses

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Asparagine-linked Oligosaccharides Protect Lamp-1 and Lamp-2 from Intracellular Proteolysis

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Induction of cell transformation by mutated 16K vacuolar H+-atpase (ductin) is accompanied by down-regulation of gap junctional intercellular communication and translocation of connexin 43 in NIH3T3 cells

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