Molecular and biochemical characterization of the E3-ligase RNF11 (RING Finger Protein 11)

RNF11 (Ring Finger Protein 11) is an evolutionary conserved, 154 amino acid RING-H2 E3-ligase, containing different functional modules. The mature protein is anchored to membranes via a double acylation and localizes to early endosome and recycling compartments. Functional studies have implicated RNF11 in a variety of biological processes, including ubiquitylation, signal transduction and endocytic trafficking, but the molecular mechanisms underlying its function are still poorly understood. Combining affinity purification coupled with quantitative mass spectrometry and molecular biology techniques, we profiled the in vivo protein interaction network of RNF11 and revealed novel insight about its function. Specifically, we identified two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of GGA protein adaptors. We found that these motifs are the molecular determinants governing RNF11 sorting at the trans-Golgi network, as well as RNF11 internalization from the plasma membrane, and its intracellular trafficking between early and recycling endosome compartments. We also showed that RNF11, by recruiting the HECT E3-ligase Itch, regulates Itch catalytic activity and drives HECT-mediated ubiquitination of GGA protein family. As our preliminary evidence suggested that a similar mechanism could affect also other trafficking machinery proteins, such as the ESCRT-0 components, we proposed for RNF11 a central role in a finely regulated system where it acts both as an adaptor and a modulator of HECT-ligase activity, controlling the ubiquitination events underlying membrane traffic. Lastly, we demonstrated that a subgroup of Ubiquitin Binding Domain (UBD) - particularly UIM – interacts directly with RNF11, without requiring its ubiquitination, at odds with “conventional” UBD binding mode. This, in addition to identify the first specific binding partner or these UBD, open novel and intriguing perspective about RNF11 binding mode and mechanistic insights into the ubiquitin-network and the biological processes RNF11 is involved in.