Abstract
The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA.
Highlights
Receptor-mediated endocytosis is a process in which eukaryotic cells selectively internalize macromolecules and a large variety of extracellular solutes
Fusion of the docked vesicle with the target membrane involves the binding of the soluble proteins ␣-soluble NSF attachment protein (SNAP) and NSF to this SNARE complex, driven by the ATPase activity of NSF
The v- and t-SNARE families would determine the specificity of vesicular transport
Summary
Animals and Reagents—Male Harlan Sprague-Dawley rats weighing 200 –250 g were maintained under a controlled lighting schedule with 12-h dark period. For the pIgR immunoprecipitation, the mouse monoclonal anti-pIgR antibody (SC-166) was used according the procedure described by Luton et al [26]; briefly, liver endosomal fractions (50 g of protein), with or without IgA, were solubilized in a freshly buffer of 1% Nonidet P-40, 125 mM NaCl, 20 mM HEPES (pH 7.4), 10 mM NaF, 2 mM sodium vanadate, and protease inhibitor mixture. Immunofluorescence Studies in Liver and in Isolated Primary Cultured Hepatocytes: Uptake of pIgA, LDL, Transferrin-FITC, and Dextran-FITC—pIgA (100 g), LDL (5 mg), or dextran-FITC (5 mg) (Mr 70,000) or 0.9% NaCl for control animals, were injected into the portal vein (except for LDL, which was injected into the femoral vein) of Harlan Sprague-Dawley (200 –250 g) rats anesthetized with isofluorane, and 2.5 or 20 min later livers were perfused with 2% paraformaldehyde-PBS (50 ml).
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