Fig. 6. Hrs-Y329/334F and Hrs-UIM are present in clathrin coats on endosomes. HeLa cells were transfected with GFP-Hrs, GFPY329/334F or GFP-UIM, processed for immunogold-labelling on cryosections as described in Materials and Methods. Cryosections were double labeled for GFP (10 nm gold) and clathrin (15 nm gold). (A-C) Overexpression of GFP-Hrs, GFP-Y329/334F and GFP-UIM leads to an increased recruitment of clathrin to endosomal vacuoles (E). All three constructs colocalise with clathrin on the peripheral endosomal membrane but label is absent from tubular extensions that emerge from the vacuole (arrows in B and not shown). (C) In contrast to A and B, cells transfected with GFP-UIM show GFP labelling at the limiting membrane as well as on internal vesicles of endosomal vacuoles. PM, plasma membrane. Scale bars: 200 nm.

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