Abstract
Purpose:
We aim to identify the elements that ensure proper subcellular targeting of outer segment resident proteins. Cyclic nucleotide gated (CNG) channels are located in the plasma membrane of photoreceptor outer segments and mediate the electrical response to light. The rod CNG-channel is composed of CNGα1 and CNGβ1 subunits that co-assemble to form a heterotetrameric channel. A previous study identified a C-terminal RVXP sequence that is required for ciliary localization of the olfactory CNGβ1b subunit. In this report, we identify a different CNGβ1 targeting sequence sufficient for its outer segment delivery.
Methods:
DNA constructs expressed in Xenopus utilized a dual promoter strategy; Xenopus opsin promoter used to express MYC-tagged human CNGβ1 constructs in rods as well as a γ-crystallin promoter to drive GFP expression in the lens. Transgenic Xenopus tadpoles were produced using standard techniques. At developmental stage 45, immunohistochemical staining was performed and subcellular localization of each construct analyzed by confocal microscopy. Primary mouse anti-MYC 9E10 (Santa Cruz) or anti-CNGβ1 (GARP 8G8) antibodies and goat secondary antibodies conjugated to Alexa Fluor 488 or 568 (Invitrogen).
Results:
In order to identify a targeting signal in the CNGβ1 subunit, we transgenically expressed membrane-bound and cytosolic fragments of the human CNGβ1 into Xenopus rods. Initially, we examined the C-terminal of CNGβ1, including the predicted RVXP motif, but determined that this sequence is not involved in outer segment localization of the CNGβ1 channel. Instead we identified a short domain on the cytoplasmic N-terminal that is sufficient for outer segment delivery of the channel. Interestingly, this targeting domain of CNGβ1 is separate from its N-terminal peripherin binding site. However, peripherin binding is capable of bringing cytoplasmic fragments of CNGβ1 to the outer segment, most likely through protein-protein interactions.
Conclusions:
These findings indicate that the rod CNGβ1 channel does not contain a C-terminal RVXP targeting sequence (as reported for olfactory CNGβ1b), but instead contains a targeting domain present in the N-terminal. This targeting domain is separate from the peripherin binding site and is sufficient for CNGβ1’s outer segment delivery.
Keywords: 648 photoreceptors •
569 ion channels •
659 protein structure/function