Technical Reference #2079

2079.

Genome-Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN; a Mitochondrial E3 that Regulates the Organelle’s Dynamics and Signaling Wei Li; Mario H. Bengtson.; Axel Ulbrich; Akio Matsuda; Venkateshwar A. Reddy; Anthony Orth; Sumit K. Chanda; Serge Batalov; Claudio A. P. Joazeiro, Scripps Research Institute, PLoS ONE, (2008)
Link To Paper

Abstract:
Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated 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-kB 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.

Materials & Methods:
Immunocytochemistry and confocal microscopy For immunocytochemistry cells were plated out on 35-mm glass bottom dishes (MatTek Corp. Ashland MA). Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min washed with PBS permeabilized with 0.2% Triton X-100 for 5 min washed four times with PBS and blocked with 3% bovine serum albumin all at room temperature. Cells were then incubated with primary antibodies for 2 h at room temperature washed three times with 0.2% Triton X-100 incubated with secondary antibodies for 30 min and washed again. Samples were mounted using Prolong Antifade (Invitrogen) and analyzed by confocal microscopy using an Olympus Fluoview 500 laser scanning confocal on an Olympus IX61 upright microscope. GFP was imaged with the 488-nm line of the Argon laser and the emission filter was a 505–525 bandpass filter. RFP was imaged with 543 nm laser line from a HeNe green laser and the emission filter was a 560–600 bandpass filter.

Microscopic Technique
Confocal microscopy

Cell Type(s)
HEK293; NIH3T3 and HeLa cells