A Proteomics-Based Approach Identifies the NEDD4 Adaptor NDFIP2 as an Important Regulator of Ifitm3 Levels

IFITMs are a family of highly related interferon-induced transmembrane proteins that interfere with the processes of fusion between viral and cellular membranes and are thus endowed with broad antiviral properties. A number of studies have shown how the antiviral potency of IFITMs is highly dependent on their steady-state levels, their intracellular distribution and a complex pattern of post-translational modifications, parameters that are overall tributary of a number of cellular partners. In an effort to identify additional protein partners involved in the biology of IFITMs, we devised a proteomics-based approach based on the piggyback incorporation of IFITM3 partners into extracellular vesicles. MS analysis of the proteome of vesicles bearing or not bearing IFITM3 identified the NDFIP2 protein adaptor protein as an important regulator of IFITM3 levels. NDFIP2 is a membrane-anchored adaptor protein of the E3 ubiquitin ligases of the NEDD4 family that have already been found to be involved in IFITM3 regulation. We show here that NDFIP2 acts as a recruitment factor for both IFITM3 and NEDD4 and mediates their distribution in lysosomal vesicles. The genetic inactivation and overexpression of NDFIP2 drive, respectively, lower and higher levels of IFITM3 accumulation in the cell, overall suggesting that NDFIP2 locally competes with IFITM3 for NEDD4 binding. Given that NDFIP2 is itself tightly regulated and highly responsive to external cues, our study sheds light on a novel and likely dynamic layer of regulation of IFITM3.