Abstract
Purpose: :
Heterologous membrane proteins tend to accumulate predominantly but not exclusively in frog rod outer segments (ROS). Adding rhodopsin C-terminal peptides containing the VXPX targeting motif drives specific and exclusive localization of these membrane proteins to frog ROS. In contrast, successful ROS-specific targeting of heterologous proteins has been problematic in mouse. We sought a strategy to increase the efficiency of ROS targeting of transmembrane proteins in mouse rods.
Methods: :
Rhodopsin C-terminal peptides of varying length were fused to a single-pass transmembrane domain or to the heptahelical 5-HT1A receptor. The constructs were expressed in transgenic Xenopus laevis rods using the REMI method and in transfected mouse rods using electroporation. Transgenic tadpoles at developmental stages 43-55 and mouse retinas 21 days post-transfection were processed for confocal microscopy.
Results: :
Addition of rhodopsin’s 15 C-terminal amino acids sufficed to direct the single-pass transmembrane protein specifically to frog ROS, but this construct partially mislocalized in mouse rods. This problem was overcome by using rhodopsin’s entire 38-residue C-terminus. We found that the 5-HT1A receptor contained targeting information specifying exclusion from ROS; in frog, this intrinsic signal could be completely overcome by adding rhodopsin’s 38-residue tail to the receptor, but in mouse ROS targeting of this construct was incomplete. Finally, we found that overexpression of Arf4, the GTPase that recognizes the VXPX motif, in mouse rods allowed fusion constructs to target similarly as in frog.
Conclusions: :
The lower ROS targeting efficiency of heterologous proteins in mouse rods can be increased by optimizing VXPX motif presentation and/or boosting Arf4 expression. This finding suggests that the lower ROS targeting efficiency in mouse rods as compared to frog rods is secondary to a lower probability of successful VXPX motif/Arf4 interactions. This is likely due to a smaller Arf4 load in mouse rods, reflecting their naturally lower flux of rhodopsin traffic.
Keywords: photoreceptors • retina: distal (photoreceptors, horizontal cells, bipolar cells)