May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Mechanism of Light–Induced Translocation of Rod Transducin: GTP–Stimulated Release From the Membrane and Passive Diffusion
Author Affiliations & Notes
  • V.Z. Slepak
    Pharmacology, University of Miami Shool of Medicine, Miami, FL
  • K.S. Nair
    Pharmacology, University of Miami Shool of Medicine, Miami, FL
  • D. Rosenzweig
    Pharmacology, University of Miami Shool of Medicine, Miami, FL
  • J. Wei
    Biochemistry, University of Washington, Seattle, WA
  • C.–K. Chen
    Biochemistry, Virginia Commonwealth University, Richmond, VA
  • J. Lem
    Ophthalmology, Tuftsâ New England Med Center, Boston,
  • A. Swaroop
    Ophthalmology, University of Michigan, Ann Arbor, MI
  • J.B. Hurley
    Biochemistry, University of Washington, Seattle, WA
  • Footnotes
    Commercial Relationships  V.Z. Slepak, None; K.S. Nair, None; D. Rosenzweig, None; J. Wei, None; C. Chen, None; J. Lem, None; A. Swaroop, None; J.B. Hurley, None.
  • Footnotes
    Support  NIH grant GM60019, EY012982, AHA Florida Affiliate Grant–in–Aid (VZS), EY 06641 (JBH), EY13811 (CKC), EY11115 (AS). DR is supported by an American Heart Association graduate fellowship.
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 3742. doi:https://doi.org/
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      V.Z. Slepak, K.S. Nair, D. Rosenzweig, J. Wei, C.–K. Chen, J. Lem, A. Swaroop, J.B. Hurley; Mechanism of Light–Induced Translocation of Rod Transducin: GTP–Stimulated Release From the Membrane and Passive Diffusion . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3742. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Transducin (Gt) is present in outer segments (OS) of dark–adapted rods, and it re–localizes to the inner compartments in light. Here we examined the molecular mechanism underlying this re–distribution.

Methods: : Mouse eyecups or retinas were incubated, in light or dark, in a culture medium under conditions permitting the control of ATP, GTP or GTP analogs in the cells. Localization of rod and cone transducin subunits in the cells was examined by immunofluorescence microscopy. Retinas were fractionated by ultracentrifugation, and the presence of Gt was determined by western blot. The following mouse models were examined: 1. Gna1–/–; Gna2+, where cone Gαt is expressed in rods; 2. Nrl–/–, containing only cone–like photoreceptors; 3. Gß5–/–; and 4. RGS9–/–. N–Acylation status of Gαt was determined by mass–spectrometry.

Results: : Re–localization of rod Gt in light occurs even in ATP–depleted rod cells. GTP is necessary and sufficient for the departure of Gt from the OS, and the hydrolysis of GTP is required for its return. Accordingly, light–induced re–distribution of rod transducin from the OS is accelerated in RGS9–/– or Gß5–/– mice in vivo. Fractionation studies showed that in WT retinas ∼50% of rod Gt dissociates from membranes upon activation, whereas only ∼10% of cone Gt dissociates both in WT and Nrl–/– retinas. Rod Gαt is acylated heterogeneously with C14:0, C14:1, C14:2 and C12 fatty acids. More than 50% of mouse rod transducin is acylated with C14:2. Cone Gαt from Nrl–/– mouse retinas is N–acylated exclusively with C14:0 myristic acid. Cone transducin does not re–localize either in wild–type or in Nrl–/– retinas. However, cone Gαt does relocalize when it is expressed in Gna1–/–; Gna2+ rods.

Conclusions: : Light–dependent re–distribution of transducin between the OS and inner compartments of rods is an energy–independent process sustained by simple diffusion. The ability of the GTP–bound form of rod transducin to diffuse out of the OS upon activation appears to be influenced by the hydrophobicity of the N–terminal fatty acyl residue on transducin alpha subunit.

Keywords: signal transduction • protein modifications-post translational • photoreceptors 
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