The Late Endosomal Pathway Regulates the Ciliary Targeting of Tetraspanin Protein Peripherin 2

Abstract

Peripherin 2 (PRPH2) is a tetraspanin protein concentrated in the light-sensing cilium (called the outer segment) of the vertebrate photoreceptor. The mechanism underlying the ciliary targeting of PRPH2 and the etiology of cone dystrophy caused by PRPH2 mutations remain elusive. Here we show that the late endosome (LE) is the main waystation that critically sorts newly synthesized PRPH2 to the cilium. PRPH2 is expressed in the luminal membrane of the LE. We delineate multiple C-terminal motifs of PRPH2 that distinctively regulate its LE and ciliary targeting through ubiquitination and binding to ESCRT (Endosomal Sorting Complexes Required for Transport) component Hrs. Using the newly developed TetOn-inducible system in transfected male and female mouse cones in vivo, we show that the entry of nascent PRPH2 into the cone outer segment can be blocked by either cone dystrophy-causing C-terminal mutations of PRPH2, or by short-term perturbation of the LE or recycling endosomal traffic. These findings open new avenues of research to explore the biological role of the LE in the biosynthetic pathway and the etiology of cone dystrophy caused by PRPH2 mutations and/or malfunctions of the LE.Peripherin 2 (PRPH2) is a tetraspanin protein abundantly expressed in the light-sensing cilium, the outer segment, of the vertebrate photoreceptor. The mechanism underlying the ciliary transport of PRPH2 is unclear. The present study reveals a novel ciliary targeting pathway, in which the newly synthesized PRPH2 is first targeted to the lumen of the late endosome (LE) en route to the cilia. We deciphered the protein motifs and the machinery that regulates the LE trafficking of PRPH2. Using a novel TetOninducible system in transfected mouse cones, we showed that the LE pathway of PRPH2 is critical for its outer segment expression. A cone dystrophy-causing mutation impairs the LE and ciliary targeting of PRPH2, implicating the relevance of LE to cone/ macular degenerative diseases.

The Primary Cilium as a Therapeutic Target in Ocular Diseases

“…In addition, peripherin 2 is a main component of the outer segment also formed in the endoplasmic reticulum and Golgi and transported to the outer segment through the cilia ( Otsu et al., 2019 ). RPGR, which is located in the ciliary transition region, serves as a scaffold protein to recruit the small GTPase Rab8A, phosphodiesterase 6D (PDE6D), and Arl3 to carry cargo to the cilia, and PDE6D can also promote the positioning of cilia by prenylating RPGR ( Murga-Zamalloa et al., 2010 ; Dutta and Seo, 2016 ).…”

The GARP Domain of the Rod CNG Channel’s β1-subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disc Rim

“…This argues that the CNGb1 cannot be delivered to the outer segment without peripherin-2 and they ought to interact before reaching this destination. A possible trafficking mechanism reconciling all of these observations was proposed in a recent study suggesting that, at least in cones, peripherin-2 is trafficked through the late endosome before entering the outer segment (Otsu et al, 2019). Likewise, the CNG channel might pass through endosomes, based on its mislocalization from the outer segment in conditional double knockout mice lacking the endocytic adaptor proteins Numb and Numb-like in rods (Ramamurthy et al, 2014).…”

Survival Motor Neuron Protein Modulates Lysosomal Function Through the Expression of Transcription Factor EB in Motoneurons

“…Immunostaining Immunostaining was performed as previously described, with minor modifications. 21) Briefly, cells were grown on 12-mm glass coverslips (No. 1-S, Matsunami Glass Industry, Osaka, Japan), coated with 0.2% gelatin, from porcine skin (Sigma Aldrich, G2500), and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS-C/M [phosphate-buffered saline (PBS), containing 0.2 mM CaCl 2 , 2 mM MgCl 2 ], for 10 min at room temperature, quenched with 50 mM NH 4 Cl, for 10 min, followed by 30 min in blocking buffer [PBS-C/M containing 0.5% bovine serum albumin (BSA), 0.5% saponin, 0.2 mg/ ml sodium azide].…”