1 Guanylate cyclase 1 relies on rhodopsin for intracellular stability and ciliary trafficking 2 Jillian N. Pearring1, William J. Spencer1,2, Eric C. Lieu1 and Vadim Y. Arshavsky1,2,* 3 1Department of Ophthalmology, Duke University Medical Center, Durham, NC 27710, USA 4 2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 5 27710, USA 6 *Correspondence author: 7 Vadim Y. Arshavsky 8 Mailing address: Duke Eye Center, 2351 Erwin Rd., Box 3802, Durham, NC 27710 9 Phone # 919-668-5391 10 [email protected] 11 1 12 Abstract 13 Sensory cilia are populated by a select group of signaling proteins that detect environmental 14 stimuli. How these molecules are delivered to the sensory cilium and whether they rely on one 15 another for specific transport remains poorly understood. Here, we investigated whether the 16 visual pigment, rhodopsin, is critical for delivering other signaling proteins to the sensory cilium 17 of photoreceptor cells, the outer segment. Rhodopsin is the most abundant outer segment 18 protein and its proper transport is essential for formation of this organelle, suggesting that such 19 a dependency might exist. Indeed, we demonstrated that guanylate cyclase-1, producing the 20 cGMP second messenger in photoreceptors, requires rhodopsin for intracellular stability and 21 outer segment delivery. We elucidated this dependency by showing that guanylate cyclase-1 is 22 a novel rhodopsin-binding protein. These findings expand rhodopsin’s role in vision from being 23 a visual pigment and major outer segment building block to directing trafficking of another key 24 signaling protein. 2 25 Introduction 26 Photoreceptor cells transform information entering the eye as photons into patterns of 27 neuronal electrical activity. This transformation takes place in the sensory cilium organelle, the 28 outer segment. Outer segments are built from a relatively small set of structural and signaling 29 proteins, including components of the classical GPCR phototransduction cascade. Such a 30 distinct functional and morphological specialization allow outer segments to serve as a nearly 31 unmatched model system for studying general principles of GPCR signaling (Arshavsky et al., 32 2002) and, in more recent years, a model for ciliary trafficking (Garcia-Gonzalo and Reiter, 33 2012; Nemet et al., 2015; Pearring et al., 2013; Schou et al., 2015; Wang and Deretic, 2014). 34 Despite our deep understanding of visual signal transduction, little is known how the outer 35 segment is populated by proteins performing this function. Indeed, nearly all mechanistic 36 studies of outer segment protein trafficking were devoted to rhodopsin (Nemet et al., 2015; 37 Wang and Deretic, 2014), which is a GPCR visual pigment comprising the majority of the outer 38 segment membrane protein mass (Palczewski, 2006). The mechanisms responsible for outer 39 segment delivery of other transmembrane proteins remain essentially unknown. Some of them 40 contain short outer segment targeting signals, which can be identified through site-specific 41 mutagenesis (Deretic et al., 1998; Li et al., 1996; Pearring et al., 2014; Salinas et al., 2013; Sung 42 et al., 1994; Tam et al., 2000; Tam et al., 2004). A documented exception is retinal guanylate 43 cyclase 1 (GC-1), whose exhaustive mutagenesis did not yield a distinct outer segment targeting 44 motif (Karan et al., 2011). 45 GC-1 is a critical component of the phototransduction machinery responsible for synthesizing 46 the second messenger, cGMP (Wen et al., 2014). GC-1 is the only guanylate cyclase isoform 3 47 expressed in the outer segments of cones and the predominant isoform in rods (Baehr et al., 48 2007; Yang et al., 1999). GC-1 knockout in mice is characterized by severe degeneration of 49 cones and abnormal light-response recovery kinetics in rods (Yang et al., 1999). Furthermore, a 50 very large number of GC-1 mutations found in human patients cause one of the most severe 51 forms of early onset retinal dystrophy, called Leber’s congenital amaurosis (Boye, 2014; 52 Kitiratschky et al., 2008). Many of these mutations are located outside the catalytic site of GC-1, 53 which raises great interest to understanding the mechanisms of its intracellular processing and 54 trafficking. 55 In this study, we demonstrate that, rather than relying on its own targeting motif, GC-1 is 56 transported to the outer segment in a complex with rhodopsin. We conducted a 57 comprehensive screen of outer segment protein localization in rod photoreceptors of 58 rhodopsin knockout (Rho-/-) mice and found that GC-1 was the only protein severely affected by 59 this knockout. We next showed that this unique property of GC-1 is explained by its interaction 60 with rhodopsin, which likely initiates in the biosynthetic membranes and supports both 61 intracellular stability and outer segment delivery of this enzyme. These findings explain how 62 GC-1 reaches its specific intracellular destination and also expand the role of rhodopsin in 63 supporting normal vision by showing that it guides trafficking of another key phototransduction 64 protein. 65 Results 66 GC-1 is the outer segment-resident protein severely down-regulated in rhodopsin knockout 67 rods 4 68 This study was initiated by testing the hypothesis that rhodopsin, by far the most abundant 69 outer segment protein whose proper transport is essential for formation of this organelle, may 70 affect trafficking and abundance of outer segment membrane proteins. This was accomplished 71 by a comprehensive examination of outer segment protein localization in rods of Rho-/- mice. 72 Normal outer segments are cylindrical structures filled with an ordered stack of several 73 hundred membrane discs (Figure 1A). In contrast, Rho-/- rods only develop small ciliary 74 extensions filled with disorganized membrane material (Figure 1A; (Humphries et al., 1997; Lee 75 et al., 2006; Lem et al., 1999)). Despite this morphological defect, two outer segment-specific 76 proteins, peripherin and R9AP, have been previously shown to reliably target to this ciliary 77 extension (Lee et al., 2006; Pearring et al., 2014). We broadened this analysis to include the 78 majority of transmembrane outer segment proteins. 79 We analyzed ten proteins, whose antibodies have been verified in the corresponding knockout 80 controls. Five of these proteins are components of the phototransduction cascade (R9AP, GC-1, 81 GC-2, CNGα1 and CNGβ1), two support disc structure (peripherin and Rom1), one is a 82 membrane lipid flippase (ABCA4), and the last two are thought to participate in photoreceptor 83 disc morphogenesis (protocadherin 21 and prominin). All experiments were performed with 84 animals sacrificed on postnatal day 21 when the rudimentary outer segments of Rho-/- rods are 85 fully formed, but photoreceptor degeneration that eventually occurs in these mice remains 86 minimal. 87 Remarkably, nine out of ten proteins were localized specifically to the ciliary extensions of the 88 Rho-/- rods. They included Rom1, ABCA4, GC-2, CNGα1, CNGβ1, protocadherin 21 and prominin 89 (Figure 1B-H), as well as previously reported R9AP and peripherin (Figure 1I,J). A striking 5 90 exception was GC-1, which displayed a punctate pattern in the outer segment layer with no 91 distinct signal in rod ciliary extensions (Figure 1K). Further analysis using a cone marker, peanut 92 agglutinin, revealed that the GC-1-positive puncta corresponds to cone outer segments (Figure 93 1L; note that cone outer segments in Rho-/- mice are smaller than normal). Faint fluorescent 94 signal outside cone outer segments was indistinguishable from non-specific background in the 95 outer segment layer of GC-1 knockout mice (Gucy2e-/-; Figure 2). Interestingly, this effect was 96 not reciprocal. As documented in a previous study (Baehr et al., 2007), the knockout of GC-1 97 was not associated with reduction or mislocalization of rhodopsin from rod outer segments 98 (Figure 2). 99 We then used quantitative Western blotting to measure the amounts of outer segment 100 proteins in the retinas of Rho-/- knockout mice. Availability of suitable antibodies allowed us to 101 analyze eight of the initial ten proteins (Figure 3). Serial dilutions of retinal lysates from WT and 102 Rho-/- mice were run on the same blot (such as examples in Figure 3A) and the relative protein 103 amounts were calculated using WT data to generate calibration curves. We found that proteins 104 retaining their normal outer segment localization (Figure 1) were all expressed at 40-80% WT 105 levels (Figure 3B). Considering how small the ciliary extensions of Rho-/- rods are, this amount of 106 protein expression is quite remarkable and suggests a high density of protein packing. 107 Once again the outlier was GC-1 whose content in Rho-/- retinas was only 10±3% (SEM, n=5) of 108 WT (Figure 3A,B). Considering that: (1) a large fraction of this GC-1 is expressed in cones (Figure 109 1L); (2) cones comprise 3% of mouse photoreceptors; and (3) cones express more GC-1 than 110 rods (Dizhoor et al., 1994), our most conservative estimate is that GC-1 in Rho-/- rods is reduced 111 by at least 95%. This reduction is not caused by a loss of the GC-1 transcript, since qRT-PCR 6 112 showed that the amount of GC-1 mRNA in Rho-/- retinas was actually twice higher than in WT 113 retinas (Figure 3 – figure supplement 1), perhaps reflecting a feedback mechanism attempting 114 to compensate for the missing GC-1 enzyme. 115 Taken together, the data reported in Figures 1 and 3 demonstrate that GC-1 is unique among 116 outer segment transmembrane proteins in its reliance on rhodopsin for intracellular stability 117 and outer segment localization. We next addressed the mechanistic basis for this phenomenon. 118 GC-1 stability and trafficking require the transmembrane core of rhodopsin but not its outer 119 segment targeting domain 120 We first asked whether the stability and localization of GC-1 in rods require rhodopsin itself or 121 the activity of rhodopsin’s trafficking pathway.