Work in our laboratory focuses on the biosynthesis, sorting and post-Golgi trafficking of rhodopsin to the rod outer segments (ROS) in retinal photoreceptor cells. Sorting into post-Golgi membranes of the appropriate proteins and lipids destined for delivery to the ROS is critical for the maintenance of photoreceptor polarity and functional integrity of retinal rods. To study the molecular mechanisms that govern trafficking to the ROS we have developed a cell-free system that reconstitutes formation of rhodopsin-bearing post-Golgi carrier membranes from the trans-Golgi network (TGN) in vitro.
The development of this cell free system to study rhodopsin trafficking has allowed new insights into these processes at the molecular level. Using this system we found that post-Golgi trafficking to the ROS depends on the membrane-bound rab proteins (members of the ras superfamily of small GTPases) and on the carboxyl-terminal domain of rhodopsin. We found that the five C-terminal residues QVS(A)PA, play a critical role in regulating rhodopsin sorting into specific post-Golgi membranes. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Since mutations that cluster within the five C-terminal amino acids of rhodopsin cause particularly severe forms of the human disease autosomal dominant retinitis pigmentosa (ADRP) our findings clearly suggest a possible underlying molecular mechanism: these mutations most likely result in abnormal post-Golgi membrane formation and mistargeting of mutant rhodopsin.
Our recent studies are focused on several aspects of the regulation of post-Golgi trafficking to the ROS. Using biochemical approaches we are identifying the protein, or the protein complex that interacts with rhodopsin at the TGN. We study the regulation of rhodopsin trafficking by small GTPases in the cell-free system and in transgenic animals. We are identifying and characterizing additional post-Golgi membrane specific proteins, determining the composition of the lipid milieu of the TGN and studying the role of phospholipids and lipid kinases in rhodopsin sorting and in ROS membrane renewal.
Deretic D, Schmerl S, Hargrave PA, Arendt A and McDowell JH. (1998) Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA. Proc. Natl. Acad. Sci. USA. 95:10620-10625.
Krappa R, Nguyen A, Burrola P, Deretic D and Lemke G. (1999) Evectins: Novel vesicular proteins that carry a plekstrin homology domain and localize to post-Golgi membranes. Proc. Natl. Acad. Sci. USA. 96:4633-4638.
Deretic D. (2000) Rhodopsin trafficking in photoreceptors using retinal cell-free system. In: Vertebrate Phototransduction and the Visual Cycle. Palczewski K, ed., Methods in Enzymology. J. N. Abelson and M. I. Simon, Ed in Chief. Academic Press, San Diego, CA. 315: 77-88.
Morel V, Poschet R, Traverso V and Deretic D. (2000) Towards the proteome of the rhodopsin-bearing post-Golgi compartment of retinal photoreceptor cells. Electrophoresis, special issue on “Organelle Proteomics”. 21:3460-3469.
Moritz OL, Tam BM, Hurd L, Peranen J, Deretic D and Papermaster DS. (2001) Mutant rab8 impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death in transgenic Xenopus rods. Mol. Biol. Cell. 12:2341-2351.
Traverso V, Bush RA, Sieving PA and Deretic D. (2002) Retinal cAMP levels during the progression of retinal degeneration in rhodopsin P23H and S334ter transgenic rats. Invest. Ophthalmol. Vis. Sci. 43:1655-1661.
Deretic D, Traverso V, Parkins N, Jackson F, Rodriguez de Turco EB and Ransom, N. (2004) Phosphoinositides, ezrin/moesin and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors. Mol. Biol. Cell. 15:359-370.
