Fig 2: The N-terminus and ß-domain of pVHL30 are required for MDM2 binding. (A) Schematic representation of pVHL30 domain composition. (B) Y2H assays revealed that none of pVHL30 domains alone bind MDM2 fragment 329–433 (n = 3). (C) Schematic representation of the MDM2 and pVHL30 fragments used in panel (D). (D) Y2H assays showed the interaction of pVHL30 1–157 region with MDM2 329–433 region (n = 3).

Fig 3: The intrinsically disordered portion of MDM2 is required for pVHL30 binding. (A) Schematic representation of MDM2 domain composition. (B) Y2H assays revealed that the C-terminal portion of MDM2 binds pVHL30. (n = 3). (C) Zoomed view and FELLS representation of the binding motif driving pVHL30/MDM2 association. Blue dots represent positively charged residues. The identified binding region partially overlaps the pseudo zinc-finger zf-RanBP. (D) Schematic representation of the C-terminal region MDM2 fragments used in panels (E,F). (E,F) Y2H assays revealed that the interaction of pVHL30 with MDM2 requires either the 329–350 or 434–451 regions of MDM2, yet the presence of the entire region (329–451) enhanced yeast growth (n = 3).

Fig 4: Network of interacting proteins. Connections between nodes represent experimental evidence for interaction, while colored bubbles are for proteins forming the network. A green edge represents the interaction between pVHL30 and p14ARF (CDKN2A), currently not yet reported in STRING, while a red line is used for the novel association of pVHL30 with MDM2 reported here.

Fig 5: pVHL interacts with MDM2 is an isoform-specific manner. (A) Left panel: Schematic representation of pVHL30 and pVHL19 domains. The two isoforms differ for the presence of an intrinsically disordered N-terminus (N). Right panel: Y2H assay showing that pVHL30 specifically associates with MDM2. C + , positive control. C-, negative control (n = 10). (B) Immunoprecipitation (IP) of total protein extracts from HEK293T cells expressing Flag-MDM2 and HA-VHL30 with an anti-Flag antibody and immunostained with anti-Flag antibody, and anti-pVHL/anti-HA antibodies, as indicated. Arrow correspond to HA-VHL30 recognized by anti-VHL antibody (n = 3). (C) IP of total protein extracts from MN-1 cells expressing pVHL30-GFP, pVHL19-GFP, and soluble GFP, as indicated, and processed for pull down with anti-GFP antibody revealed endogenous MDM2 pull down only in the presence of pVHL30-GFP. Endogenous MDM2 was detected with a specific antibody. Asterisks indicate from left to right: pVHL30-GFP, pVHL19-GFP, and GFP both in input and Ip panels (n = 2). (D) IP of total protein extracts from HEK293T cells with either an anti-MDM2 antibody and IgG mouse as control or anti-pVHL antibody and IgG rabbit as control showed specific reciprocal pull down of endogenous proteins. Asterisks indicate the specific band corresponding to MDM2 protein (n = 3).