Rhodopsin Biosynthesis & Transport
Rhodopsin is the major protein of the rod outer segment (ROS), a cellular domain which captures light and initiates the visual response. Rhodopsin is made in the rod inner segment (RIS) and is transported from there to the ROS on small membrane spheres called vesicles. We are studying how the cell forms these vesicles and transports them only toward the outer segment and not to the other end of the cell. Recent studies of retinal degeneration indicate that certain blinding disorders are associated with a disorder of this transport pathway. To study this pathway in depth, we have initiated these studies in frog retinas because their rod cells are very large and easily viewed in the light microscope. We use the GFP tag to make the proteins visible by fluorescence microscopy. If we shine bright blue light on the retinas and put a green barrier filter in the eyepieces, only the green light comes back out of the eye and is captured by the camera. We also counterstain the retinas with a red-fluorescing marker to outline the outer segments and the sides of the rods.

The images on this page show the difference in distribution of GFP when it is expressed alone or as part of rhodopsin in the major (red) rods of X. laevis retinas.


Using the XOP-GFP or XOP-GFP-CT constructs, the GFP is localized to the rod inner segment (IS), nucleus and synapse. The smaller amount seen in the rod outer segment (OS) reflects the smaller cytoplasmic volume of that compartment.

When the GFP is inserted in the rhodopsin sequence between residues 333 and 334, its distribution shifts to the outer segment (Right side). Remarkably, very little of the protein is visible in either the lateral plasma membrane or the synapse indicating that this hybrid molecule is transported appropriately and that the addition of GFP did not affect transport significantly. It's localization reflects that the GFP is now membrane bound and the localization is determined by the destination of the membrane protein to which it is attached.
The rhodopsin-GFP-CT constructs above were prepared by our collaborator on this project, Dr. Tomoko Nakayama of the UCHC Department of Biostructure and Function.

In the next pages we illustrate how alteration of sequences affects the outer segment sorting/retention signal.
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