Interaction of arrestin with enolase1 in photoreceptors.
OBJECTIVE
Arrestin is in disequilibrium in photoreceptors, translocating between inner and outer segments in response to light. The purpose of this project was to identify the cellular component with which arrestin associates in the dark-adapted retina.


METHODS
Retinas were cross-linked with 2.5 mM dithiobis(succinimidylpropionate) (DSP), and arrestin-containing complexes purified by anion-exchange chromatography. Tandem mass spectrometric analysis was used to identify the protein components in the complex. Enolase localization in photoreceptors was assessed by immunohistochemistry. Confirmation of interacting components was performed using immunoprecipitation and surface plasmon resonance (SPR). Enolase activity was also assessed in the presence of arrestin1.


RESULTS
In retinas treated with DSP, arrestin cross-linked in a 125-kDa complex. The principal components of this complex were arrestin1 and enolase1. Both arrestin1 and -4 were pulled down with enolase1 when enolase1 was immunoprecipitated. In the dark-adapted retina, enolase1 co-localized with arrestin1 in the inner segments and outer nuclear layer, but remained in the inner segments when arrestin1 translocated in response to light adaptation. SPR of purified arrestin1 and enolase1 demonstrated direct binding between arrestin1 and enolase1. Arrestin1 modulated the catalytic activity of enolase1, slowing it by as much as 24%.


CONCLUSIONS
The results show that in the dark-adapted retina, arrestin1 and -4 interact with enolase1. The SPR data show that the interaction between arrestin1 and enolase1 was direct, not requiring a third element to form the complex. Arrestin1 slowed the catalytic activity of enolase1, suggesting that light-driven translocation of arrestin1 may modulate the metabolic activity of photoreceptors.
Department of Ophthalmology, University of Florida, Gainesville, FL 32610-0284, USA. wcsmith@ufl.edu
W Clay
WC
Smith
Susan
S
Bolch
Donald R
DR
Dugger
Jian
J
Li
Isi
I
Esquenazi
Anatol
A
Arendt
Del
D
Benzenhafer
J Hugh
JH
McDowell
D000818:Animals; D019393:Arrestin / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D003623:Dark Adaptation; D007150:Immunohistochemistry; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry; D014982:Xenopus laevis
D019393:Arrestin; D003432:Cross-Linking Reagents; D010751:Phosphopyruvate Hydratase