Abstract
Purpose: :
UNC119/HRG4 is a protein with sequence similarity to PrBP/Δ/Pde6Δ. A homolog of C. elegans UNC-119, it is expressed ubiquitously in multiple tissues, and abundantly in photoreceptor inner segments and synaptic terminals. We previously identified UNC119 as a novel acyl-binding protein with specificity for the transducin alpha subunit (Tα). The purpose of this study is to study interaction of UNC119 with Tα.
Methods: :
Isothermal titration microcalorimetry (ITC), GTPase assay, co-crystallization of lauroyl-GAGASAEEKH (Tα peptide) with UNC119, immunohistochemistry, diffusion model of UNC119/Tα-GTP.
Results: :
Isothermal titration calorimetry of UNC119 with an acylated N-terminal peptide of Tα shows tight interaction (Kd = 0.3 uM). Reconstitution of purified transducin with depleted ROS membranes and UNC119 reveals that UNC119 inhibits the endogenous GTPase activity of Tα. Co-crystallization of UNC119 with acylated Tα peptide demonstrates that the Tα lipid chain is buried deeply into UNC119's hydrophobic cavity formed by an immunoglobulin-like β-sandwich fold. The UNC119 cavity is lined predominantly by hydrophobic residues (mostly Phe and Tyr) that mediate interaction with the lauroylated Tα peptide primarily by Van der Waals forces, consistent with properties of a lipid binding site.Interaction of UNC119 with full-length Tα requires the TαGTP bound form. Translocation of Tα to the inner segment during illumination is independent of UNC119, but full return to the outer segment during darkness is UNC119-dependent. Formation of the diffusible complex TαGTP/UNC119 requires GTP/GDP exchange on membranes in the inner segment in the absence of its GEF (rhodopsin). We suggest that slow GDP/GTP exchange and slow formation of TαGTP/UNC119 in the inner segment is a rate-limiting step in return of transducin to the outer segments in darkness.
Conclusions: :
We propose a model for the return of transducin to the outer segment by diffusion. The enzymatically switchable sink in the inner segments is controlled by slow GDP/GTP exchange in the absence of the GEF, rhodopsin, and low levels of UNC119 relative to transducin.
Keywords: photoreceptors • protein modifications-post translational • protein structure/function