Figure 1.
HD-PTP Binds to the Smad Regulators SARA and Endofin at the Endosome
(A) Top: domain organization of HD-PTP (CC, coiled-coil domain; PRR, proline-rich region; PTP, protein tyrosine phosphatase domain). Bottom: domain organization of endofin and SARA (FYVE, Fab1, YOTB, Vac1, and EEA1 zinc-finger domain; SBD, Smad binding domain; PRR, proline-rich region). In gray are the minimal HD-PTP interacting regions on endofin and SARA identified by the Y2H screen; black indicates the N-terminal 22-residue regions predicted to form α helices, with sequences shown below and identical residues highlighted in bold.
(B) Y2H assays show that endofin and SARA interact with HD-PTPFL (full-length) or HD-PTPBro1-CC, but not with AlixBro1-V. Yeast cells were transformed in triplicate with the identified prey constructs and full-length endofin or SARA bait constructs, and selected for interactions on QDO plates.
(C) HA-HD-PTP co-immunoprecipitates endogenous endofin and SARA from cell lysates.
(D and E) Co-expression of endofin-myc (D) or SARA-myc (E) redistributes HA-HD-PTP onto enlarged myc-labeled endosomes (indicated by arrows). Left panels are example images; right panels are quantitation from three independent experiments (mean ± SD). Scale bars, 10 μm.