Abstract
Abstract: :
Purpose: AIPL1 has been established recently as being essential for rod PDE biosynthesis/transport. Earlier studies suggest that AIPL1 is undetectable in adult cones, and that AIPL1 plays an essential role in the processing of farnesylated proteins. These observations raise the question as to what role, if any, AIPL1 might play in adult cone photoreceptors. This study sought to clarify this important issue, since a rod defect alone cannot explain the LCA phenotype. Methods:Rabbit and chicken antibodies against the N–terminal 200 residues of murine cone PDE, which are least conserved with rod PDE, were generated and affinity–purified. Corresponding regions of rod PDE α– and ß–subunit were synthesized as recombinant proteins and were used to deplete any cross–reactivity with rod PDE. Retinas of an AIPL1 knockdown mutant, in which AIPL1 was expressed at 20% of WT level, were analyzed by western blotting and by immunofluorescence. Results: By Western blotting, cone PDE level in the mutant was reduced to an extent proportional to the reduction in AIPL1 expression. By immunofluorescence, cone PDE in the mutant was present at a much lower level in the outer segments but was similarly present in the cell body compared to the WT, suggesting a reduced transport to the outer segments. No other cone–specific proteins (e.g., cone opsins) were affected. Conclusions: 1). AIPL1 is essential for the biosynthesis/transport of both cone and rod PDEs. 2). Since cone PDE is not farnesylated, farnesylation does not appear to be an essential feature that AIPL1 recognizes in a client protein. Rather, conserved sequence motifs in the rod and cone PDEs likely provide the basis for functional interaction with AIPL1. 3). AIPL1 is presumed to be present in adult cones. 4). The in vivo role of AIPL1 reported here fully explains the LCA phenotype of human patients with loss of AIPL1. We propose that AIPL1 functions as a specialized chaperone acting at the late stages of rod and cone PDE biosynthesis, such as in a quality control pathway and in targeting the client proteins to the photoreceptor outer segments.
Keywords: retinal degenerations: cell biology • transgenics/knock-outs • chaperones