Differential roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI trafficking

The formation of coat protein complex I (COPI)–coated vesicles is regulated by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor 1 (Arf1), which in its GTP-bound form recruits coatomer to the Golgi membrane. Arf GTPase-activating protein (GAP) catalyzed GTP hydrolysis in Arf1 triggers uncoating and is required for uptake of cargo molecules into vesicles. Three mammalian ArfGAPs are involved in COPI vesicle trafficking; however, their individual functions remain obscure. ArfGAP1 binds to membranes depending on their curvature. In this study, we show that ArfGAP2 and ArfGAP3 do not bind directly to membranes but are recruited via interactions with coatomer. In the presence of coatomer, ArfGAP2 and ArfGAP3 activities are comparable with or even higher than ArfGAP1 activity. Although previously speculated, our results now demonstrate a function for coatomer in ArfGAP-catalyzed GTP hydrolysis by Arf1. We suggest that ArfGAP2 and ArfGAP3 are coat protein–dependent ArfGAPs, whereas ArfGAP1 has a more general function

Figure S1: Supplemental images of TGFβ2 KO coloboma phenotypes from TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

(A) Serial frontal sections (E18.5, H&E) of a TGFβ2 KO embryo from mixed genetic background. The optic fissure margins did not fuse but grew inwards. Scale bars represent 200µm. (B) Serial frontal sections (E18.5, H&E) of a TGFβ2 KO embryo from mixed genetic background. In addition to the usual coloboma phenotype, there is a dorsal coloboma. Scale bars represent 200µm. (C) Serial frontal sections (E18.5, H&E) of a TGFβ2/GDNF double KO embryo. In addition to the usual coloboma phenotype, there is a dorsal coloboma. Scale bars represent 200µm.ted fish as control

Figure S2: Establishment of a TGF signalling reporter in zebrafish from TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

(A) Construct for the TGFβ signalling reporter. Multimerised Smad Binding Elements (SBEs) in combination with a minimal promoter (MP) drive membrane localised GFP (GFPcaax). (B) Bright field, fluorescent and merged images of a zebrafish larva expressing the TGFβ signalling reporter construct (21.5 hpf). Note the expression domains in the forebrain (encircled) and the tail. (C) SB431542 reduces TGFβ reporter activity, DMSO treated fish as control. Treatment was administered from 24 hpf to 48 hpf. The inhibitor was renewed during the time of the treatment