Fig. 4: MAG3 interacts with the plant-specific proteins WPP1 and WPP2.

a Immunoblots (left) and silver staining (right) of pulled-down products with anti-GFP antibody from seedlings of the transgenic plant expressing either GFP or GFP-MAG3. Arrowheads indicate full-length GFP-MAG3. b Mass spectrometry-based proteomics of the pulled-down products with anti-GFP antibody from different organs of the transgenic plants identified two WPP domain proteins (WPP1 and WPP2) with high scores as well as MAG3. Scores were calculated using the Mascot program (Matrix Science). n.d. not detected. c Yeast two-hybrid analysis of the strain expressing MAG3 lacking the transmembrane domain (MAG3ΔTMD) fused with the GAL4 DNA binding domain (BD) and each of WPPs (WPP1, WPP2, and WPP3) fused with the GAL4 activation domain (AD). Three independent transformants were incubated on SD/–Leu/–Trp/–His/–Ade/+Aureobasidin A/ + X-α-Gal medium (–LWHA +AbA +X-α-Gal) or SD/–Leu/–Trp medium (–LW). Negative controls (empty vector) were also examined. d Representative VAEM images of cotyledon epidermal cells of two transgenic plants (proMAG3:GFP-MAG3 proWPP1:WPP1-mCherry mag3-5 and proMAG3:GFP-MAG3 proWPP2:WPP2-mCherry mag3-5), showing close localization of MAG3 with WPP1 or WPP2 (arrowheads). Arrows show WPP1 and WPP2 detected on plasma membrane (see Supplementary Fig. 5 and Supplementary Video 4). e Representative VAEM images of cotyledon epidermal cells of wpp1-1 wpp2-1 and the respective control line (WPP1 WPP2) expressing GFP-MAG3 and mCherry-SYP32 (cis-Golgi marker), showing no effects of deficiency of both WPP1 and WPP2 on MAG3 signal patterns (arrowheads). f The MAG3 protein levels in seedlings (arrowhead) of wpp1-1, wpp2-1, and wpp1-1 wpp2-1 were similar to that of the wild-type (Col-0). g No accumulation of storage protein precursors (pro12S globulin and pro2S albumin) in seeds of wpp1-1, wpp2-1, and wpp1-1 wpp2-1, as in the wild type (Col-0). h A hypothetical model of the MAG3 function in the dynamic capture-and-release of ERESs by Golgi stacks at the ER-Golgi interface. COPII-bound ERESs (green) are captured by a Golgi stack in an ER cavity, where COPII vesicles bud from ERESs and then fuse with cis-Golgi cisternae. After cargo transport, ERESs are released by Golgi stacks for recycling. The coiled-coil ER membrane protein MAG3 (blue), in cooperation with WPPs (orange), facilitates the cargo transport from the ER to Golgi stacks. Adapted from Takagi et al. ²⁰.