Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver

Abstract

Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.

Fig 1. Comparison of rat cLPM proteomics with previous rat liver and brush border membrane proteomic studies.

A: Representation of unique and overlapping proteins between rat cLPM compared with previous proteomics studies conducted on rat liver: cLPM this study, total [24], microsomes [25], plasma membrane [26] B: Representation of unique and overlapping proteins between rat cLPM and brush broader membrane proteome: cLPMthis study, kidney brush border membrane [27], total [24], plasma membrane[26].

Fig 2. RT-PCR and Western blot analysis on P4- and P5-ATPases.

(A) Representation of relative mRNA expression in rat liver of the P4- and P5-ATPases by semi quantitative RT-PCR analysis. The expression is given is relative to the house keeping genes actin and GAPDH as described in Materials and Methods. Data are the mean of two independent determinations (biological replicates) with 3 technical replicates each. (B) Western blot analysis of P4-ATPases in different liver subcellular fractions. The lanes were loaded with 1. homogenate 2. microsomes, 3. cLPM 4. bLPM, 5.mitochondria. For detecting the P4-ATPases and the canalicular marker APN, the following amount of protein was loaded for each lane: ATP8B1: 100 μg; ATP9A: 150 μg; ATP11A: 100 μg, ATP11C: 50 μg; APN: 150 μg

Fig 3. Immunohistochemical localization of ATP8A1 in non-perfused rat liver 5μm cryosections.

(A) ATP8A1. (B) Aminopeptidase-N localized on the canalicular membrane (C) Colocalization of ATP8A1 and aminopeptidase-N illustrates the expression of ATP8A1 at canalicular membrane of rat hepatocytes. Scale bar: 20 μm

Fig 4. Immunohistochemical localization of ATP9A in non-perfused rat liver 5μm cryosections.

(A) ATP9A. (B) BSEP expressed at the canalicular membrane of rat hepatocytes. (C) Colocalization of ATP9A and BSEP shows ATP9A expression at the canalicular membrane of rat hepatocytes. Scale bar: 20 μm

Fig 5. Immunohistochemical localization of ATP11A in perfused rat liver 5μm cryosections.

(A) ATP11A. (B) Expression of RAB7, a late endosome marker. (C) Colocalization of ATP11A and RAB7 shows considerable overlap demonstrating ATP11A in a late endosomal compartment. Scale bar: 20 μm

Fig 6. Immunohistochemical localization of ATP11A in perfused rat liver 5μm cryosections.

(A) ATP11A. (B) Expression of EEA1, an early endosome marker. (C) Colocalization of ATP11A and EEA1 shows partial overlap demonstrating ATP11A in an early endosomal compartment. Scale bar: 20 μm