Summary The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn DENN domain functions as a guanine nucleotide exchange factor for Rab35 to regulate endosomal trafficking. Loss of Rab35 activity causes an enlargement of early endosomes, inhibits MHCI recycling, and prevents the early endosomal recruitment of EHD1, a common component recycling tubules on endosomes. Our data are the first to reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3-phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3-phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3-phosphate metabolism is integrated into the cellular network.

The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.Protein domains are modular cassettes with conserved folds that are often found in otherwise unrelated proteins. From an evolutionary point of view, modular domains are readily joined in new combinations, creating novel connections and cellular pathways (1). The DENN (differentially expressed in normal and neoplastic cells) domain is a poorly characterized protein module conserved throughout evolution, with DENN domain proteins found in species as diverse as humans, Caenorhabditis elegans, Arabidopsis thaliana, and Schizosaccharomyces pombe. There are 18 genes encoding DENN domain-containing proteins in humans. For some, the DENN domain is the only recognizable feature; for others, the DENN domain is found alongside other modular domains. The observation that several DENN domain proteins interact with Rab GTPases provided the first insight into the potential function of the domain (2). Rabs, with ϳ70 members in humans, are the largest family of small GTPases. They cycle between an inactive GDP-bound state and an active GTP-bound state. In the active state, they recruit effectors that control multiple aspects of membrane trafficking (3, 4). A breakthrough in our understanding of the DENN domain came with the observation that the DENN domain from the connecdenn family of proteins interacts directly with Rab35 and functions as a guanine nucleotide exchange factor (GEF) 3 for this GTPase (5-7). GEFs activate Rabs by mediating the exchange of GDP for GTP. As Rabs have diversified throughout evolution (9 Rabs/Ypts in S. pombe, 30 in C. elegans, and ϳ70 in humans, for example), so have DENN domain proteins, with 1 in S. pombe, ϳ5 in C. elegans, and 18 in humans. Thus, DENN domains may have evolved as generalized GEFs for Rabs, and in fact, all subfamilies of DENN domain proteins appear to possess Rab-directed GEF activity (8). Moreover, because at least some DENN domains interact with one Rab while mediating GEF activity toward a second, DENN domains may be at an interface between different Rab pathways. Here, we will provide an overview of all DENN domain proteins encoded in the human genome and will describe exciting new insights confirming that DENN domain-bearing proteins are an important class of membrane trafficking molecules and key regulators of Rab GTPases. Identification of the DENN DomainChow and Lee (9) originally cloned an open reading frame that they named DENN based on its variable mRNA expression levels in tissues and cell lines. The DENN prote...