3C). These observations suggest that BSEP is internalized through

a Rab5a- and dynamin-dependent process. The C-terminal tail of BSEP encompassing residue 1284-1321 contains a canonical Tyr-based signal (Y1310-Y-K-L-V) that might overlap Selleck Midostaurin a leucine-based (Leu1301-M) signal. To assess these signals, we appended the seven amino acids (GAYYKLV) to the Tac molecule with an eight-alanine amino acid linker region to investigate the ability of this motif to internalize. We found that Tac-8AGAYYKLV was able to internalize into punctuate structures (Fig. 4). Mutations of Y1310Y1311 or L1313V1314 to alanines abolished the internalization of the Tac chimera and caused the protein to be retained at the plasma membrane (Fig. 4). These data demonstrated that the Y1310Y1311 and L1313V1314 amino acids are important for internalization. SB203580 research buy In order to investigate the relative contribution of the tyrosine-based motif compared with the leucine-based motif within the 38–amino acid C-terminal end of BSEP, we mutated Y1310Y1311 (Tac-YY), L1303M1304 (Tac-LM), or both (Tac-YYLM) to alanine residues and observed their internalization by immunofluorescence and cell ELISA. Compared with TacCterm, Tac-YY remained on the cell surface after internalization for 20 minutes at 37°C (Fig. 5A). However, the Tac–LM internalized to a similar extent as

TacCterm, indicating that these two residues do not contribute to internalization of this construct (Fig. 5A). In addition, mutating both putative motifs resulted in loss of internalization compared with TacCterm, MCE公司 similar to the Tac–YY (Fig. 5A). Quantitation of internalization of these Tac chimeras was then determined using a cell ELISA internalization assay. In HEK293T cells, 40% of cell surface TacCterm was internalized in 20 minutes, resulting in a rate of ∼2%/min (Fig. 5B). In contrast, Tac alone was internalized at a rate of ∼0.5%/min (Fig. 5B). The efficiency of internalization was completely abolished in the Y1310Y1311

mutant. Mutation of L1303M1304 did not lead to a significant decrease in internalization, confirming that the leucine-based motif does not contribute significantly to endocytosis. Therefore, the Y1310Y1311 is the predominant signal for internalization. Transfection of HEK293T cells with full-length BSEP with or without the tyrosine mutations was then carried out in order to confirm the importance of this motif in endocytosis. Immunofluorescence showed that GFP–BSEP was expressed on the cell surface in both the wild-type and mutant transfected cells (Fig. 6A). Cell surface biotinylation was used to label the BSEP before internalization and membrane stripping. Strepavidin bead pull-down demonstrated that full-length wild-type GFP–BSEP that had been expressed on the plasma membrane could be efficiently endocytosed (Fig. 6B). Densitometry of the blots revealed that this internalization occurred at a rate similar to that demonstrated with TacCterm (Supporting Fig. 2).