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. 2012 Jun;55(6):1901-11.
doi: 10.1002/hep.25523. Epub 2012 Apr 25.

A C-terminal tyrosine-based motif in the bile salt export pump directs clathrin-dependent endocytosis

Ping Lam  1 , Shuhua Xu, Carol J Soroka, James L Boyer
Affiliations

A C-terminal tyrosine-based motif in the bile salt export pump directs clathrin-dependent endocytosis

Ping Lam et al. Hepatology. 2012 Jun.

Abstract

The liver-specific bile salt export pump (BSEP) is crucial for bile acid-dependent bile flow at the apical membrane. BSEP, a member of the family of structurally related adenosine triphosphate (ATP)-binding cassette (ABC) proteins, is composed of 12 transmembrane segments (TMS) and two large cytoplasmic nucleotide-binding domains (NBDs). The regulation of trafficking of BSEP to and from the cell surface is not well understood, but is believed to play an important role in cholestatic liver diseases such as primary familial intrahepatic cholestasis type 2 (PFIC2). To address this issue, BSEP endocytosis was studied by immunofluorescence and a cell surface enzyme-linked immunosorbent assay (ELISA) endocytosis reporter system using a chimera of the interleukin-2 receptor α (previously referred to as Tac) and the C-terminal tail of BSEP (TacCterm). An autonomous endocytosis motif in the carboxyl cytoplasmic terminus of BSEP was identified. We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif (1310) YYKLV(1314) (YxxØ). When expressed in HEK293T cells, TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y(1310) Y(1311) amino acids in TacCterm and in full-length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that mediate ATP-dependent transport of broad substrate specificity.

Conclusion: Our results indicate that constitutive internalization of BSEP is clathrin-mediated and dependent on the tyrosine-based endocytic motif at the C-terminal end of BSEP.

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Figures

Figure 1
Figure 1
Identification and analysis of putative tyrosine-based and leucine-based motifs in BSEP. (A) Sequence alignment of BSEP from ten different species. The C-terminal 36-amino acid of BSEP contain a putative tyrosine-based motif (Y1310YKLV) and a leucine-based (THEEL1301M) motif. The latter has been predicted to be a casein kinase II site. (B) Schematic representation of the Tac-chimeras that were constructed as described in Materials and Methods. Tac chimeras (TacCterm and Tac-8AGAYYKLV) are constructs with the arrangement of Tac (open bar) and the C-terminal cytoplasmic tail of BSEP (filled bar) as illustrated. The BSEP C-terminal sequences are indicated with numbers representing the original BSEP sequence. Tac-8AGAYYKLV is the minimal BSEP construct consisting of eight alanines (8A) inserted between the Tac fragment and the short BSEP sequence, YYKLV, to generate Tac-8AGAYYKLV and different mutations as described in the text. TM refers to the transmembrane segment of Tac.
Figure 2
Figure 2
Internalization of TacCterm in HEK293T cells. (A) HEK293T cells were transiently transfected with TacCterm and internalization was detected after antibody labeling at 4°C and shifting to 37°C. Whereas only a small number of punctuate structures were detected in control Tac transfected cells, cytoplasmic puncta were readily evident in TacCterm, indicating internalization of the construct. Internalized TacCterm first presents (5–10 min) in a number of small vesicles and later (40 min) appears in larger, perinuclear vesicles.
Figure 3
Figure 3
Inhibition of internalization by dominant negative (DN) Rab5a-DSRed, N133I, and DN dynamin, K44A. (A) HEK293T cells were co-transfected with Rab5a (top) or Rab5aDN (bottom) and TacCterm construct. After 18 hrs of transient expression, the cell surface was labeled with anti-Tac antibody at 4° C, and then allowed to internalize at 37°C for 20 min. Endocytosed TacCterm (green) is colocalized with Rab5a-DSRed on swollen endosomes (top). However, expression of Rab5aDN-DSRed, N131I, partially prevented the internalization of surface TacCterm, as shown by accumulation of vesicles and small tubules at the periphery of the cell (bottom) Bar=5μm. (B) Quantitation of internalization of TacCterm after co-transfection with DN dynamin, K44A, was performed using a cell ELISA, as described in Material and Methods. TacCterm is internalized approximately 5 fold more than control Tac and this internalization of TacCterm was partially reduced after co-transfection with K44A dynamin. Tac control transfected with K44A dynamin was not significantly different than control Tac alone. N=3. Astericks indicate significant difference in percent internalization between Tac and TacCterm with p<0.05 using student two tailed t-test.
Figure 4
Figure 4
Tac-8A-GAYYKLV is sufficient for the internalization in HEK293T cells. The Tac-8AGAYYKLV was constructed with eight alanines (8A) followed by the tyrosine-based motif (GAYYKLV) fused to the C-terminal end of the Tac protein. After 20 min at 37°C Tac-GAYYKLV has been internalized into punctuate structures similar to those seen with TacCterm construct. The internalization was abolished in the mutant TAC-8AGAAAKLV, where both tyrosine residues were mutated to alanine residues or in the mutant TAC-8AGAYKAA, where the lysine and valine residue was mutated to alanine residues. Bar=5μm
Figure 5
Figure 5
The tyrosine-containing motif is essential for the internalization of TacCterm. (A) To investigate the role of the tyrosine-based (YYKLV) or leucine-based (LM) motif within the C-terminal end of BSEP, alanine substitutions were made in these amino acids and constructs were designated as follows: 1. Tac-YY (Y1310Y1311 ), 2. Tac-LM (L1303M1304) and 3. Tac-YYLM. Anti-Tac labeling at 4°C and internalization for 20 min was performed as previously described. Note the lack of internalization for the Tac-YY and Tac-YYLM mutants. Bar=5μm. (B) The amount of internalization from the cell surface was quantitated by cell ELISA as described in the Materials and Methods. Tac was internalized 13.1+3.1% and TacCterm was internalized 41.1+7.6% after 20 min at 37°C. Mutating Y1310Y1311 to alanine residues (Tac-YY) inhibited internalization completely (15.3+4.5%). Mutating L1303M1304 to alanine residues (Tac-LM) did not affect internalization with 36.3+2.3% internalized after 20 min. Mutating all four residues (Tac-YYLM) resulted in complete inhibition of internalization (12.7+3.0%). Results are means ± SD for three separate experiments performed in quadruplicate. Astericks indicate significant difference in percent internalization between TacCterm and Tac-YY or Tac-YYLM with p<0.05 using student two tailed t-test.
Figure 6
Figure 6
Mutation of the tyrosine-based motif in full length BSEP prevents endocytosis (A) HEK293T cells were transiently transfected with full length WT GFP-BSEP or Mut (A1310A1311) GFP-BSEP, fixed with 4% paraformaldehyde, and GFP fluorescence observed on a confocal microscope. Both cells showed cell surface expression of the GFP constructs. Bar=10 μm. (B and C) GFP-BSEP expressed on the cell surface was biotinylated as described in Methods and the cells were warmed to 37°C to follow the endocytic internalization. At the specified time points GFP-BSEP remaining on the cell surface was stripped with MESNA and the cells were lysed. The biotinylated protein internalized from the cell surface was subjected to streptavidin agarose (SA) pull down. Total cell lysates and SA bound proteins were separated by PAGE, transferred to nitrocellulose membrane and blotted with antibody to GFP or βactin. (B) WT GFP-BSEP was endocytosed in a time dependent manner. (C) No internalized Mut GFP-BSEP was detected even after prolonged exposure of the blot, confirming that the C-terminal tyrosine motif is important in BSEP endocytosis. Whole cell lysates indicate that the expression of the GFP-BSEP constructs was similar in all dishes and blotting with antibody to βactin showed equal loading in all lanes.
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