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

Khan AR (2012) Mapping the Interactions between a RUN Domain from DENND5/Rab6IP1 and Sorting Nexin 1. PLoS ONE 7(4): e35637. doi:10.1371/journal.pone.0035637 Mapping the Interactions between a RUN Domain from DENND5/Rab6IP1 and Sorting Nexin 1 Humberto Fernandes 0 Edward Franklin 0 Florence Jollivet 0 Katharina Bliedtner 0 Amir R. Khan 0 Anna Maria Delprato, BioScience Project, United States of America 0 School of Biochemistry and Immunology, Trinity College , Dublin , Ireland 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. - Funding: This work was supported by Science Foundation Ireland grant number 07.IN1.B975 to ARK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Eukaryotic cells rely on an intricate trafficking system to shuttle proteins, lipids, and other vesicular cargo between sub-cellular compartments. Trafficking involves protein/protein and protein/ lipid interactions leading to vesicle formation, tethering, docking and fusion. These steps are regulated to provide specificity and preserve cellular structure and organelle identity, such as the Golgi apparatus, endosomes and lysosomes [13]. The specificity between donor and acceptor membranes is widely believed to be mediated by the Rab family of small GTPases, which comprise the largest member of the Ras superfamily [4]. Active Rabs are noncovalently associated with GTP and localize to distinct sub-cellular compartments via prenylated C-terminal tails [5,6]. Recruitment of effector proteins, which recognize the active conformation, leads to the regulation of various steps in vesicle delivery. Inactivation of Rabs follows hydrolysis of GTP, aided by GTPase activating proteins (GAPs), and Rabs are subsequently extracted from the membrane into the cytosolic fraction by GDP dissociation inhibitor [7]. Rab6 regulates anterograde and retrograde traffic at the level of Golgi via interactions with numerous and unrelated effector proteins [8,9]. Rab6A and Rab6A are ubiquitously expressed in cells, whereas expression of a third isoform Rab6B is restricted to brain tissue [10]. A fourth isoform, Rab6C, has recently been shown to encode a brain-specific retrogene with an unusual GTPbinding motif that localizes to the centrosome and regulates cell cycle progression [11]. One of the most widely studied effectors of Rab6 is DENND5/Rab6IP1 protein (Differentially Expressed in Neoplastic versus Normal Cell; alternatively called Rab6-Interacting Protein 1) [12]. DENND5 was initially identified by yeast two-hybrid assays as an effector of Rab6 [13]. The N-terminal half of the 1287-residue DENND5A isoform is composed of a series of the eponymous DENN domains that appear to function as a GDP/GTP exchange factor (GEF) for Rab39 [14]. The Cterminal half of the effector is composed of two RUN domains (RUN1 and RUN2) separated by a PLAT domain. In previous work, we determined the crystal structure of Rab6A in complex with the tandem RUN1-PLAT domains of DENND5A [15]. The structure revealed the molecular basis for Rab6-mediated recruitment of DENND5 to Golgi, as well as the orientation of the lipophilic loops of the PLAT domain, relative to the Rabbinding interface. Despite numerous cellular and structural studies of Rab6 effectors, the role of the RUN2 domain of DENND5 remains unknown. RUN domains, named after RPIP8 (Rap2 interacting protein 8), UNC-14 and NESCA (new molecule containing SH3 at the carboxyl-terminus) [16], are widely occurring modules that appear to have diverse roles in cell signaling [17]. Rap2Interacting Protein X (RPIPX) also contains a RUN domain and belongs to a family of effectors that bind to the small GTPase Rap2 [18,19]. The uncomplexed crystal structure of the RUN domain of RPIPX has been determined [20]. However, RUN domains appear to have roles beyond small GTPase signaling [21]. Recently, an interaction between DENND5 and sorting nexin 1 (SNX1) has been reported by a genome-wide yeast two-hybrid screen and GST pulldowns [22]. SNX1 forms a transient complex with the retromer in mammalian cells to drive vesicle transport between early endosomes and the trans-Golgi network [2325]. Human SNX1 consists of an N-terminal sorting nexin (SNX) region, a central PX (Phox-homology) domain, and a C-terminally situated BAR domain (Bin, amphiphysin and Rvs) that binds to and/or induces membrane curvature via interactions with the lipid bilayer [2628]. Here we report that the RUN2 domain of DENND5 binds to the N-terminal SNX region of SNX1 by surface plasmon resonance analyses. DENND5 Expression and Purification Cloning, expression and purification of Rab6 and an engineered RUN1-PLAT (RPdel) construct has been published previously [29]. In brief, a truncated version of human Rab6a encompassing residues 8 to 195 (Q72L mutant), with a thrombin cleavable Nterminal His-tag was used in this study. An engineered variant of mouse DENND5A containing a loop deletion between residues 813835 (inclusive) of RUN1, henceforth referred to as RPdel, was also used [29]. RPdel denotes the RUN-PLAT tandem domains of DENND5A. This protein was expressed as an N-terminal Histagged protein containing a tobacco etch virus (TEV) cleavable Nterminal His-tag. Both proteins were expressed in E. coli individually or co-expressed and purified to homogeneity. Cloning, expression and purification of the RUN1-PLATRUN2 (RPRdel; residues 7161278) region of DENND5A, was performed using similar strategies [29], with the exception of the reverse primer (Table 1). Cloning of the fragment was derived from a synthetic gene comprising the entire coding region of DENND5 (Geneart AG), with or without the cDNA corresponding to the loop 813835 of RUN1. However, both the wild-type RPR protein and RPRdel were insoluble when expressed alone in E. coli. Co-expression with Rab6a increased the solubility of the three-domain constructs, therefore the complex Rab6a/RPRdel was (...truncated)