Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

Abstract

BackgroundOptineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.ResultsWe show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking.ConclusionsOur results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.

Optineurin mediates negative regulation of Rab8 function by TBC1D17, a GTPase activating protein

“…1H,I). Previously, it has been shown that optineurin is present in vesicles positive for TfR (Nagabhushana et al, 2010;Park et al, 2010). Hence, we examined the distribution of TfR in the cells co-expressing optineurin and TBC1D17.…”

“…It is involved in regulating many cellular functions such as vesicular trafficking from the Golgi to plasma membrane (Sahlender et al, 2005), endocytic trafficking (Au et al, 2007;Nagabhushana et al, 2010) and signaling leading to NF-kB activation (Nagabhushana et al, 2011;Zhu et al, 2007). Mutations in optineurin are associated with certain glaucomas and amyotrophic lateral sclerosis (Maruyama et al, 2010;Rezaie et al, 2002).…”

Toward an integrative view of Optineurin functions

“…TGN46 (marker for the Golgi) positive vesicles and recycling endosomes, while an UBD-mutated form of Optn (D474N) was redistributed into a cytosolic-like staining pattern. 42,78 The observation that the ubiquitin-binding activity of Optn is necessary for its localization to the Golgi apparatus suggests that, in addition to its interaction with Rab8, interaction with an ubiquitinated protein is important for this localization. 42 This protein could be Htt, since it is constitutively ubiquitinated and binds to a region of Optn encompassing its UBD.…”

“…24,80 The ubiquitin-binding function of Optn is also important for antiviral immune response, selective autophagy of cytosolic bacteria and Transferrin receptor (TfR) recycling. 43,55,78 However, the role of Ub-binding activity in some Optn-mediated functions, such as the transport of lysosomal proteins, remains to be determined. Since ubiquitination of NEMO was shown to be important for NFκB activation, ubiquitination of Optn may also be important for its subcellular localization and functions.…”

Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein

“…3). These data indicate that ␣-tocopherol is trafficked through the recycling endosomes: vesicles that replenish the plasma membrane with recycled receptors and some endocytic cargo and are controlled by Rab8 and its downstream effectors (51,53). To our surprise we found that in live cells, ␣-tocopherol is also located in lipid droplets, marked by the neutral lipid stain Nile red (Fig.…”