Isolating Photoreceptor Compartment-Specific Protein Complexes for Subsequent Proteomic Analysis

  • Gregory H. GrossmanEmail author
  • Gayle J. T. Pauer
  • George Hoppe
  • Stephanie A. Hagstrom
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 723)


Tulp1 is a photoreceptor-specific protein that may perform two distinct functions, one in the inner segment (IS) and another in the synapse. To differentiate the roles of Tulp1 in the photoreceptor, we developed a methodology for the capture of compartment-specific complexes for subsequent protein analysis. Using in vivo perfusion, we crosslinked proteins to conserve endogenous protein complexes. Laser microdissection was used to collect three compartment tissue samples from retinal sections: the IS, the outer plexiform layer (OPL) containing the photoreceptor synapses, and the inner plexiform layer (IPL), serving as a Tulp1-free control tissue. Compartment-specific as well as whole retinal samples were homogenized and subjected to Western blot analysis. Our analysis showed that a band at approximately 78 kDa labels native Tulp1 in the wt crosslinked tissue, but not tulp1 −/− crosslinked tissue. Two additional bands were detected at ∼180 and ∼280 kDa in wt homogenate, indicating the presence of Tulp1 complexes. Finally, a band matching the 280 kDa band was present in the IS-isolated sample, but not the OPL-isolated sample, indicating an IS compartment-specific Tulp1 complex.


Photoreceptor Retinal degeneration Rhodopsin Tulp1 Mouse mutant Trafficking 



This study was supported by National Institute of Health Grants EY16072 and EY15638 (SAH), Foundation Fighting Blindness (SAH), Fight for Sight (GHG), Research to Prevent Blindness (RPB) Center Grant, RPB Sybil B. Harrington Special Scholar Award (SAH), and Hope for Vision (SAH).


  1. Banerjee P, Kleyn PW, Knowles JA et al (1998) TULP1 mutation in two extended Dominican kindreds with autosomal recessive retinitis pigmentosa. Nat Genet 18:177–179PubMedCrossRefGoogle Scholar
  2. Boughman JA, Conneally PM, Nance WE (1980) Population genetic studies of retinitis pigmentosa. Am J Hum Genet 32:223–235PubMedGoogle Scholar
  3. Bunker CH, Berson EL, Bromley WC et al (1984) Prevalence of retinitis pigmentosa in Maine. Am J Ophthalmol 97:357–365PubMedGoogle Scholar
  4. Grossman GH, Pauer GJ, Narendra U et al (2009) Early synaptic defects in tulp1−/− mice. Invest Ophthalmol Vis Sci 50:3074  –3083PubMedCrossRefGoogle Scholar
  5. Gu S, Lennon A, Li Y et al (1998) Tubby-like protein-1 mutations in autosomal recessive retinitis pigmentosa. Lancet 351:1103–1104PubMedCrossRefGoogle Scholar
  6. Hagstrom SA, Duyao M, North MA et al (1999) Retinal degeneration in tulp1−/− mice: vesicular accumulation in the interphotoreceptor matrix. Invest Ophthalmol Vis Sci 40:2795–2802PubMedGoogle Scholar
  7. Hagstrom SA, North MA, Nishina PL et al (1998) Recessive mutations in the gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa. Nat Genet 18:174–176PubMedCrossRefGoogle Scholar
  8. Hagstrom SA, Adamian M, Scimeca M et al (2001) A role for the Tubby-like protein 1 in rhodopsin transport. Invest Ophthalmol Vis Sci 42:1955–1962PubMedGoogle Scholar
  9. Ikeda S, Shiva N, Ikeda A et al (2000) Retinal degeneration but not obesity is observed in null mutants of the tubby-like protein 1 gene. Hum Mol Genet 9:155–163PubMedCrossRefGoogle Scholar
  10. Paloma E, Hjelmqvist L, Bayes M et al (2000) Novel mutations in the TULP1 gene causing autosomal recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci 41:656–659PubMedGoogle Scholar
  11. Schmitt-Ulms G, Hansen K, Liu J et al (2004) Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues. Nat Biotechnol 22:724  –731PubMedCrossRefGoogle Scholar
  12. Smalheiser NR (1996) Proteins in unexpected locations. Mol Biol Cell 7:1003–1014PubMedGoogle Scholar
  13. Xi Q, Pauer GJ, West KA et al (2003) Retinal degeneration caused by mutations in TULP1. Adv Exp Med Biol 533:303–308PubMedCrossRefGoogle Scholar
  14. Xi Q, Pauer GJ, Marmorstein AD et al (2005) Tubby-like protein 1 (TULP1) interacts with F-actin in photoreceptor cells. Invest Ophthalmol Vis Sci 46:4754–4761PubMedCrossRefGoogle Scholar
  15. Xi Q, Pauer GJ, Ball SL et al (2007) Interaction between the photoreceptor-specific tubby-like protein 1 and the neuronal-specific GTPase dynamin-1. Invest Ophthalmol Vis Sci 48:2837–2844PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Gregory H. Grossman
    • 1
    Email author
  • Gayle J. T. Pauer
    • 1
  • George Hoppe
    • 1
  • Stephanie A. Hagstrom
    • 1
    • 2
  1. 1.Department of Ophthalmic ResearchCole Eye Institute, Cleveland ClinicClevelandUSA
  2. 2.Department of OphthalmologyCleveland Clinic Lerner College of Medicine of Case Western Reserve UniversityClevelandUSA

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