Fig. 1.

ARRDC1 interacts with and recruits TSG101 to the cell membrane via a conserved PSAP motif. (A) Schematic representation of the ARRDC1 protein, indicating relevant structural domains: the N-terminal arrestin domain (gray box), the C-terminal PSAP motif (filled box), and PPXY motifs (open boxes). Also depicted are the ARRDC1 fragments identified in the yeast two-hybrid screen using the UEV domain of TSG101 as the bait. (B) Alignment of fragments of ARRDC1 orthologs showing conservation of the PSAP motif. (C) Western blot analysis of the interaction between FLAG-tagged wild-type (wt) ARRDC1 or the PSAP mutant (PAAP) and HA-tagged TSG101. Anti-FLAG immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected with the indicated constructs were examined by immunoblotting using the indicated antibodies. (D) Western blot analysis of the interaction between HA-tagged wild-type or a mutant (M95A) TSG101 and ARRDC1, as in C. (E) Localization of mCherry-TSG101 fusion protein in HEK293T cells cotransfected with GFP, ARRDC1-GFP, or its PSAP mutant (PAAP-GFP). Cells were fixed and examined by confocal microscopy. More than 20 cells expressing both ARRDC1-GFP and mcherry-TSG101 were examined, and all showed a similar localization pattern. Representative images are shown in the figure.