The Rab8A-GDP variants were loaded with the fluorescent GDP analogue 2′/3′- N-methylanthraniloyl -GDP (mantGDP).
Co-precipitation of FLAG-Rab26WT was observed with EGFP-Rab26 WT and even more efficiently with the GDP-preferring variant Rab26TN whereas co-precipitation with the nucleotide-empty variant (Rab26NI) was reduced and binding to the GTP-preferring variant (Rab26QL) was abolished.
The following mutants were included: a GDP-locked variant of Rho3, in which the conserved (among Rho3 proteins) Thr27 was replaced with Asn (Rho3T27N), a GTP-locked variant of Rho3 (Rho3G22V) in which the conserved Gly22 was replaced with Val, and an effector domain mutant Rho3 (Rho3E48V) in which the conserved Glu48 was replaced with Val.
Note that occasional puncta were observed for the GDP-preferring variant Rab26TN that, however, showed no overlap with LC3B (arrowhead).
It is somewhat surprising that, along with the GDP-bound variant, wild-type Rab26 also appears to oligomerize (albeit to a lesser extent).
As shown in Figure 2B, co-precipitation of FLAG-tagged Rab26 was only observed when cells were transfected with either wild-type EGFP-Rab26 or with a GDP-preferring variant (Rab26T77N, henceforth referred to as Rab26TN).
Expression of GDP- and GTP-locked variants of Arl6 in a human cell line does not alter cell growth or gross morphology but significantly changes the proportion of cells possessing cilia and the length of assembled cilia.
The UpSafe-DownSafe filters were used to assure that the regions immediately upstream (UpSafe) or downstream (DownSafe), or both upstream and downstream (BothSafe), of a marker variant were free of any other variants across the GDP.
The Rab26 variants (Rab26wt, GTP-preferring Rab26Q250L, and GDP-preferring Rab26T204N), were expressed using elav-Gal4.
Using bacterially expressed His-tagged CDC-42 variants that mimic the GTP- and GDP-bound states (Q61L and T17N, respectively), we found that the SRGP-1 GAP domain also specifically interacts with the active form of CDC-42.
The octoploid Global Discovery Panel (GDP) and F. iinumae F1D sample were used for variant discovery.
