Document Detail

Priority targeting of glycosyl-phosphatidylinositol-anchored proteins to the bile-canalicular (apical) plasma membrane of hepatocytes. Involvement of 'late' endosomes.
MedLine Citation:
PMID:  2171497     Owner:  NLM     Status:  MEDLINE    
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.
N Ali; W H Evans
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  271     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1990 Oct 
Date Detail:
Created Date:  1990-11-15     Completed Date:  1990-11-15     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  193-9     Citation Subset:  IM    
National Institute for Medical Research, Mill Hill, London, U.K.
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MeSH Terms
Bacterial Proteins
Bile Canaliculi / chemistry*,  enzymology,  ultrastructure
Blotting, Western
Cell Membrane / chemistry*,  enzymology
Fluorescent Antibody Technique
Glycolipids / metabolism*
Intracellular Membranes / chemistry
Liver / enzymology,  ultrastructure*
Membrane Proteins / analysis*,  metabolism
Phosphatidylinositol Diacylglycerol-Lyase
Phosphatidylinositols / metabolism*
Phosphoinositide Phospholipase C
Phosphoric Diester Hydrolases / metabolism
Rats, Inbred Strains
Grant Support
//Wellcome Trust
Reg. No./Substance:
0/Bacterial Proteins; 0/Glycolipids; 0/Glycosylphosphatidylinositols; 0/Membrane Proteins; 0/Phosphatidylinositols; 58-85-5/Biotin; 9013-20-1/Streptavidin; EC 3.1.4.-/Phosphoric Diester Hydrolases; EC Phospholipase C; EC Diacylglycerol-Lyase

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