Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets.
BACKGROUND
Spartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.


RESULTS
In this study, we show that spartin is not a substrate for AIP4 activity and that spartin's binding to AIP4 significantly increases self-ubiquitination of this E3 ligase, indicating that spartin disrupts the AIP4 autoinhibitory intramolecular interaction. Correspondingly, spartin has a seven times higher binding affinity to the WW region of AIP4 than the binding of the WW region has to the catalytic homologues of the E6-associated protein C-terminus (HECT) domain, as measured by enzyme-linked immunosorbent assay. We also show that spartin recruits AIP4 to lipid droplets and promotes ubiquitination of lipid droplet-associated protein, adipophilin, which regulates turnover of lipid droplets.


CONCLUSIONS
Our findings demonstrate that spartin acts as an adaptor protein that activates and recruits AIP4 E3 ubiquitin ligase to lipid droplets and by this means regulates the level of ubiquitination of adipophilin and potentially other lipid-associated proteins. We propose that this is one of the mechanisms by which spartin regulates lipid droplet turnover and might contribute to the pathology of SPG20.
Hooper
Christopher
C
Department of Molecular Pharmacology and Therapeutics, Loyola Chicago University, Maywood, IL, USA.
Puttamadappa
Swamy S
SS
Loring
Zak
Z
Shekhtman
Alexander
A
Bakowska
Joanna C
JC
D018797:Cell Cycle Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism / Q000502:physiology*; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D000070780:Perilipin-2; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D015419:Spastic Paraplegia, Hereditary / Q000503:physiopathology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
D018797:Cell Cycle Proteins; D008565:Membrane Proteins; C000602982:PLIN2 protein, human; D010455:Peptides; D000070780:Perilipin-2; D011506:Proteins; D012097:Repressor Proteins; C464787:SPART protein, human; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases