Priority targeting of glycosyl-phosphatidylinositol-anchored proteins to the bile-canalicular (apical) plasma membrane of hepatocytes. Involvement of ‘late’ endosomes

Abstract

1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the results suggest that a junction where exocytic and endocytic traffic routes meet occurs in a 'late' endocytic compartment.

Cellular function and molecular structure of ecto-nucleotidases

“…This includes rapid lateral motility, cellular sorting to the apical membrane surface of polarized cells, clustering in lipid rafts and also transmembrane signaling [250,252]. Accordingly, eN is sorted to the apical membrane of hepatic cells [253,254].…”

Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies

“…One interpretation of this result is that certain proteins are transferred directly from the Golgi apparatus to the bile-canalicular plasma membrane domain, thereby avoiding a detour to the sinusoidal plasma membrane. The demonstration that at least six glycosylphosphatidylinositol-anchored proteins were routed directly to the bile-canalicular surface, two of these via a 'late' endosomal compartment (Ali & Evans, 1990) …”

Dissection of the multifunctional “receptor-recycling” endocytic compartment of hepatocytes

“…For example, glycosylphosphatidylinositol (GPI)-anchored proteins such as 5Ј-nucleotidase or alkaline phosphatase are first delivered to the basolateral membrane before being transported to their functional destination. 3 The existence of a direct route 4,5 for adenosine triphosphate-binding cassette transporters has now been shown. 6 Finally, several lines of evidence indicate that the predominant (constitutive) recycling route for internalized membrane constituents is the direct vesicular shuttle from the peripheral tubulo-vesicular endosomes back to the plasma membrane.…”