Identification of Substrate Proteins of Molecular Chaperones CCT and PHLP in Retinal Photoreceptors

Abstract

Purpose: The chaperonin CCT is a large ATPase complex found in all eukaryotic cells that folds nascent proteins in its inner cavity. The known CCT substrates vary in their structure and function, however, many of them in specialized mammalian cells remain unknown. It has been demonstrated that the folding and assembly of the Gβ₁γ₁ subunit complex of the heterotrimeric G protein, transducin, which is engaged in phototransduction, is conducted by CCT and also by phosducin-like protein (PHLP). Our goal was to identify other protein substrates of these molecular chaperones in retinal photoreceptors.

Methods: The “short” splice isoform of phosducin-like protein, PHLPs, which is known to form a high-affinity complex with CCT, was overexpressed with a FLAG-tag in mouse photoreceptors and in HEK 293 cells. PHLPs and any proteins bound to it were captured with anti-FLAG agarose, further purified by native gel electrophoresis, and identified by mass spectroscopy. Any structural and functional changes evoked by PHLPs expression in the photoreceptors were monitored by electron microscopy and electroretinography.

Results: The isolated high molecular weight complex predominantly contained the intact CCT. Besides PHLPs, ~ 25 other proteins co-purified with CCT, and they were regarded as potential encapsulated substrates. Gβ₁ appeared to be the most abundant substrate type in photoreceptors. Gβ₃, Gβ₅ and Gβ₂ were also present, however, in the smaller amounts. No Gγ or RGS proteins normally associated with mature G-protein β subunits could be detected. In HEK 293, CCT predominantly co-precipitated filamin A, cytoplasmic actin, and ufm1-specific protease 2. Thyroid hormone receptor-associated protein 3, elongation factor 1-alpha, histones H4 and H3.1, cytoplasmic actin, and Gβ₂ were among the common substrates identified in both photoreceptors and HEK 293 cells. Furthermore, HEK 293 cells tolerated PHLPs well, whereas in photoreceptors, it caused shortening of the outer segments and a profound decline in visual responses.<br />

Conclusions: Our study identified G-protein β subunits as the primary substrates of the molecular chaperones, CCT and PHLP, in retinal photoreceptors, but demonstrated a rather different substrate specificity in HEK 293 cells.