SIMPLE interacts with NEDD4 and TSG101: Evidence for a role in lysosomal sorting and implications for Charcot-Marie-Tooth disease

Abstract

Mutation of the SIMPLE gene (small integral membrane protein of the lysosome/late endosome) is the molecular basis of Charcot-Marie-Tooth disease type 1C (CMT1C), a demyelinating peripheral neuropathy. Although the precise function of SIMPLE is unknown, prior reports suggest it localizes to the lysosome/late endosome. Furthermore, murine Simple interacts with Nedd4 (neural precursor cell expressed, developmentally downregulated 4), an E3 ubiquitin ligase that is important for regulating lysosomal degradation of plasma membrane proteins. To bring insights into the biochemical function of human SIMPLE, we confirmed that human SIMPLE interacts with NEDD4 and also report a novel interaction with tumor susceptibility gene 101 (TSG101), a class E vacuolar sorting protein. TSG101 is known to function downstream of NEDD4, sorting ubiquitinated substrates into multivesicular bodies (MVBs), which then deliver their cargo into the lysosomal lumen for degradation. Given the interaction with NEDD4 and TSG101, and the localization of SIMPLE along the lysosomal degradation pathway, we hypothesize that SIMPLE plays a role in the lysosomal sorting of plasma membrane proteins. We examine three CMT1C-associated SIMPLE mutations and show that they do not affect the interaction with NEDD4 or TSG101, nor do they lead to altered subcellular localization.

Mutations associated with Charcot–Marie–Tooth disease cause SIMPLE protein mislocalization and degradation by the proteasome and aggresome–autophagy pathways

“…Instead, SIMPLE is a substrate protein for degradation by the lysosome, as we can only detect the presence of SIMPLE in the lysosome and late endosome upon lysosome inhibition. Our finding that SIMPLE is an early endosomal membrane protein is consistent with the reported interaction of SIMPLE with the endosomal sorting complex required for transport (ESCRT) subunit TSG101 (Shirk et al, 2005) and suggests that SIMPLE regulates protein sorting at the early endosome.…”

“…First, mutation-induced mislocalization away from the early endosomal membrane would remove SIMPLE protein from its site of action, thereby contributing to CMT pathogenesis by a lossof-function mechanism (Kim et al, 2002;Lupo et al, 2009;Roberts et al, 2010). Although the cellular function of SIMPLE is unknown, it has been proposed that SIMPLE might participate in protein sorting at the early endosome (Shirk et al, 2005). Loss of endosomal function of SIMPLE triggered by CMT1C mutations might cause deregulated trafficking of myelin proteins and/or other membrane proteins, resulting in Schwann cell dysfunction and demyelination.…”

Lysosomal killing ofMycobacteriummediated by ubiquitin-derived peptides is enhanced by autophagy

“…In addition to this ''classic'' route for protein breakdown, it is also known that ubiquitin regulates trafficking of proteins in the endocytic pathway. Monoubiquitinated proteins, most notably integral membrane proteins, are imported into multivesicular bodies (MVBs) and delivered to lysosomes for degradation (15,16). However, lysosomally degraded proteins are thought to be deubiquitinated before import into the lysosome.…”

“…In previous experiments demonstrating the transference of cytosolic cargo into lysosomes in macrophages infected with Leishmania, we found that this transfer was enhanced upon the induction of autophagy and was routed through autophagosomes, which appeared comparable ultrastructurally with MVBs (17). Moreover, cells deficient in autophagy accumulate aggregates of ubiquitinated proteins in the cytosol (16,18,19).…”

Cellular LITAF Interacts with Frog Virus 3 75L Protein and Alters Its Subcellular Localization

“…LITAF is predicted to encode a 161-amino-acid protein with distinct N and C termini (6). The N terminus of LITAF contains proline-rich binding domains for several cellular proteins, including neural precursor cell expressed, developmentally downregulate 4 (Nedd4) (3,7), tumor susceptibility gene 101 (TSG101) (8), and Itch (9), which function along a conserved pathway of lysosomal protein degradation. The C terminus of LITAF contains an arrangement of cysteine residues resembling a RING finger domain (4).…”