Optineurin (OPTN) is an autophagy receptor protein that has been implicated in glaucoma and amyotrophic lateral sclerosis. OPTN-mediated autophagy is a complex process involving many autophagy-regulating proteins. Autophagy plays a critical role in removing damaged organelles, intracellular pathogens, and protein aggregates to maintain cellular homeostasis. We identified Ypt1 as a novel interaction partner of OPTN by performing a large-scale yeast-human two-hybrid assay. Coimmunoprecipitation assay showed that OPTN interacted with Rab1, the mammalian homolog of yeast Ypt1, in N2a mouse neuroblastoma cell line. We confirmed this interaction by confocal microscopy showing intracellular colocalization of the two proteins. We observed that a zinc finger domain of OPTN is important for Rab1a binding. Rab1a activity is also required for the binding with OPTN. The role of the OPTN-Rab1a complex in neuronal autophagy was determined by measuring the translocation of microtubule-associated protein light chain 3-EGFP to autophagosomes. In N2a cells, OPTN-induced autophagosome formation was inhibited by Rab1a knockdown, indicating the important role of OPTN-Rab1a interaction in neuronal autophagy processes. Similarly, in N2a cells overexpressing Rab1a, serum starvation-induced formation of autophagosome was enhanced, while OPTN knockdown reduced the Rab1a-induced autophagy. These results show that the OPTN-Rab1a complex modulates autophagosome formation in neuroblastoma cells.
Pubmed ID: 28843006 RIS Download
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