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. 2014 Oct 15;9(10):e109824. doi: 10.1371/journal.pone.0109824

Figure 4. VGLUT1 interacts with the E3 ubiquitin ligases AIP4/Itch and Nedd4.

Figure 4

(A) Rat brain extracts were incubated with GST or GST fusions of WW domains from proteins identified in the array screen. No specific binding to VGLUT1 was detected. (B) GST fusions of the VGLUT1 C-terminus (VG1), or VGLUT1 mutants lacking polyproline domains (ΔPP1, ΔPP2, ΔPP1&2), or point mutants disrupting the WW domain-binding consensus sequence (ΔWW/PAXA, ΔWW/PAXY), or all three SH3 consensus sequences in PP1 (ΔSH3/1,2,3) were incubated with rat brain lysates. Immunoblots were probed with antibody to AIP4/Itch, Nedd4, WWP1, or WWP2. Specific binding of AIP4/Itch to VGLUT1 is significantly reduced in the absence of PP1 or by disruption of the WW domain (ΔPP1&2: 0.00622±0.02337 a.u.; ΔPP1, 0.04012±0.02316 a.u.; ΔPP2, 0.8217±0.2670 a.u.; ΔWW/PAXA, 0.06691±0.08685 a.u.; ΔWW/PAXY, 0.1194±0.0668 a.u.; ΔSH3/1,2,3, 0.9896±0.2678 a.u.). Deletion of PP1 also abrogates binding of Nedd4 to VGLUT1 (ΔPP1&2, 0.08213±0.04285 a.u.; ΔPP1, 0.04586±0.03081 a.u.; ΔPP2, 0.6636±0.1280 a.u.). Band intensities were normalized to VG1. nd: not detected. Top panels show representative immunoblots, lower panels show the averaged quantification of band intensity from at least three independent experiments. *p<0.05, ***p<0.001, one-way ANOVA with Bonferroni's post-test. (C) Cultured rat cortical neurons transfected with HA-VGLUT1 and AIP4/Itch, were incubated with DSP crosslinker and immunoprecipitated with rat anti-HA antibodies. AIP4/Itch specifically co-immunoprecipitates with HA antibody. HA-VGLUT1 was detected with mouse anti-HA antibody. (D) Cultured rat cortical neurons transfected with HA-VGLUT1, 3x-FLAG-ubiquitin, and AIP4/Itch, were immunoprecipitated with rat anti-HA antibodies as in (C). Immunoprecipitates were probed with FLAG antibody to detect ubiquitination. Two bands of approximately 58 and 74 kD were specifically recognized by antibody to FLAG when immunoprecipitation was carried out with antibody to HA, but not IgG. Mouse anti-HA antibody was used to detect HA-VGLUT1. FT: flow through.