1B) The localization of the TacCterm was followed by live cell l

1B). The localization of the TacCterm was followed by live cell labeling at 4°C with an antibody specific for the extracellular domain of Tac, followed by shifting to 37°C. After 10 minutes, TacCterm showed plasma membrane localization with small amounts localized to peripheral vesicles (Fig. 2, bottom). Internalization from the plasma membrane continued over the 60 minutes with an increase in the punctuate vesicular fluorescent pattern PF-562271 and, in addition, some shifting to a perinuclear location resembling a recycling endosomal compartment. Minimal

internalization from the plasma membrane was seen in the cells transfected with the Tac reporter alone (Fig. 2, top). These data were confirmed in COS-7 and HeLa cells (data not shown). These observations suggest that endocytic sorting signals in the C-terminus of BSEP are functional. Immunofluorescence experiments suggested that the internalized TacCterm was localized to the endosomal compartments (data not shown). In the early endocytic pathway, Rab5 regulates clathrin-coated vesicle–mediated transport from the plasma membrane to the early endosomes as well as homotypic early endosome fusion.32, 33 Therefore, we compared the effect of cotransfection with the Rab5a and Rab5a dominant-negative construct (Rab5a DN, I133N) on the internalization of

TacCterm in order to determine whether these vesicles were internalized via a clathrin-dependent pathway. TacCterm colocalized with Rab5a-DsRed in swollen endosomes in cotransfected cells (Fig. 3A, top). In contrast, when Rab5a DN was cotransfected, KU-57788 in vivo there appeared to be less internalization of TacCterm into the endocytic compartment (Fig. 3A, bottom). This Rab5a DN mutant has reduced guanosine triphosphatase (GTPase) activity and is a potent stimulator of homotypic fusion between early endosomes.34 Western blotting and cell enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that cotransfection with Rab5a resulted in slightly, but not significantly, less total and surface TacCterm (Supporting Fig. 1A,B).

Internalization of TacCterm was slightly higher in cells transfected with Rab5a and slightly lower in the presence of Rab5a DN compared with TacCterm alone, although neither were statistically different Astemizole (Supporting Fig. 1C). These results suggest that TacCterm most probably enters the early endosomal vesicles following a clathrin-dependent pathway. Clathrin-dependent and a subset of clathrin-independent endocytosis requires the activity of dynamin, an adenosine triphosphatase (ATPase) responsible for pinching vesicles from the plasma membrane and therefore driving cargo internalization into carrier vesicles.35, 36 To determine whether TacCterm internalization was dynamin dependent, a dominant-negative dynamin mutant (K44A-GFP) was transfected with TacCterm into HEK293T cells.

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