title: A Novel Approach to Identify Photoreceptor Compartment-Specific Tulp1 Binding Partners.
abstract: Photoreceptors (PRs) are highly polarized and compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. The PR-specific protein, Tulp1, is localized to the IS and synapse and is hypothesized to be involved in protein trafficking. To better understand the molecular processes that regulate protein trafficking in PRs, we aimed to identify compartment-specific Tulp1 binding partners. Serial tangential sectioning of Long Evans rat retinas was utilized to isolate the IS and synaptic PR compartments. Tulp1 binding partners in each of these layers were identified using co-immunoprecipitation (co-IP) with Tulp1 antibodies. The co-IP eluates were separated by SDS-PAGE, trypsinized into peptide fragments, and proteins were identified by liquid chromatography tandem mass spectrometry. In the IS, potential Tulp1-binding partners included cytoskeletal scaffold proteins, protein trafficking molecules, as well as members of the phototransduction cascade. In the synaptic region, the majority of interacting proteins identified were cytoskeletal. A separate subset of proteins were identified in both the IS and synapse including chaperones and family members of the GTPase activating proteins. Tulp1 has two distinct PR compartment-specific interactomes. Our results support the hypothesis that Tulp1 is involved in the trafficking of proteins from the IS to the OS and the continuous membrane remodeling and vesicle cycling at the synaptic terminal.
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, 44195, Cleveland, OH, USA. ebkel@ccf.org.
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Ebke
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, 44195, Cleveland, OH, USA. pauerg@ccf.org.
authors.forename: Gayle J T
authors.initials: GJ
authors.lastname: Pauer
authors.affiliation: Proteomics Core Services, Lerner Research Institute, Cleveland Clinic, 44195, Cleveland, OH, USA. willarb@ccf.org.
authors.forename: Belinda
authors.initials: B
authors.lastname: Willard
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, 44195, Cleveland, OH, USA. hagstrs@ccf.org.|Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 44195, Cleveland, OH, USA. hagstrs@ccf.org.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Hagstrom
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D047468:Immunoprecipitation; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D020318:Rats, Long-Evans; D055211:Retinal Photoreceptor Cell Inner Segment / Q000378:metabolism*; D055214:Retinal Photoreceptor Cell Outer Segment / Q000378:metabolism; D013569:Synapses / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D005136:Eye Proteins; C491353:Tulp1 protein, mouse; C000606664:Tulp1 protein, rat