DENN domains are found in multiple protein products encoded by 16 human genes including myotubularin related (MTMR) 5 and 13, DENN/MADD, suppressor of tumorgenicity 5, Rab6-interacting protein 1 (Rab6IP1), connecdenn, and a number of uncharacterized proteins. The presence of a single DENN domain protein in Schizosaccharomyces pombe
indicates that the DENN domain is evolutionary ancient and bioinformatics suggests it is present in all eukaryotes (Harsay and Schekman, 2007
). Mutations in DENN domains and their parent proteins cause human diseases (Azzedine et al., 2003
; Del Villar and Miller, 2004
; Lichy et al., 1992
; Senderek et al., 2003
; Volterra and Meldolesi, 2005
). Notably, Charcot-Marie-Tooth 4B2 neuropathy is caused by deletion of the dDENN in MTMR 13 (Senderek et al., 2003
). Even in Arabidopsis thaliana
, a point mutation in the DENN domain of SCD1 impairs exocytic vesicle trafficking crucial for cytokinesis and polarized cell expansion and causes sterility (Falbel et al., 2003
). Thus, DENN domain proteins mediate crucial functions throughout evolution.
In several cases, DENN domain proteins have been linked to Rabs but no common mode of interaction has been gleaned. Rab6IP1 binds to Rab6 and Rab11 in their GTP-bound form but also binds GDP-bound Rab6 (Miserey-Lenkei et al., 2007
). DENN/MADD selectively binds through its uDENN to GTP-bound Rab3 (Niwa et al., 2008
). However, DENN/MADD also functions as a GEF for Rab3 (Wada et al., 1997
). Notably, the GEF activity has only been observed with full-length protein and it is argued that the GEF activity resides outside of the DENN domain (Coppola et al., 2002
). We now demonstrate that the purified connecdenn DENN domain has intrinsic GEF activity specific for Rab35. Our study is the first to assign an enzymatic activity to the DENN domain and it will be interesting to see if other DENN domains function as GEFs towards other Rabs.
We propose a model for connecdenn function in which the protein is recruited to nascent CCVs through C-terminal interactions with AP-2 while the DENN domain anchors the protein to the vesicle membrane and functions as a platform for Rab35 recruitment. Transition of the endocytic carrier from PtdIns(4,5)P2 to PtdIns(3)P, the PtdInsP enriched on endocytic vesicles could signal to the DENN domain to exert its GEF function. Connecdenn would then dissociate from the vesicle before fusion with EEA1-positive endosomes and in fact, we observe minimal co-localization of connecdenn and EEA1 (data not shown). Activated Rab35 remains on the vesicles for delivery to early endosomes, where its effector protein(s) control the recruitment of EHD1 for cargo recycling.
Over expression studies have linked Rab35 to the fast transferrin recycling pathway from early endosomes to the PM (Kouranti et al., 2006
), a function previously assigned to Rab4 (Daro et al., 1996
; van der Sluijs et al., 1992
). However, Deneka et al. (2003)
showed that KD of Rab4 enhanced transferrin recycling and our studies reveal that Rab35 KD does not affect transferrin trafficking. Thus, the Rab-based protein machinery for this transport pathway is yet to be defined.
KD of Rab35 and connecdenn impairs recycling of MHCI. MHCI is internalized in a clathrin-independent pathway that merges with EEA1-positive endosomes (Naslavsky et al., 2003
). From there, MHCI is either delivered to late endosomes/lysosomes or traffics to recycling endosomes for recycling back to the PM in transferrin-negative, Rab22-positive tubular structures (Weigert et al., 2004
). In addition, these tubules contain Rab11 and EHD1, which also regulate the recycling of clathrin-dependent cargo from recycling endosomes (Caplan et al., 2002
; Grant et al., 2001
; Lin et al., 2001
; Weigert et al., 2004
). Clathrin-independent and -dependent cargo thus utilize in part common machineries to recycle from recycling endosomes to the PM (Weigert et al., 2004
). Weigert et al. (2004)
further describe MHCI-positive tubules that originate from transferrin-positive endosomal structures but the protein machinery involved in their formation remained undefined. Our studies demonstrate that Rab35 activation is crucial for EHD1 recruitment to EEA1-positive endosomes and for efficient MHCI recycling. Together, these data suggest that MHCI recycling from distinct intracellular locations is likely mediated by a common protein machinery but that the spatial organization of this machinery is determined by pathway-specific Rab GTPases. Moreover, these pathways seem, at least in part, to be cargo-specific. β1-integrin has been shown to follow the MHCI trafficking route from the PM to recycling endosomes and back (Jovic et al., 2009
; Jovic et al., 2007
; Powelka et al., 2004
), however, KD of Rab35 or connecdenn does not impair the trafficking of this cargo. This is reminiscent of recent data from the Corvera group, which demonstrated differences in the early endosomal processing of endocytic vesicles dependent on the cargo they carried (Leonard et al., 2008
). Incoming transferrin is efficiently routed to recycling endosomes whereas EGF is retained in EEA1-positive endosomes for delivery to the degradative pathway due to higher levels of Rab5 present on EGF-delivering vesicles (Leonard et al., 2008
), underscoring the importance of Rabs for sorting decisions within the endosomal network.
In summary, our results reveal an enzymatic activity for a DENN domain and given the ancient nature of this module, it is likely that this activity functions broadly throughout eukaryotes. Moreover, our results indicate that distinct Rabs GTPases use a common protein machinery to control cargo selective recycling from different sites in the endosomal system.