Journal article
Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin
PloS one, Vol.5(7), e11547
07/12/2010
DOI: 10.1371/journal.pone.0011547
PMCID: PMC2902519
PMID: 20634958
Abstract
Background
Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu50 to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.
Methodology/Principal Findings
Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu157 to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.
Conclusions/Significance
The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.
Details
- Title: Subtitle
- Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin
- Creators
- BumChan Park - Johns Hopkins UniversityHongyu Ying - University of Illinois ChicagoXiang Shen - University of Illinois ChicagoJeong-Seok Park - Johns Hopkins UniversityYe Qiu - University of Illinois ChicagoRajalekshmy Shyam - University of Illinois Urbana-ChampaignBeatrice Y. J. T. Yue - Johns Hopkins University
- Resource Type
- Journal article
- Publication Details
- PloS one, Vol.5(7), e11547
- DOI
- 10.1371/journal.pone.0011547
- PMID
- 20634958
- PMCID
- PMC2902519
- NLM abbreviation
- PLoS One
- ISSN
- 1932-6203
- eISSN
- 1932-6203
- Publisher
- Public Library of Science
- Alternative title
- Optineurin Affects Trafficking
- Language
- English
- Date published
- 07/12/2010
- Academic Unit
- Anatomy and Cell Biology
- Record Identifier
- 9984949264002771
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