Abstract
Purpose: :
Phosducin-like protein 1 (PhLP1) is a cofactor of the eukaryotic chaperonin complex, CCT, implemented in the folding of a number of proteins including heterotrimeric G protein β subunits. Nevertheless, the physiological significance of CCT/PhLP1 in vertebrate photoreceptors, which produce large amounts of the visual G protein, transducin, has remained largely unexplored, and this fact has prompted these studies.
Methods: :
The subcellular localization of PhLP1 and CCT in the retina was determined using Western blot analysis of serial retinal sections. The innate activity of PhLP1 in photoreceptors was suppressed using its dominant-negative mutant, PhLPs, generated by the deletion of the first 83 amino acids in mouse PhLP1. This mutant was expressed in mouse photoreceptors under the control of rhodopsin promoter, and the function and viability of the photoreceptors was monitored using ERG and light microscopy. The levels of protein in the retina were determined using Western blotting.
Results: :
Having found that both PhLP1 and CCT were expressed in rods and, therefore, likely to play an important role in photoreceptor biosynthetic pathways, we generated several transgenic mouse lines, expressing different levels of PhLPs. All mice were characterized by various rates of retinal degeneration due to the loss of photoreceptors. Mice with the higher levels of PhLPs expression failed to fully develop photoreceptors, and lost most of them by age P09. The retinas of mice expressing less PhLPs looked normal until age P12, while losing half of photoreceptors by age P25. This line was used for the analysis of photoreceptor protein levels. It was determined that prior to the onset of photoreceptor degeneration, the levels of transducin and β subunits and phosducin in these retinas had been significantly reduced, in contrast to rhodopsin, PDE, and β-tubulin, whose levels remained unchanged.
Conclusions: :
These studies provide new insights into the role of molecular chaperones in vertebrate photoreceptors and reveal a new type of retinal degeneration caused by their malfunction. We identified transducin and β subunits as primary substrates of the chaperonin CCT and potential culprits of rod degeneration.
Keywords: photoreceptors • protein structure/function • signal transduction