The overall goal of this proposal is to elucidate the scope of intracellular trafficking pathways responsible for the delivery of signaling and structura proteins into the light-sensitive organelle of photoreceptor cells, the outer segment. Almost everything we know about outer segment protein delivery relates to the visual pigment, rhodopsin, whereas the mechanisms responsible for the outer segment trafficking of other resident proteins remain largely unknown. During the next grant cycle, we propose to conduct detailed studies of two outer segment-resident proteins, peripherin and PRCD.
Aim 1 will focus on the trafficking mechanism for the photoreceptor disc rim protein peripherin-2/RDS. We will employ live imaging, biochemical fractionation and quantitative mass spectrometry to elucidate whether peripherin and rhodopsin are packaged into the same or different transport vesicles and whether this packaging is performed by the same or different accessory proteins.
Aim 2 will focus on PRCD, a novel constitutive component of photoreceptor discs whose mutations are associated with retinal degeneration in human patients and multiple breeds of dogs. Our proposed experiments will combine basic functional characterization of PRCD with investigation of its prevalent C2Y mutation that leads to PRCD mislocalization from the discs to other photoreceptor compartments. A particular emphasis will be placed on identifying PRCD interacting partners, which will benefit both of these directions. The proposed Aims are relevant to understanding the most basic issues in photoreceptor cell biology and are key for understanding the causes of many types of photoreceptor degeneration associated with defects in protein sorting, targeting and trafficking.

Public Health Relevance

The studies proposed in this application address the molecular and cellular mechanisms responsible for the maintaining the light-sensitive compartment of photoreceptor cells, the outer segment. Because of adverse effects of daily light exposure, the building materials of the outer segment have to be replaced approximately every ten days. This requires an enormous flow of highly organized protein trafficking from the intracellular biosynthetic machinery to this compartment. Dysfunction of outer segment trafficking causes some of the most prevalent and severe types of inherited degenerative diseases of the retina, highlighting the importance of understanding how protein sorting and trafficking are performed in these cells. Elucidating these mechanisms is essential for developing strategies for disease prevention and future therapeutic interventions.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01EY012859-16A1
Application #
8761605
Study Section
(BVS)
Program Officer
Neuhold, Lisa
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Duke University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
City
Durham
State
NC
Country
United States
Zip Code
27705
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Pearring, Jillian N; Lieu, Eric C; Winter, Joan R et al. (2014) R9AP targeting to rod outer segments is independent of rhodopsin and is guided by the SNARE homology domain. Mol Biol Cell 25:2644-9
Salinas, Raquel Y; Baker, Sheila A; Gospe 3rd, Sidney M et al. (2013) A single valine residue plays an essential role in peripherin/rds targeting to photoreceptor outer segments. PLoS One 8:e54292
Skiba, Nikolai P; Spencer, William J; Salinas, Raquel Y et al. (2013) Proteomic identification of unique photoreceptor disc components reveals the presence of PRCD, a protein linked to retinal degeneration. J Proteome Res 12:3010-8
Pearring, Jillian N; Salinas, Raquel Y; Baker, Sheila A et al. (2013) Protein sorting, targeting and trafficking in photoreceptor cells. Prog Retin Eye Res 36:24-51
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Kosloff, Mickey; Travis, Amanda M; Bosch, Dustin E et al. (2011) Integrating energy calculations with functional assays to decipher the specificity of G protein-RGS protein interactions. Nat Struct Mol Biol 18:846-53
Herrmann, Rolf; Lee, Bowa; Arshavsky, Vadim Y (2011) RGS9 knockout causes a short delay in light responses of ON-bipolar cells. PLoS One 6:e27573
Gospe 3rd, Sidney M; Baker, Sheila A; Kessler, Christopher et al. (2011) Membrane attachment is key to protecting transducin GTPase-activating complex from intracellular proteolysis in photoreceptors. J Neurosci 31:14660-8
Gospe 3rd, Sidney M; Baker, Sheila A; Arshavsky, Vadim Y (2010) Facilitative glucose transporter Glut1 is actively excluded from rod outer segments. J Cell Sci 123:3639-44

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