Determining the Guanine Exchange Factor (GEF) and Guanine Accelerating Protein (GAP) for rhodopsin trafficking protein rab11a

Abstract

Purpose: Rab11a is a small GTPase, which we have shown to be vital for proper rhodopsin trafficking to the outer segment (OS). It alternates between an active, GTP-bound and inactive, GDP-bound form via its respective guanine exchange factor (GEF) and guanine accelerating protein (GAP). While the GEF and GAP for rab11a have yet to be determined in terminally differentiated rod photoreceptors, but we hypothesize the GEF to be DENND4A and the GAP to be evi5.

Methods: Transgenic Xenopus laevis tadpoles were prepared expressing EGFP-fused human rab11a under the opsin promoter. GFP-fusions express locked nucleotide bound rab11a, mimicking when it is GTP-bound (Rab11^Q70L ), GDP-bound(Rab11^S25N) and also an empty nucleotide binding pocket (Rab11^N124I) mimic. Eyes were sectioned and used for immunohistochemcial staining, labeling for DENND4A and evi5. Pull-down assays were performed using His-tagged evi5 bound to a His-Cobalt column. Soluble GST-rab11a fusion proteins were affinity purified, applied to the column. Following wash steps proteins were eluted from the column and analyzed by western blot.

Results: Based on results from immunohistochemical analysis, DENND4A and evi5 are expressed in X. laevis retina. The localization of these proteins differs depending on the nucleotide bound state of rab11a. Transgenic rab11a is expressed throughout the rod cell, as is evi5 and DENND4A. Rab11a^Q70L, the GTP-bound mimic, localizes to the myoid region, whereas evi5 is solely in the outer segment and DENND4A is throughout the cell, colocalizing at the myoid region. Rab11a^N124I, the empty nucleotide binding pocket mimic, both DENND4A and evi5 found both in the outer segments. Rab11a^S25N, DENND4A and evi5 localize throughout the cell. Analysis of pull-down assays demonstrate that evi5 can successfully pull-down rab11a while in its active form, Rab11a^Q70L.

Conclusions: Our data show that DENND4A and evi5 are expressed in X. laevis retina, and localization is altered by the nucleotide bound state of rab11a. His-tagged evi5 can effectively pull-down Rab11a^Q70L, whereas it could not pull-down Rab11a^WT, indicating rab11a must be in its active conformation for evi5 interaction. These data support our hypothesis for the potential GEF and GAP for rab11a to be DENND4A and evi5.