Figure 2. Poh1 facilitates aggresome clearance by producing unanchored ubiquitin chains.

(A) Immuno-purified FLAG-HDAC6 produced in 293T cells was incubated with unanchored ubiquitin chains (K63-linked Ub 1–7) or ubiquitinated protein (ub-protein, See Methods for details). The input and bound fractions were analyzed by immuno-blotting with the indicated antibodies. (B) A549 cells were transfected with control or Poh1-specific siRNA and treated with MG132 (5 μM, 24 hr) to induce aggresome or followed by MG132 washout as indicated. Aggresomes (marked by arrows) were detected and quantified as described in Fig 1a. Values represent the mean ± S.E.M. n = 3. (C) Poh1 knockdown (KD) cells were treated with MG132 to induce aggresomes followed by MG132 washout in the presence of DMSO or 2.5 μM of nocodazole (Noc.) for 24h. Cells containing aggresomes were quantified and averaged from three independent experiments in the histogram. Note that nocodazole treatment disrupted microtubule networks (α-tubulin, green) but had no effect on aggresome clearance. (D) A549 cells stably expressing shRNA-resistant wild type (wt) or catalytically inactive (CI) H113/115A mutant Poh1 were infected with Poh1-shRNA lentivirus. The percentage of cells containing an aggresome after MG132 washout was analyzed and quantified as described in (B). (E) Poh1 KD cells were pre-treated with MG132 to induce aggresome formation. 3h after MG132 was removed, cells were microinjected with indicated ubiquitin species or BSA mixed with fluorescence-conjugated dextran. The presence of aggresomes was analyzed 21h post-injection. Injected cells were identified by dextran (green, top panels) and marked by white dotted lines (bottom panels). Aggresomes were identified by staining with an antibody specific for K48-linked ubiquitin (red, clone Apu2) and marked by white arrowheads in injected cells. Note that only Poh1 KD cells injected with the K63-linked ubiquitin chains do not contain aggresomes. Non-injected cells retained aggresomes under all conditions (yellow arrowheads) and served as an internal control. Right panel shows the quantification from three independent experiments. 50 to 100 injected cells were scored in each experiment. *, p < 0.01. Error bars indicate ± S.E.M. Scale bar = 25 μm. (F) Control and Poh1 KD A549 cells were treated with 5 μM MG132 for 24h, or MG132 followed by 24h washout in the presence or absence of 10 mM 3-MA as indicated. Detergent insoluble fractions were isolated from whole cell lysates and resolved by SDS-PAGE, followed by immuno-blotting with a K48- or K63- specific ubiquitin antibodies as indicated. Actin is used as a loading control. See also Figure S2, S3, S4 and S8.