Mapping the Interactions between a RUN Domain from DENND5/Rab6IP1 and Sorting Nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses

Control SPR experiments to map the binding segment of SNX1.

<p>(A) Injection of equivalent 15 µM amounts of SNX-PX and PX domains are superimposed. A CM5 chip coupled with RPRdel was used in these experiments. (B) Injection of 30 µM SNX-PX protein onto a CM5 chip coupled with Rab6/RPdel. Note that the magnitude of the binding, as evidenced by the <i>y</i>-axis (RU), is 10-fold smaller. These representative control experiments show that SNX-PX binds stably to DENND5-coupled chips that include the RUN2 domain. Data from the control experiments could not be processed and fit to conventional kinetics or equilibrium binding models using the evaluation software.</p

Immune co-localization studies of Rab6 and DENND5.

<p>(A) transfection and overlay with GFP-DENND5del (green) and GM130 (Cy3; red). The black and white panels are the same image without colour, revealing the expression of each protein in the transfected cells. (B) Transfection with GFP-DENND5del and wild-type Rab6A (mCherry; red), with the overlap apparent from the yellow colour. (C) Transfection with YFP-DENND5 (wild-type) and mCherry-Rab6A (wild-type), revealing co-localization in Golgi compartments.</p

Purification of various SNX1 fragments by gel filtration chromatography.

<p>Lanes from SDS-PAGE gels (inset) correspond to the bars across the peaks.</p

The domain organization of DENND5 and SNX1.

<p>Constructs used in the mapping studies are shown below the domains. The engineered loop deletion in DENND5 between α3 and α4, denoted by Δ (residues 813–835), rendered the protein soluble and facilitated its purification.</p

Surface plasmon resonance analyses of SNX1 binding to DENND5.

<p>The SNX-PX protein fragment was injected onto a Rab6/RPRdel coupled CM5 chip at various concentrations ranging from 0 to 15 µM.</p

Fitting of SPR data using Biacore evaluation software.

<p>The end-points of the various injections were plotted against protein concentration and the hyperbolic curve was fit to a 1∶1 binding model. The vertical line represents the estimate of K_D at half-maximal binding of the ligand and analyte.</p

Purification of Rab6/DENND5 complexes by gel filtration chromatography.

<p>(A) Elution of Rab6/RPRdel. (B) elution of Rab6/RPdel. The bars denote the samples analyzed by SDS-PAGE (inset).</p