Isolation of Heavy Endosomes from Dog Proximal Tubules in Suspension

Abstract

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42-44 kDa and 20-24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as "heavy endosomes" (buoyant density D = 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.

GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

“…10, January 1999 123 other set of experiments, RhoGDI (Boivin and Beliveau, 1995), a soluble protein that has been shown previously to exist both in the cytosol as well as in the membrane (Marshansky et al, 1996), is also detected more in the cytosol (70%) as compared with the membrane (30%) fractions ( Figure 4B). On the other hand, transmembrane proteins GS28 (Subramaniam et al, 1995) and rbet1 are detected only in the membrane fraction (Figure 4, C and D).…”

Renal Vacuolar H+-ATPase

“…Modelling of acidification rates reported from several intracellular organelles has confirmed that these acidification rates can only be achieved by the presence of an anion conductance that may be modulated by voltage (210). In many intracellular organelles and at cell plasma membranes, a parallel Cl Ϫ conductance provides an electrical shunt compensating for the positive charge transferred by the pump and thus dissipating the electrical gradient (345,346,355,384,385,392).…”

“…The presence of this parallel Cl Ϫ conductance has been found in most intracellular organelles that are acidified by vacuolar H ϩ -ATPases such as lysosomes (396), endosomes (24, 345,346,427,439,479,580), the transGolgi network (TGN) (129,574), and neurotransmitter storage vesicles (348,487). In most cases, the presence of an anion conductance was inferred from the higher rate of acidification of the organelles in the presence of chloride, or the generation of a higher transmembrane potential in its absence.…”

“…The chloride dependence of plasma membrane H ϩ -ATPase is, however, a much more complex issue because of the expression of numerous other conductances or electrogenic transporters in apical and basolateral membrane domains. In the kidney, it is beyond doubt that vacuolar H ϩ -ATPase-dependent acidification is Cl Ϫ dependent in endosomal fractions (24,238,345,346,427,439) and in brush-border membrane vesicles (270, 288,343,441,442). However, the role of chloride in apical H ϩ -ATPase-dependent proton secretion has been examined in only a few studies.…”

“…Many of the membranebound compartments that comprise the secretory and endocytotic pathways in epithelial and nonepithelial cells are acidified via the action of a vacuolar H ϩ -ATPase (188,345,346,354,355). As discussed above in this review, development of maximal acidification of these structures depends on the presence of a parallel anion (usually chloride) conductance in the same membrane to dissipate the positive potential that would otherwise result from accumulation of protons within the vesicles (24, 343, 479).…”