FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte
Introduction
Bile flow is one of the critical functions of the liver for excretion of drugs and lipids as well as endogenous and exogenous waste products [1], [2]. To comply with the demands of an ongoing bile flow, the hepatocytes and intrahepatic bile duct epithelial cells (cholangiocytes) express polarized transport systems at the basolateral and apical plasma membrane domains [3], [4]. Defective bile secretion results in cholestasis with accumulation of bile salts and other cholephiles within hepatocytes and blood plasma. Cholestasis may occur because of hereditary or acquired impairment of selective canalicular bile secretory processes or as a secondary consequence of chronic hepatocyte or bile duct damage [5]. A number of inheritable human disorders leading to cholestasis are associated with mutations in genes that encode hepatocyte transport proteins [6]. Progressive familial intrahepatic cholestasis type 1 (PFIC1; Ref. [7], [8]) and benign recurrent intrahepatic cholestasis (BRIC; Ref. [9], [10], [11]) are two such autosomal recessive inherited disorders, which are characterized by an impaired bile secretion [12]. These disorders initially manifest with similar clinical and biochemical features but they greatly differ in severity. Both are characterized by normal serum γ-glutamyltransferase activity and cholesterol levels, high concentrations of serum primary bile acids and low concentrations of biliary bile salts (especially chenodeoxycholic acid). BRIC is characterized by recurrent episodes of intrahepatic cholestasis, lasting weeks to months and starting in childhood or adulthood. The cholestatic attacks resolve spontaneously without leaving detectable damage in the liver. A distinguishing feature of PFIC1 is the progressive cholestasis starting in early childhood and leading to end-stage liver disease before reaching adulthood. The molecular basis of these forms of intrahepatic cholestasis was determined by positional cloning [13], [14]. PFIC1 and BRIC are caused by mutations in a single gene at chromosome 18q21, FIC1 [14], now officially renamed ATP8B1. Recently, we established that a third syndrome characterized by intrahepatic cholestasis, Greenland familial cholestasis, is also associated with a mutation in FIC1 [15]. FIC1 is the first characterized human member of the P4 subfamily of P-type ATPases; the proteins within this subfamily were suggested to function as aminophospholipid translocases [14], [16], [17], [18]. However, the aminophospholipid translocase activity of one of the other members of this subfamily (Drs2p from yeast) was questioned [19]. Thus, the molecular function of FIC1 is presently unclear. Besides its physiological role the subcellular localization within the liver also remains elusive. FIC1 mRNA is expressed in many epithelial tissues, including small intestine, pancreas, kidney and to a lesser extent in the liver [14]. The aim of the present study was to localize the FIC1 protein within the human and rodent liver to provide a basis for subsequent studies on the function of this protein.
Section snippets
Antibodies
cDNA fragments encoding different FIC1 cytoplasmic domains were individually subcloned into expression vector pQE30 (Qiagen, Düsseldorf, Germany) to create His6-tagged fusions. The sequences of the FIC1-C31, FIC1-C32 and FIC1-C6 fusion peptides correspond to the amino acid residues 539-748, 468–748 and 1164–1251, respectively of FIC1. These FIC1 sequences shared no significant sequence homology to other known liver membrane proteins. These His6-tagged fusion proteins were purified using Ni2+
Generation and characterization of FIC1 antibodies
To determine the subcellular localization of human FIC1 protein in the liver, polyclonal antibodies were raised against two distinct cytoplasmic domains of this protein. cDNA fragments encoding these domains were subcloned in a His6-affinity tag expression vector. To perform immunization of the rabbits, the His6-tagged fusion peptides FIC1-C31, FIC1-C32 and FIC1-C6 were overexpressed and purified from an Escherichia coli expression strain (Fig. 1a). After immunization, the raised antisera
Discussion
The intrahepatic cholestasis observed in the two clinically distinct disorders PFIC1 and BRIC is caused by mutations in a single gene, FIC1. The function of the FIC1-encoded protein is presently unclear. So far, the question of the localization of FIC1 in the liver has only been addressed in preliminary reports. However, these reports were in conflict with each other since either a localization in the cholangiocytes [28] or in the canalicular membrane [29] was suggested. Here, polyclonal
Acknowledgements
We express our appreciation to Carin Nibbering for stimulating discussions and to Dr R.J. Thompson for providing the liver biopsy material from the PFIC1 patient. We thank Titia Rolf and Ellen van Beurden for excellent technical assistance. EFE is supported by a grant from the Anton Meelmeyer Foundation (Academic Medical Center Amsterdam). SWCvM is supported by grant WS98-12 of the Dutch Digestive Disease Foundation; LWJK is the recipient of Ster grant SW006 of the University Medical Center
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2014, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :ATP8B1 is a P4 ATPase and is a phospholipid flippase that translocates phosphatidylserine from the exoplasmic to the cytosolic leaflet of the plasma membrane [3,4]. ATP8B1 is expressed in the apical membrane of many epithelial cell types, including hepatocytes, cholangiocytes and enterocytes [5,6]. In vitro studies have indicated that ATP8B1 exists as a heterodimer with its β-subunit CDC50A; heterodimerization is pivotal for endoplasmic reticulum (ER) exit and for activity of the protein [3,7–9].
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These authors contributed equally to this work.