The role of spartin and its novel ubiquitin binding region in DALIS occurrence

Abstract

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains--a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively--have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin's UBR and defined the UBR's amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63-linked ubiquitin chains but does not bind Lys-48-linked ubiquitin chains. Finally, we demonstrate that spartin's role in DALIS formation depends on key residues within its UBR.