Fig. 4: HECTD3 interacts with and ubiquitylates p62.

A Co-immunoprecipitation of endogenous p62, UbcH5b and exogenously expressed Flag-HECTD3 in HEK293T cells. IgG was used as the negative control for anti-p62 Ab. S and L mean short and long exposure. B HECTD3 and UbcH5b directly interact with p62, as measured by GST pulldown assays. Purified GST fused HECTD3 and UbcH5b proteins directly pulled down purified His-p62 in vitro. C Schematic representation of the domain architecture of the HECTD3 protein. D The DOC domain (216–393 aa) of HECTD3 is sufficient for p62 binding, as determined by the GST pulldown assay. Different GST-fused HECTD3 protein fragments were co-expressed with p62-Flag in HEK293T cells. GST was used as the negative control. E Schematic representation of GST-tagged p62 and its truncated mutants. F The PB1 domain (1–121 aa) of p62 is required for interactions with HECTD3, as determined by the GST pulldown assay. HEK293T cells were cotransfected with Flag-HECTD3 and GST-p62 or its truncated mutants. G HECTD3 ubiquitinates p62 in an E3 ligase activity-dependent manner in vitro. Recombinant purified His-p62 was incubated with UbE1, UbcH5b, HA-Ub, ATP, and HECTD3 or its catalytic inactive mutant C823A. His-p62 was pulled down using the Ni²⁺-NTA beads and ubiquitinated p62 protein was detected using anti-HA Ab. H HECTD3 ubiquitinates p62 at K420. HEK293T cells were cotransfected with HECTD3 or HECTD3-C823A and p62-Flag or p62-K420R-Flag for 24 h. The cell lysates were subjected to immunoprecipitation using the anti-Flag M2 beads under a denaturing condition, followed by WB using the indicated Abs. I HECTD3 ubiquitinates p62 with K29 and K-63 linked polyubiquitin chains. HECTD3, p62-Flag and HA-Ub (WT; K0; K63 only; K63R; K29 only; or K29R mutants) were expressed in HEK293T cells as indicated. The ubiquitinated p62 was immunoprecipitated using the anti-Flag M2 beads and probed with anti-HA Ab.