Nedd4 Family-interacting Protein 1 (Ndfip1) Is Required for the Exosomal Secretion of Nedd4 Family Proteins

Abstract

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50 -90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.The ability to dispose of unwanted proteins is an important function during cellular homeostasis in health or disease. The best studied pathway for disposing of unwanted proteins involves the addition of ubiquitin chains to target proteins followed by degradation of the complex in the proteasome. A major class of enzymes involved in target recognition in this pathway is the E3 ligases with HECT (homology to the E6-associated protein C terminus domain) domains (1). Nedd4 and Nedd4-2 are archetypal members of this family, with ability to bind and ubiquitinate proteins containing PPXY motifs (2, 3). In the nervous system, Nedd4-mediated ubiquitination is required for down-regulating voltage-gated K ϩ and Na ϩ channels (4, 5), axon-guidance proteins (6), and TrkA neurotrophin receptor (7). In addition, Nedd4 family proteins (e.g. Nedd4, Nedd4-2, and Itch) can also ubiquitinate target proteins that are bound to Nedd4 adaptors. One such adaptor is Ndfip1 (Nedd4 family-interacting protein 1) originally identified in a screen for Nedd4-binding partners (8, 9). Ndfip1 contains three transmembrane domains and is localized in the Golgi and postGolgi vesicles. Through their transmembrane domains, Ndfip1 can bind membrane proteins and in doing so, either recruit or inhibit the interaction of membrane proteins with cytosolic Nedd4. In the brain, both Ndfip1 and Nedd4 are co-expressed in the same neuron, and their interaction has been shown to be crucial for increasing the survival of cortical neurons during injury (10).Besides proteasomal...

Post-translational add-ons mark the path in exosomal protein sorting

“…Moreover, the PSAP domains of small integral membrane protein of the lysosome/late endosome (SIMPLE) also bind TSG101. SIMPLE has a PPxY motif that interacts with NEDD4 to be ubiquitinated, and di-Leu and YKRL motives, which are signatures of endocytic functions [14,49]. Mutations in these motifs impair the secretion of SIMPLE to exosomes, altering MVBs biogenesis [49].…”

“…However, its presence is necessary for the import of Nedd4, Nedd4-2 and ITCH to exosomes [14,78]. Alanine replacement on di-Leu (LL) and YKRL motifs of SIMPLE reduces it secretion into exosomes, as well as a mutation on its PTAP motif, which controls SIMPLE binding to TSG101 for incorporation into ILVs.…”

“…Regarding cargo sorting, EVs receive cargoes through both ESCRT-dependent and independent mechanisms [13,14]. The existence of heterogeneous MVBs populations becomes apparent when different components of endosomal trafficking or the ESCRT pathway are silenced or overexpressed, and when exosomal secretion is disturbed and not all exosomal markers are affected to the same extent [10,15].…”

Cellular Up-regulation of Nedd4 Family Interacting Protein 1 (Ndfip1) using Low Levels of Bioactive Cobalt Complexes

“…Ndfip1 is a transmembrane protein that is localized to the Golgi and post-Golgi vesicles such as endosomes (4,5). Ndfip1 functions as an adaptor protein for the Nedd4 family of ubiquitin ligases that target proteins for both degradation and trafficking (4).…”

Drosophila Ndfip is a novel regulator of Notch signaling

“…17,19 Also, Ndfip1 promotes the localization of Nedd4, Nedd4-2 and Itch to exosomes that arise from a late endosomal origin. 22 dNdfip expression, however, did not significantly alter the localization of the E3s (Figure 3). Furthermore, similar to mammalian Ndfips, 15,17 dNdfip is ubiquitinated by Nedd4 E3s, such as dNedd4 and Su(dx) (Supplementary Figure S1).…”