Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

Abstract

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

“…The mechanisms which underlie the sorting of lipids and proteins in the biosynthetic pathway are not well understood, but proteins in rafts acquire detergent-insolubility (one of the biochemical hallmarks for raft localization) during their transit through the Golgi apparatus (42,43). Depletion of cholesterol or disruption of sphingolipid synthesis has been shown to result in mislocalization of apical proteins to the basolateral domain (44,45) thus underscoring the importance of the lipid component in vesicular traffic. Rafts also appear to preferentially segregate proteins into receptor/signaling modules at the plasma membrane.…”

Trans-Golgi Network and Subapical Compartment of HepG2 Cells Display Different Properties in Sorting and Exiting of Sphingolipids

“…This fact gains considerable significance by recent observations that several hepatic apical proteins may reach the apical membrane by direct transport (14,15,44), which might link sphingolipid sorting to raft assembly. In this regard, questions have been raised concerning the ability of NBD lipids to partition into rafts, triggered in artificial liposomal systems, given their relative water solubility (45).…”

Caveolin-1 is enriched in the peroxisomal membrane of rat hepatocytes

“…The pellets (1-1.5 mg protein) were subjected to extraction by 1% Triton X-100 (Sigma-Aldrich) or 1% Lubrol WX (Sigma-Aldrich) for the isolation of Triton X-100-or Lubrol WX-resistant rafts, respectively, according to established methods. 25 Equal volume fractions of the gradient were analyzed by western blotting.…”