Biochemical and structural studies of UNC119 and PrBP/δ: two-lipid binding proteins necessary for protein transport in photoreceptor cells

The mammalian photoreceptor is a dynamic polarized cell, in which protein transport and translocation are continuously taking place. The ability of these cells to accurately and efficiently localize protein components is essential for survival and proper functioning. Post-biosynthesis protein transport from IS to OS and translocation of transducin from OS to IS and back again, in response to light and dark, respectively, test the transport systems in these cells. Additionally, localization of membrane-associated proteins offers a challenge, as lipidated proteins must move freely within the hydrophilic intracellular environment until reaching a destination membrane. As a result, photoreceptors are an ideal model for studying intracellular membrane-associated protein trafficking. Chapter 1 reviews photoreceptor structure and describes the need for accurate and efficient transport mechanisms in these cells. Eukaryotic prenylation and acylation are examined providing context for discussing the biosynthesis, transport, and light-induced translocation of transducin. Lastly, UNC119, which interacts with and is critical in mediating transducin's translocation, is reviewed. Chapter 2 focuses on the biochemical and structural characterization of the UNC119-transducin-α interaction. The co-crystal structure of UNC119-Lauroyl- GAGASAEEKH at 2.0Å resolution offers definitive evidence for this interaction. Wildtype and Unc119-/- mouse models in conjunction with C. elegans unc-119 mutants provide in vivo evidence that UNC119 is required for G protein trafficking in sensory neurons. These data provide the first detailed description of a specific UNC119 function offering insight into the mechanism underlying transducin light-induced translocation. Chapter 3 touches on the generation of an UNC119-Tα-GMPPNP complex, but moves away from light-induced translocation and describes the role PrBP/δ plays in postbiosynthesis transport of PDE6. A general review of PrBP/δ and PDE6 offers a basic foundation for understanding how these two proteins interact and what role this interaction plays. While efforts focused on generating protein crystals of a PrBP/δ-PDE6 complex were unsuccessful, collaboration with Ted Wensel's group yielded a cryo-EM reconstruction of the complex and its features and implications are discussed. Chapter 4 concludes this work with discussions of the aforementioned findings and future directions that might prove useful in further understanding the role UNC119 plays in G protein trafficking and how the PDE6-PrBP/δ complex assembles.