A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis - PubMed (original) (raw)

A, sequence comparison between Sec2p and Rabin8. Asterisks indicate identical amino acids. Double dots indicate amino acids of high similarity, and single dots indicate amino acids of low similarity. Amino acids 300–305 (SLYNEF) are identical in Sec2p and Rabin8. B, comparison of the binding of Rab11 and Sec15 to wild-type Rabin8 and the Rabin8Δ(300–305) mutant. The Coomassie Blue-stained gel shows the Rabin8 and Rabin8Δ(300–305) fusion proteins coupled to glutathione-Sepharose (upper panels). Rab11 expressed as a NusA-His6-tagged fusion protein (lower right panel) had had weaker binding to the Rabin8 mutant. In vitro translated Sec15 bound more strongly to the Rabin8 mutant (lower left panel). C, dose-dependent interaction of Sec15 with Rabin8Δ(300–305). Various amounts of in vitro translated [35S]methionine-labeled Sec15 were incubated with 0.5 μ

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GST-Rabin8Δ(300–305). Sec15 binding was standardized to 1 μl of 35S-labeled Sec15 input (defined as 1 arbitrary unit (A.U.)). Bound Sec15 is plotted against input amounts. Error bars indicate S.D. (n = 3). D, affinity of Rabin8-Rab11(Q70L) interaction. GST-Rabin8 (0.5 μ

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) coupled to glutathione-Sepharose was incubated with various concentrations of NusA-His6-Rab11(Q70L) in the binding assay. Bound NusA-His6-Rab11(Q70L) was detected by Western blotting using anti-His6 antibody. The amount of bound Rab11(Q70L) was standardized to 50 pmol of purified Rab11(Q70L) (defined as 1 arbitrary unit). The dissociation constant (Kd) was calculated by fitting the data using SigmaPlot software with a single rectangular hyperbola equation: B = B_max_X/(Kd + X), where B is bound and X is free Rab11. The Kd for the Rabin8-Rab11(Q70L) interaction was ∼54 μ

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. E, the Rabin8Δ(300–305) mutant binds to Rab8 more strongly than Rabin8. Wild-type Rab8a and Rab8a(T22N) were expressed as GST fusion proteins and conjugated to glutathione-Sepharose. Rabin8 and Rabin8Δ(300–305) were in vitro translated in the presence of [35S]methionine. The binding of Rabin8 to Rab8a and Rab8a(T22N) in the presence of GDPβS was examined. Left panels, Coomassie Blue-stained gel showing GST fusion proteins used in the binding reaction; right panels, input (5%) and bound Rabin8 and Rabin8Δ(300–305). Compared with wild-type Rabin8, the Rabin8Δ(300–305) mutant bound better to Rab8 and Rab8(T22N). F, analysis of the release of [3H]GDP from Rab8 catalyzed by Rabin8 or Rabin8Δ(300–305). The circles indicate Rab8 only; the squares indicate Rab8 in the present of wild-type Rabin8; and the triangles indicate Rab8 in the present of Rabin8Δ(300–305). Rabin8Δ(300–305) was more potent than wild-type Rabin8 in promoting GDP release from Rab8 (p < 0.01, n = 3).