The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells. Here, using 125I-labeled anti-secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 degrees C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.

Free-flow electrophoresis (FFE) was used to investigate the intracellular compartments involved in fluid-phase marker, fluoresceine isothiocyanate (FITC)-dextran, transport in the isolated perfused rat liver. One to 2 min after uptake at 37 degrees C, FITC-dextran was found in endosomes with the same electrophoretic mobility as early sorting endosomes labeled either by the hepatocyte-specific marker asialoorosomucoid (ASOR) or by transferrin that enters all liver cells. Labeling at low temperature (16 degrees C) blocked transport of ASOR and dextran in early endosomes. With increasing internalization time (3-13 min) at 37 degrees C, FITC-dextran-labeled compartments co-localized with late, ASOR-containing endosomes. Since localization of FITC-dextran in late transcytotic compartments was not observed upon FFE separation, it is concluded that the majority of internalized markers is directed to lysosomes. The FITC-label did not account for the predominant lysosomal targeting of the dextran, since [3H]dextran-labeled endosomes exhibited an identical FFE pattern. Taken together, these data indicate that the fluid-phase marker dextran is transported through intracellular compartments with identical characteristics as endosome subcompartments of the receptor-mediated lysosomal route.

Coated vesicle fractions from a variety of tissues have been found to contain a vacuolar proton ATPase. Since these fractions contain both plasma membrane-and Golgi-derived coated vesicles, we sought to determine speciftcally whether endocytic coated vesidles from rat liver contain an active vacuolar proton ATPase. Endocytic vesicles (coated vesides and endosomes) were selectively labeled with pHsensitive endocytic tracers (fluorescein isothiocyanate-dextran or -asialoorsomucoid). Coated vesicles were then separated from endosomes by sucrose density gradient centugation. Although the endosomal fractions were found to exhibit significant ATP-dependent acidification activity, highly purified coated vesidles conining pH-senstive endocytic tracers were unable to generate a pH gradient in response to ATP addition. The coated vesicles could be passively acidified, however, by creating potassium diffson potentials, indicating that they were in fact capable of mntaining proton gradients. Moreover, signcant ATP-dependent acidification acvity was observed when the coated vedsce fractions were assayed using the nonselective externally added pH probe acridine orange. Thus, it appears that rat liver endocytic coated vesicles do not contain a functional proton pump. The active vacuolar proton ATlase found in these fractions instead reflected the presence of Golgi-derived coated vesicles or contaminating membranes.Many intracellular organelles of the endocytic and biosynthetic pathways maintain an acidic internal pH due to the activity of a vacuolar proton ATPase (V-ATPase). The main characteristics of this multisubunit V-ATPase are its electrogenicity and specific inhibition by N-ethylmaleimide and bafilomycin (1). Thus far, coated vesicles, endosomes, lysosomes, trans-Golgi elements, endocrine secretory granules, and cholinergic synaptic vesicles have been shown to be acidic (2, 3). Since these organelles maintain distinct pH values in vivo, their capacities for ATP-driven acidification must be regulated. While acidification in vitro has been shown to be regulated by the membrane potential (1, 3, 4), it is also possible that pH regulation is also controlled by regulating the activity or intracellular targeting of the V-ATPase itself.This raises the issue how functional proton pumps are delivered to endosomes and lysosomes. The most common view is that coated vesicles, which have long been known to possess a V-ATPase (5, 6), internalize proton pumps from the plasma membrane and deliver them to early endosomes and, thus, to late endosomes and lysosomes. It is possible, however, that V-ATPases are targeted to endosomes from the biosynthetic pathway via Golgi-derived coated vesicles. Since conventional preparations of coated vesicles contain both endocytic and Golgi-derived vesicles, the fact that these fractions have been found to contain ATP-dependent acidification activity does not establish the intracellular origin of the endosomal proton pump. Moreover, since coated vesicle acidification is typically monitored using...

Endocytosis at reduced temperature has been used to define and characterize endosome subpopulations. Thus, the temperature sensitivity of endosome subpopulations involved in transport to lysosomes and transcytosis in rat hepatocytes was analyzed applying endosome labeling in the isolated perfused rat liver with route-specific ligands in combination with temperature shift protocols. Free-flow electrophoresis (FFE) that separates membranes and organelles based on their surface charge was then applied to isolate functional endosomes. Using asialoorosomucoid (ASOR) and polymeric immunoglobulin A (pIgA) as specific ligands of the lysosomal and transcytotic route, respectively, two distinct endosome subpopulations along either pathway were separated by FFE. Upon a short (1-3 min) internalization at 37 degrees C, 125I-ASOR and fluorescein isothiocyanate (FITC)-pIgA were colocalized in common early endosomes. Following a 5-10 min chase of the ligands at 37 degrees C endosomes labeled with 125I-ASOR were separated from endosomes labeled with FITC-pIgA, indicative of two distinct late compartments along the lysosomal and transcytotic route. Internalization at 16 degrees C resulted in accumulation of both ligands in common early endosomes and, consequently, in inhibition of transport to lysosomes and transcytosis. When 125I-ASOR or 125I-pIgA were first chased into late compartments at 37 degrees C and the temperature was subsequently lowered to 16 degrees C, biliary secretion of 125I-ASOR-derived counts was arrested, while biliary output of 125I-pIgA continued. In summary, ASOR en route to lysosomes can be blocked in early as well as in late endosomes at 16 degrees C, while biliary secretion of pIgA cannot be prevented by temperature reduction once the ligand had been transferred from early to late compartments.

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