Genome-Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling

TLDR

Specificity of protein ubiquitylation is conferred by E3 ubiquitin ligases, and the E3 MULAN had previously been linked to NF‑κB activation, suggesting a connection between mitochondrial dynamics and mitochondria‑to‑nucleus signaling. The authors generated genome‑wide E3 cDNA and RNAi expression libraries to functionally screen the largely uncharacterized E3 superfamily. An imaging‑based screen of these libraries identified the mitochondrial RING‑finger protein MULAN/FLJ12875 as an E3 that regulates mitochondrial trafficking and morphology. The screen annotated ~617 E3s, revealed MULAN as a mitochondrial protein with two transmembrane domains and a cytosolic RING finger essential for its activity, and suggested a novel ubiquitin‑mediated mechanism integrating mitochondria into cellular signaling.

Abstract

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.