Abstract
Purpose:
The C-terminus of rhodopsin mediates several intermolecular interactions essential for its trafficking and for the formation of rod outer segments (OS). To identify previously unrecognized interactions we employed a pull-down approach using native rhodopsin purified from mouse retinas of wild type and knock-in mutant rhodopsin strains. To study rab11a in intact photoreceptors, we expressed EGFP fusions of rab11a in Xenopus photoreceptors.
Methods:
Affinity chromatography was used to isolate proteins binding to the C-terminus of rhodopsin. Rhodopsin binding proteins were identified by in-gel trypsin digestion and mass spectroscopy, and were confirmed by Western blotting. Protein localization was determined by immunohistochemistry of retinal sections and by production of transgenic X. laevis tadpoles expressing EGFP-rab11a and various guanosine nucleotide-binding mutants (S25N, Q70L, N124I).
Results:
Rab11a was identified as a rhodopsin binding protein in mouse rod OS preparations. Reciprocal pull-down experiments confirmed the interaction of rab11a with wild type rhodopsin but not with Q344ter nor rhodopsin-EGFP. Expression of EGFP-rab11a in transgenic tadpoles revealed a punctate distribution in the inner segment (IS) and a diffuse distribution in the OS. Expression of a GTP-locked variant of rab11a, Q70L, resulted in a reduced OS localization with concentrations at the Golgi and at the IS - OS junction. Expression of the GDP-locked variant S25N had the almost opposite distribution of Q70L. Retinal degeneration was only rarely seen with expression of the S25N variant, and never with the Q70L or rab11a. Expression of N124I rab11a which binds neither GDP nor GTP, resulted in an axonemal OS distribution as opposed to the diffuse distribution seen in the other constructs.
Conclusions:
Our results show rab11a is associated with rhodopsin trafficking not only from the Golgi to the apical IS, but from the IS to the disk membranes of the OS. The existence of concentrated pools of rab11a in the apical IS apparent with rab11a and Q70L rab11a but not with S25N implies this structure is a recycling endosome, likely analogous to the pericentriolar recycling endosome seen in other cell types. Our results also suggest GDP-rab11a associates with rod disk membranes and may remain resident after disk formation.
Keywords: 648 photoreceptors •
625 opsins •
698 retinal development