Abstract
Hepatocytes must transport newly synthesized apical membrane proteins from the basolateral to the apical plasma membrane. Our earlier morphological study showed that the apical proteins share a late (subapical) part of the transcytotic pathway with the well characterized polymeric immunoglobulin A receptor (Barr, V. A., and Hubbard, A. L. (1993) Gastroenterology 105, 554-571). Starting with crude microsomes from the livers of [35S]methionine-labeled rats, we sequentially immunoadsorbed first vesicles containing the endocytic asialoglycoprotein receptor and then (from the depleted supernatant) vesicles containing the polymeric IgA receptor. Biochemical characterization indicated that early basolateral and late endosomes were present in the first population but not in the second. Neither Golgi-, apical plasma membrane (PM)-, nor basolateral PM-derived vesicles were significant contaminants of either population. Both vesicle populations contained 35S-labeled receptor and 35S-labeled-dipeptidyl peptidase IV. Importantly, the elevated relative specific activity of the dipeptidyl peptidase (% of 35S-labeled/% immunoblotted) in the second population indicated that these vesicles must transport newly synthesized dipeptidyl peptidase IV. A distinct kind of vesicle was immunoadsorbed from a "carrier-vesicle fraction"; surprisingly, these vesicles contained little 35S-receptor and virtually no dipeptidyl peptidase IV. These results, together with previous kinetic data from in vivo experiments, are consistent with a computer-generated model predicting that newly synthesized dipeptidyl peptidase IV is delivered to basolateral endosomes, which also contain newly synthesized polymeric immunoglobulin A receptor. The two proteins are then transcytosed together to the subapical region.
Highlights
The plasma membrane (PM)1 of polarized epithelial cells is separated into distinct domains that have different functions and compositions [2, 3]
In contrast to the polymeric IgA receptor (pIgA-R), many apical PM proteins either have very short cytoplasmic tails with no obvious sorting signals (e.g. dipeptidyl peptidase IV (DPP IV) and aminopeptidase N; Refs. 30 –35) or have no cytoplasmic sequences whatsoever (e.g. 5Ј nucleotidase, a glycophosphatidylinositol (GPI)-anchored protein) [36, 37]. All of these proteins are transcytosed in hepatocytes [6, 7]. Do they travel with pIgA-R? In an earlier study, we found that bile duct ligation (BDL) slowed the transport of vesicles to the apical PM, and led to accumulation of newly synthesized pIgA-R and apical PM proteins in a common subapical tubulovesicular compartment [1]
The specific activity of the DPP IV was low in all of the samples that were immunoadsorbed from carrier vesicle fractions (CVF). (The quantification of DPP IV in the SC166 samples from CVF is given in Fig. 10.) We examined immunoadsorptions from animals labeled for 90 min, to look for transcytotic vesicles that might have already passed through a compartment found in the 110-min CVF
Summary
The plasma membrane (PM) of polarized epithelial cells is separated into distinct domains that have different functions and compositions [2, 3]. All newly synthesized PM proteins studied so far are transported from the Golgi first to the basolateral PM domain. We found that bile duct ligation (BDL) slowed the transport of vesicles to the apical PM, and led to accumulation of newly synthesized pIgA-R and apical PM proteins in a common subapical tubulovesicular compartment [1]. This result supports the idea that apical PM proteins share at least some of the pIgA-R pathway.
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