Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant.
BACKGROUND
Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-kappaB 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.


RESULTS
We 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.


CONCLUSIONS
Our 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.
Nagabhushana
Ananthamurthy
A
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007, India.
Chalasani
Madhavi L
ML
Jain
Nishant
N
Radha
Vegesna
V
Rangaraj
Nandini
N
Balasubramanian
Dorairajan
D
Swarup
Ghanshyam
G
D019943:Amino Acid Substitution; D017209:Apoptosis; D018797:Cell Cycle Proteins; D002460:Cell Line; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005901:Glaucoma / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011990:Receptors, Transferrin / Q000378:metabolism*; D035582:Transcription Factor TFIIIA / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D026901:Membrane Transport Proteins; C409460:OPTN protein, human; D011990:Receptors, Transferrin; D035582:Transcription Factor TFIIIA; D025801:Ubiquitin; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins