Abstract
Purpose :
Transducin is a peripheral membrane protein that undergoes light-dependent translocation between the rod outer segment (ROS) and cell body. The mechanisms underlying this translocation have been the subject of intense investigation, but remain unclear. We tested the hypothesis that hydrophobic and electrostatic membrane interactions, due to lipid moieties and surface charges on proteins and disc membranes, play a role in governing peripheral membrane protein distribution by setting membrane affinity.
Methods :
A myristoyl (MYR) and/or farnesyl (FAR) transferase motif was added to EGFP with a 15aa polybasic (+), acidic (-) or neutral (0) charge domain in between. Each probe was placed under the control of the XOP promoter and expressed in Xenopus laevis rod photoreceptors via REMI transgenics. Retinas were harvested and analyzed via live cell confocal microscopy and multiphoton fluorescence relaxation after photoconversion (mpFRAP). Diffusion coefficients were calculated from mpFRAP data. Two-tailed student T-test was used for statistical analysis.
Results :
The mobilities of the probes varied as follows: MYR(0)EGFP(0)FAR << MYR(+)EGFP ≤ MYR(0)EGFP ≤ EGFP(0)FAR << MYR(-)EGFP ≤ EGFP(-)FAR <<<EGFP. Mutating the lipid transferase motif resulted in probes with mobilities comparable to EGFP alone. Myristoylated proteins were enriched in the ROS, despite differences in charge and mobility. The distribution and mobility of farnesylated proteins varied based on charge character. The dually lipidated probe was distributed throughout the rod.
Conclusions :
The mobility of peripheral membrane proteins in the ROS depends on adjacent charge character. The data suggest that positive charge and lipidation work together to increase membrane affinity, while negative charge reduces the membrane affinity provided by the lipid. Dually lipidated proteins had the lowest mobility, indicating an additional lipid moiety conveys greater membrane affinity than adjacent positive charge. Ultimately, our results support the hypothesis that hydrophobic and electrostatic interactions play a role in setting the membrane affinity, however the distribution differs based on lipid identity.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.