Abstract
Purpose :
The outer segment of rod photoreceptors is composed of two distinct membrane subdomains: discs and plasma membrane. We are interested in understanding how membrane proteins are trafficked to these two subdomains. One resident outer segment plasma membrane protein is the cyclic nucleotide-gated (CNG) channel. It was previously reported that in peripherin-2 knockout (Rds) mice the CNGβ1 subunit is absent from rods, suggesting that CNGβ1 stability or trafficking to the outer segment requires peripherin-2 (Prph2). We recently showed that the CNG channel travels by a conventional pathway through the Golgi, while it is established that Prph2 uses an unconventional pathway bypassing the Golgi. Together, these data suggest that either Prph2 and CNGβ1 interact before segregating into separate pathways or the Prph2/Rom-1 complex known to traffic through the Golgi facilitates CNGβ1 delivery. We investigated both possibilities to understand how Prph2 is engaged in CNG channel delivery to the outer segment.
Methods :
Rds, Rom1-/-, and C57Bl6J mice were analyzed. DNA constructs were introduced in Rds rods by standard in vivo electroporation. Fixed agarose retinal sections were immunofluorescently stained and imaged by confocal microscopy. Retinal lysates were deglycosylated using PNGase F and EndoH enzymes and analyzed by Western blot
Results :
We overexpressed a MYC-tagged CNGβ1 in Rds rods and found it was trapped in internal membranes, but CNGβ1 localization was restored to the outer segment when full-length FLAG-tagged Prph2 was expressed. These data are consistent with a trafficking defect and not protein instability. To determine whether the CNG channel is trafficked with the Prph2/Rom1 complex, we stained Rom1-/- retinas and found the CNG channel was properly localized in the outer segment. This suggests CNGβ1 and Prph2 are engaged before pathway segregation, leading us to investigate which molecular feature of Prph2 is required for CNGβ1 delivery. We expressed Prph2/Rhodopsin chimeras containing either the Prph2 C-terminus or tetraspanin core and show that both Prph2 chimeras are capable of trafficking CNG to the outer segment.
Conclusions :
We conclude that Prph2, and not the Prph2/Rom1 complex, is necessary and sufficient for CNGβ1 trafficking to the outer segment. Additionally, we determine that both the Prph2 tetraspanin core and C-terminus can facilitate CNGβ1 outer segment delivery.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.