The Outer Segment Serves as a Default Destination for the Trafficking of Single-Pass Membrane Proteins in Photoreceptors

Abstract

Purpose: : To investigate the differential intracellular targeting of two related membrane proteins, R9AP and syntaxin 3, one residing primarily within the outer segment and the other excluded from this specialized compartment.

Methods: : The Xenopus R9AP and syntaxin 3 proteins (fused to either GFP or a Myc-tag) as well as various mutants of each were expressed in transgenic Xenopus laevis rods under control of the 5.5 Kb Xenopus opsin promotor. Transgenic tadpoles at developmental stages 43-55 were fixed, sectioned, and the subcellular localization of the transgenes was analyzed by confocal microscopy.

Results: : We found that so long as R9AP was membrane associated it targeted primarily to outer segments. Syntaxin 3 on the other hand has targeting information encoded within its SNARE domain that mediates its exclusion from outer segments and removal of this information redirected the localization of syntaxin 3 into the outer segment. Similarily, we found that the removal of targeting information from membrane proteins normally targeting to either mitochondria or the endoplasmic reticulum resulted in redirection of these proteins to the outer segment.

Conclusions: : Our data reveal a pattern where membrane proteins lacking specific targeting information are delivered to the outer segment, which is likely to reflect the enormous appetite of this organelle for new material necessitated by its constant renewal. We propose that R9AP (and any other membrane proteins lacking targeting information) reach the outer segment by incorporation into existing transport carriers such as those carrying rhodopsin. An important implication of this finding is that every protein residing outside the outer segment must have a means to avoid this "default" trafficking flow.

Keywords: photoreceptors • transgenics/knock-outs • microscopy: light/fluorescence/immunohistochemistry