Abstract
While it is well established that acidic pH in endosomes plays a critical role in mediating the orderly traffic of receptors and ligands during endocytosis, little is known about the bioenergetics or regulation of endosome acidification. Using highly enriched fractions of rat liver endosomes prepared by free flow electrophoresis and sucrose density gradient centrifugation, we have analyzed the mechanism of ATP-dependent acidification and ion permeability properties of the endosomal membrane. This procedure permitted the isolation of endosome fractions which were up to 200-fold enriched as indicated by the increased specific activity of ATP-dependent proton transport. Acidification was monitored using hepatocyte and total liver endosomes selectively labeled with pH-sensitive markers of receptor-mediated endocytosis (fluorescein isothiocyanate asialoorosomucoid) or fluid-phase endocytosis (fluorescein isothiocyanate-dextran). In addition, changes in membrane potential accompanying ATP-dependent acidification were directly measured using the voltage-sensitive fluorescent dye Di-S-C3(5). Our results indicate that ATP-dependent acidification of liver endosomes is electrogenic, with proton transport being accompanied by the generation of an interior-positive membrane potential opposing further acidification. The membrane potential can be dissipated by the influx of permeant external anions or efflux of internal alkali cations. Replacement externally of permeable anions with less permeable anions (e.g. replacing Cl- with gluconate) diminished acidification, as did replacement internally of a more permeant cation K+ with less permeant species (such as Na+ or tetramethylammonium). ATP-dependent H+ transport was not coupled to any specific anion or cation, however. The endosomal membrane was found to be extremely permeable to protons, with protons able to leak out almost as fast as they are pumped in. Thus, the internal pH of endosomes is likely to reflect a dynamic equilibrium of protons regulated by the intrinsic ion permeabilities of the endosomal membrane, in addition to the activity of an ATP-driven proton pump.
Highlights
Thispellet (P4),or alternatively P3, were used to prepare an enriched Golgi fraction, which was found to be enriched in endosomes, by flotation in discontinuous sucrose density gradients (18): P3 or P4 were resuspended by 10 strokes in atightfitting Dounce homogenizer in TEA buffer, and 2.0 M sucrose was added to a final concentration of 1.15 M. 15 ml of the resuspended pellets were placed in 38-ml centrifuge tubes, overlayed with 7 ml each of 1.0, 0.86, and 0.25 M sucrose, and centrifuged in a SW28 rotor (Beckman) at 80,000 X g., for 200 min
For separation of endosomes by free flow electrophoresis (FFE), the G1 fraction was concentrated by centrifugation (100,000 X g, 1 h) andresuspended in TEA buffer to a final concentrationof 1mg of protein/ml (19).As described in detail for the isolation of endosomes from tissue culture cells (12, 13), the sample was subjected to gentle trypsin treatment by incubation for 5 min at 37 'C with 0.2% L-1-tosylamido-2-phenylethylchloromethyl ketone-trypsin
Alkaline phosphodiesterase I was enriched (25-fold) in the shifted endosome fraction, and presumably reflected that fraction of plasma membrane ectoenzymes whichexist as intrinsiccomponents of endocytic vesicle membrane in rat liver (25, 26). This suggestion is supported by the fact that theenrichment of alkaline phosphodiesterase I in the G1 fraction by FFE was comparable (4-fold) to that observed for the endosome markers FITC-ASOR and FITCdextran
Summary
Dependent acidification in endosomes is electrogenic and is Receptor-mediated endocytosis is the major pathway for controlled by the activity of the H’-ATPase itself, the cellular uptake of nutrients, hormones, enzymes, immu- and by the ion permeability characteristics of the endonoglobulins, toxins, and viruses. In most cases, these ligands soma[1] membrane. The abbreviations usedare: NEM,N-ethylmaleimide;AMPPNP, adenyl-5”yl imidodiphosphate; HEPES, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; FFE, free flow electrophoresis; TEA, triethanolamine;FITC-dextran, fluorescein isothiocyanate-derivatized dextran; ASOR, asialoorosomucoid; FCCP, carbonylcyanide p trifluoromethoxyphenylhydrazone;ER, endoplasmicreticulum; CHO, Chinese hamster ovary; TMA, tetramethylammonium hydroxide
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