Supplementary Figure 3: Identification of PSMD4 as binding partner of UBE3A by yeast two-hybrid screening. | Nature Neuroscience

Supplementary Figure 3: Identification of PSMD4 as binding partner of UBE3A by yeast two-hybrid screening.

From: Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome

Supplementary Figure 3: Identification of PSMD4 as binding partner of UBE3A by yeast two-hybrid screening.The alternative text for this image may have been generated using AI.

a. Screening for UBE3A interacting proteins. Mouse UBE3A-Iso3 lacking the C- terminal 6 amino acids was fused in frame to the LexA DNA-binding domain and a custom yeast two-hybrid screen (Hybrigenics) was performed against 69 million independent mouse adult brain cDNA clones, fused to the Gal4 activation domain. Twenty-four high confidence clones, which activated multiple reporter genes, encoding four different proteins were isolated: PSMD4 (also known as Rpn10 or S5a), UbcH7 (cognate E2 of UBE3A), and two novel UBE3A binding proteins, NSUN2 and RPH3A. b. Mapping of the binding sites on UBE3A with the four identified proteins. The full-length mUBE3A-Iso3 (FL) interacts strongly with PSMD4 and NSUN2 while the interaction with RPH3A and UbcH7 is relatively weak. Deletion of the UBE3A AZUL domain (Δ76, the domain required for nuclear localization of mUBE3A-Iso3) results in loss of interaction with PSMD4 while interaction with the other three binding partners is unaffected. Upon further deletion the interaction with RPH3A (Δ274) and NSUN2 (Δ466) is also lost, showing that the three binding partners, PSMD4, NSUN2 and RPH3A, interact with different regions in the N-terminal domain of UBE3A. All N-terminal deletion constructs harbor the HECT domain, which is the binding site for UbcH7 and therefore, the interaction with UbcH7 is unaffected. Strength of interaction was determined by spotting serial dilutions of yeast cells co-transformed with bait and prey plasmids on solid medium plates without histidine or without histidine and containing 10 mM 3-AT (3-amino triazol; inhibitor of the HIS3 gene product), or without histidine and containing 20 mM 3-AT or on medium lacking adenine. ‘–,’ indicates no growth on His- plates (no interaction); +, growth on His- plates (weak interaction); ++, growth on His- +10 mM 3AT plates (medium strength interaction); +++, growth on His- + 20 mM 3AT (strong interaction); ++++, growth on plates lacking adenine(Ade-) (very strong interaction). c. Graphical representation of the Selected Interaction Domain (SID) of PMSD4, based on the shared overlap of the 6 independently identified PMSD4 clones in the two-hybrid screen (see panel A). Indicated are the von Willebrand factor type A domain (VWA; green) and the two Ubiquitin Interacting Motifs (UIMs) of PMSD4. The SID encompasses approximately half of the second UIM and most of the C- terminal tail (amino acids 288-352).

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