Figure 3.

Assembly of the Arf4-mediated ciliary membrane-targeting complex at the trans-Golgi: the Arf GEF GBF1 is an effector of Rab6

(a) Schematic of GBF1. DCB-HUS (AA 1–710) and Sec7-HDS1 (AA 695–1066) are indicated. (b) GST-DCB-HUS, or GST (Ctrl), were incubated with recombinant human Rab6 bound to GTPγS, or with Rab6Q72L or T22N mutants. Bound Rab6 was detected by immunoblotting. The GST fusion proteins were detected with anti-GST antibody. Arrowheads point to the GST-fusion proteins used in pulldowns. (c) GST-DCB-HUS, GST-Sec7-HDS1, or GST (Ctrl), were incubated with Rab6 bound to GTPγS, or with Rab6Q72L mutant and bound Rab6 was detected as above. (d) Frog retinas were pulse-labelled for 60 min and retinal PNS was incubated for 30 min with 50 µM peptides, as indicated in the panel, prior to a 2 h cell-free chase; photoreceptor membranes were fractionated into Golgi, TGN and RTCs as described [6,98], and radiolabelled proteins analysed by SDS-PAGE and autoradiography ([35S]-Rh). The Rab6 effector peptide mimicking switch I arrested rhodopsin in the Golgi, where Rab6 and GBF1 are localized [23,93] and potently and specifically inhibited its uptake into RTCs. The Rab7 switch I and the Rab6 SF3 peptide had no effect. (e) The ribbon model of Rab6. Switch I is coloured blue, and switch II is red. Modified from ref [110]. SF3 domain is indicated.