Abstract
Purpose :
It has long been recognized that rod photoreceptor arrestin (Arr1) forms oligomers, dimers and tetramers, in vitro and it has been speculated that oligomer formation serves various biological functions, including light-dependent intercompartment transport and storage. The significance of Arr1 oligomerization in living photoreceptors, however, remains unknown. To address this question, we develop a model system for examining Xenopus Arr1 (xArr1) oligomerization in vivo, in Xenopus laevis rods. Characterized xArr1 oligomerization in vitro was carried out to support this effort.
Methods :
Recombinant bovine Arr1 (bArr1) and xArr1 were expressed in E. coli and purified via affinity columns and Gel Filtration. Analytical ultracentrifugation (AUC) of each was performed at various concentrations to examine oligomerization. AUC data was analyzed in SEDFIT and the results were plotted in MATLAB. xArr homology modeling was done with the Modeler interface in Chimera (UCSF) using the crystal structure of bArr1 (PDB: 1CF1 chain A) as a template. Sequence alignments were produced in MegAlign.
Results :
Like bArr1, xArr1 self-associates in a concentration dependent manner. However, where bArr1 formed dimers and higher order oligomers, xArr1 formed dimers only. AUC analysis of xArr1-EGFP showed that a C-terminal EGFP fusion does not appreciably impact self-association in vitro. Similar to mammalian Arr1s, inositol-hexakisphosphate (IP6) also altered the self-association of xArr1. Homology modeling using the crystal of bArr1 as a template suggests an extremely similar fold for xArr1. Sequence alignments indicate high levels of conservation for the C-C interface and the contacts indicated for mammalian Arr1 oligomerization. Some differences were found in the xArr1 N-domain and residues close to the NN interface of mammalian Arr1 tetramers.
Conclusions :
Surprisingly, we found Xenopus Arr1 forms dimers but not tetramers, indicating a potentially significant difference in the function of Arr1 oligomerization across species. An EGFP fusion does not affect self-association of xArr1. Finally, IP6 binding at the rhodopsin interface resulting in oligomer dissociation is conserved in xArr1.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.