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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: J Neurochem. 2013 Nov 20;129(2):256–263. doi: 10.1111/jnc.12501

The GAFa domain of phosphodiesterase-6 contains a rod outer segment localization signal

Pallavi Cheguru 1, Zhongming Zhang 1,#, Nikolai O Artemyev 1,2,*
PMCID: PMC4054601  NIHMSID: NIHMS539380  PMID: 24147783

Abstract

Phosphodiesterase – 6 (PDE6) is a peripheral membrane protein synthesized in the inner segment of photoreceptor cells. Newly synthesized PDE6 is transported to the outer segment (OS) where it serves as a key effector enzyme in the phototransduction cascade. Proper localization of PDE6 in photoreceptors is critically important to the function and survival of photoreceptor cells. The mechanism of PDE6 transport to the OS is largely unknown. In this study, we investigated potential OS targeting signals of PDE6 by constructing PDE5/PDE6 chimeric and mutant proteins and analyzing their localization in rods of transgenic Xenopus laevis. We found that efficient OS localization of chimeric isoprenylated PDE enzymes required the presence of a targeting motif within the PDE6 GAFa domain. Furthermore, the GAFa-dependent localization signal was sufficient to target EGFP-GAFa fusion protein to the OS. Our results support the idea that effective trafficking of the peripheral membrane proteins to the OS of photoreceptor cells requires a sorting/targeting motif in addition to a membrane-binding signal.

Keywords: retina, rods, protein trafficking, PDE6, GAF domain, targeting signal

Introduction

The rod photoreceptor cell is an excellent system to study protein trafficking (Pearring et al. 2013). Rods are polarized cells with a specialized ciliary compartment, outer segment (OS), containing all the phototransduction machinery, and housekeeping compartment, inner segment (IS), where the phototransduction proteins are synthesized. A slender connecting cilium (CC) links the IS with the OS. Opposite to the OS is the synaptic terminal that senses hyperpolarization produced by light-induced changes in the conductance of the OS plasma membrane. Constant renewal of disc membranes in ROS requires continuous synthesis of phototransduction components in the IS, sorting and transport through the CC to the OS. In particular, rhodopsin trafficking in rods has been extensively investigated and is perhaps one of the best understood targeting pathways (Tam et al. 2000, Moritz et al. 2001, Deretic 2006, Deretic & Wang 2012). Rhodopsin transport to the CC starts with the protein sorting into rhodopsin-bearing vesicles at the TGN. It involves recognition of the two targeting signals VxPx and FR by a small GTPase Arf4 and its GAP protein ASAP1 (Deretic 2006, Deretic & Wang 2012). Defects in rhodopsin trafficking are a common cause of retinitis pigmentosa (Deretic 2006). Targeting signals have been demonstrated in several other OS integral membrane proteins including peripherin/rds and retinal guanylate cyclase GC1 (Tam et al. 2004, Karan et al. 2011). Correct transport of GC1 appears to require the entire cytoplasmic domain (Karan et al. 2011) and the interaction with the RD3 protein (Azadi et al. 2010). Yet, the existence of targeting sequences remains unclear for the majority of the OS proteins with transmembrane domains. Furthermore, membrane proteins lacking specific targeting signals may be delivered to the OS by a “default” route such as co-transport with abundant rhodopsin-carrying vesicles (Baker et al. 2008). Many of the key OS signaling proteins are peripheral membrane proteins anchored to disc membrane by lipid modifications. Among them are N-acylated transducin-α (Gαt) and recoverin, S-acylated (palmitoylated) photoreceptor retinol dehydrogenase (prRDH or RDH8), isoprenylated cGMP-phosphodiesterase catalytic subunits PDE6αβ (PDE6AB), transducin-γ (Gγ1) and rhodopsin kinase. However, the mechanisms of OS targeting of peripheral membrane proteins remain largely obscure (Pearring et al. 2013, Baker et al. 2008, Luo et al. 2004, Karan et al. 2008, Karan et al. 2010). Membrane association is thought to be essential but not sufficient for effective OS localization (Luo et al. 2004). Gαt lacking the N-acylation was severely mislocalized to the IS in mutant mice (Kerov et al. 2007). Efficient OS targeting was found to require membrane binding of prRDH through S-acylation of conserved C-terminal Cys residues and the rhodopsin-like V/IxPx sorting sequence at the very C-terminus (Luo et al. 2004).

Very little is known about the trafficking of PDE6 (Pearring et al. 2013, Karan et al. 2008, Karan et al. 2010). Yet, proper localization of PDE6 in photoreceptors is critically important to the function and survival of rods and cones. Lack of functional rod PDE6 in the ROS leads to elevation of cGMP levels and causes rapid RD in animal models and humans (Farber & Lolley 1974, Bowes et al. 1990, Pittler & Baehr 1991, Ramamurthy et al. 2004, Liu et al. 2004). Mutations in the PDE6A and PDE6B genes are responsible for a significant fraction of recessive RP (McLaughlin et al. 1995, Dryja et al. 1999), whereas mutations in PDE6C cause autosomal recessive achromatopsia (ACMH) in humans (Chang et al. 2009, Thiadens et al. 2009, Grau et al. 2011). Following prenylation in the cytosol, PDE6 catalytic subunits bind ER membranes and undergo CAAX-box processing with the cleavage of –AAX and carboxymethylation of the prenylated Cys residue (Karan et al. 2008, Zhang & Casey 1996, Gelb et al. 2006, Christiansen & Ramamurthy 2012). The lipid modifications of PDE6 do not allow diffusion of PDE6 in the cytosol (Muradov et al. 2009), and the protein is apparently transported from the ER membranes to the OS by vesicular transport (Karan et al. 2008, Christiansen & Ramamurthy 2012). Prenyl-binding protein PrBP/δ is capable of solubilizing PDE6 and may assist the protein transfer to vesicles (Zhang et al. 2012). However, PDE6 sorting into vesicles, the nature of these vesicles, their targeting to the base of the cilium, and subsequent PDE6 transport to the OS are unknown. Transgenic Xenopus laevis is a valuable tool to study protein trafficking in rod photoreceptors. Previously, we have demonstrated that the EGFP-fused cone PDE6C expressed in X. laevis rods under control of Xenopus opsin promoter is correctly targeted to the OS (Muradov et al. 2009). In the OS, EGFP-PDE6C is concentrated at the disc rims and colocalizes with endogenous frog rod PDE6 (Muradov et al. 2009). Here, we utilize this system to examine the OS localization signals of PDE6.

Methods

Cloning of PDE6, PDE5/6 chimeric and mutant constructs

The generation of the pXOP-EGFP-PDE6C vector for expression of the EGFP-fused human cone PDE6 in rod of transgenic X. laevis has been described previously (Fig. 1) (Muradov et al. 2009). Chimera 5-6ct (Fig. 1), containing residues 1-847 of bovine PDE5 joined with the C-terminal residues 817-858 of human PDE6C was constructed in a two-step PCR procedure. In the first step, the PDE6C-817-858 sequence was amplified from the pXOP-EGFP-PDE6C template with a hybrid forward primer coding PDE5-840-847/PDE6-817-824 and a reverse primer containing the stop codon and an XmaI site. This PCR product was used as a reverse primer in the second PCR amplification with a forward primer containing a NotI site and coding the N-terminus of PDE5. The pFastBacHTb-PDE5 vector was used as template in the second PCR reaction (Granovsky et al. 1998). This resulting PCR product was then cloned into the modified pXOP(−508/+41)EGFP using NotI/XmaI sites. Chimera 6-5-6ct (PDE6C-1-451/PDE5-512-847/PDE6C--817-858) was constructed by PCR-amplification of the PDE5-512-847/PDE6C--817-858 sequence with flanking EcoRV/XmaI sites from the 5-6ct template and cloning the PCR product into the EcoRV/XmaI digested pXOP-EGFP-PDE6C plasmid (Fig. 1). Chimera 5-6-6ct (PDE5-1-509/PDE6-450-858) was constructed by PCR-amplification of the PDE5-1-509 sequence with flanking NotI/EcoRV sites from the 5-6ct template and cloning the PCR product into the NotI/EcoRV digested pXOP-EGFP-PDE6C plasmid (Fig. 1). To obtain chimera 6ga-5-6ct (PDE6C-1-246/PDE5-327-847/PDE6C--817-858) (Fig. 1), PDE6C-1-246 sequence was amplified from the 6-5-6ct template with the introduction of the NheI site at the 3′ end. This PCR product was cloned into the NotI/NheI sites of the 5-6ct plasmid. To obtain the 6ga construct (Fig. 1), PDE6C-1-225 was amplified from the pXOP-EGFP-PDE6C plasmid with the introduction of the XmaI site at the 3′ end. This PCR product was cloned into the NotI/XmaI restricted pXOP-EGFP-PDE6C. A quadruple I761A/P762A/M763A/F782A mutant of PDE6C (PDE6C-4A) was generated using QuikChange site-directed mutagenesis (Agilent) (Fig. 1). All constructs were confirmed by automated DNA sequencing at University of Iowa DNA Core Facility. DNA was purified using a Qiagen Midiprep kit, digested with XhoI to linearize the plasmids, and re-purified with Qiagen Gel Purification kit with final elution in water.

Figure 1. Chimeric and mutant PDE5/PDE6 constructs to probe PDE6 trafficking in X. laevis rods.

Figure 1

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PDE5 and PDE6 share similar domain organization (GAFa, GAFb, catalytic domain). These domains were swapped in PDE5/6 chimeras. The constructs (except 6ga) contained the C-terminus of PDE6C (aa 817-858) (6ct) with the –CAAX(-CLML) prenylation/processing box essential for PDE6 expression and localization. PDE6-4A denotes the PDE6C quadruple mutant I761A/P762A/M763A/F782A in which the four key residues interacting with the Pγ C-terminus are replaced with Ala.

Generation of transgenic Xenopus laevis tadpoles

All experimental procedures involving the use of frogs were carried out in accordance with the protocol approved by the University of Iowa Animal Care and Use Committee and compliant with the ARRIVE guidelines. Transgenic tadpoles were produced using the method of restriction enzyme mediated integration (REMI) (Kroll & Amaya 1996). Tadpoles were maintained at 18-22°C until 2 weeks (~stage 50) and then sacrificed for further analysis.

Live cell imaging and cryo-sectioning

Transgenic tadpoles expressing green fluorescence in retinal rod cells were screened using an MZ16F Leica fluorescence microscope using GFP filter. Tadpoles were initially anesthetized for 5 min in 0.02% Tricaine and transferred to Ringer buffer (10 mM HEPES (pH 7.5), 110 mM NaCl, 2 mM CaCl2, 2.5 mM KCl and 1 mM MgCl2). Retinas were extracted from the eyeball and minced in 60 μl of Ringer buffer on a glass slide using two 30-G needles. EGFP fluorescence in living cells was imaged immediately using an LSM 510 confocal microscope (Zeiss). Simultaneously, anesthetized tadpoles were fixed in 4% paraformaldehyde for 1hr. Later, tadpoles were incubated in 30% sucrose in PBS and 30% sucrose and OCT (1:1) solution, each for 1hr. Tadpole heads were dissected and embedded in OCT and frozen at −80°C until use. Cryo-sections were made using Leica Microm Cryostat HM505E and stored at −80°C until use.

Staining and confocal microscopy

Frozen sections were thawed to room temperature and washed in PBS twice for 5min. For staining rod OS, sections were incubated in 0.2% Triton in PBS for 30 min and stained with 2 μg/ml Alexa Fluor Wheat Germ Agglutinin 594 conjugates (Molecular Probes, Invitrogen) (WGA) dissolved in 0.2% Triton-PBS for 30min. WGA is commonly used to visualize rod OS (Luo et al. 2004, Karan et al. 2011). Sections were washed twice with 0.2% Triton-PBS and twice with PBS only. For nuclear counter-staining, sections were treated with 5μg/ml RNase A (Thermo Scientific) in PBS for 5 min followed by wash with PBS twice, 5min each. Later, sections were incubated with 1 μM TO-PRO3 (Invitrogen) solution in PBS. Slides were washed briefly in PBS and mounted using Vectashield mounting medium (Vector Labs, Burlingame, CA). Sections were imaged using a LSM510 confocal microscope (Zeiss)

Protein extraction and Immunoblotting

Eyeballs were excised from tadpoles at 2 weeks of age and stored at −80°C until use. Typically, around 100 eyeballs were homogenized with pestle in 1.5 ml tubes (Beckman Microfuge Polyallomer) using 200 μl of Buffer A (20 mM Tris-HCl (pH 7.5), 120 mM NaCl, 1 mM MgSO4, 1 mM 2-mercaptoethanol, and Complete-Mini EDTA-free protease inhibitor tablets (Roche)). The homogenate was centrifuged at 20,000×g and 4°C for 20 min, and supernatant was termed as isotonic extract. The resulting pellet was resuspended in 200 μl of Buffer B (10 mM Tris-HCl (pH 7.5), 1 mM 2-mercaptoethanol and Complete-Mini EDTA-free protease inhibitor tablets (Roche)). The solution was centrifuged at 70,000×g and 4°C for 1hr. This supernatant was termed as hypotonic extract. Samples were subjected to SDS-PAGE in 4-12% pre-cast gels (Invitrogen) and electro-transferred onto nitrocellulose membrane using iBLOT dry transfer method (Invitrogen). Membranes were incubated with anti-GFP B-2 monoclonal antibody (Santa Cruz Biotech) (1:1000 dilution) at 4°C overnight. The antibody-antigen complexes were detected using anti-mouse antibodies conjugated to horseradish peroxidase (Santa Cruz Biotech) (1:10,000 dilution) and ECL Prime reagent (Amersham Pharmacia Biotech).

Results

PDE6 sequence contains specific OS targeting motifs

Human EGFP-PDE6C has been shown to localize to the OS in rods of transgenic X. laevis (Muradov et al. 2009). The characteristic striated pattern of EGFP-PDE6C distribution in the OS is observed by live photoreceptor cell imaging or imaging of fixed cryosections of transgenic retina (Fig. 2A,B). PDE6C is thought to be geranylgeranylated, and the prenyl moieties are necessary for its membrane association (Karan et al. 2008, Christiansen & Ramamurthy 2012). Our analysis of PDE6C trafficking was based on construction of chimeric enzymes between PDE6 and PDE5 (Fig. 1). Phosphodiesterase 5 (PDE5) is a cytosolic enzyme with relatively low catalytic activity and no lipid modifications. PDE5 shares similar domain organization with PDE6. Both enzymes contain two N-terminal regulatory GAF domains, termed for their presence in cGMP-regulated PDEs, adenylyl cyclases, and the E. coli protein Fh1A (Conti & Beavo 2007). Conserved PDE catalytic domains are located in the C-terminal part of the proteins. Low resolution electron microscopy structures display similar molecular shapes and sizes for PDE6 and PDE5 (Kameni Tcheudji et al. 2001). Thus, PDE5 was selected as an appropriate template for these studies.

Figure 2. Localization of PDE6C in transgenic X. laevis rods.

Figure 2

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EGFP-PDE6C localizes to the OS in the striated peripheral pattern. (A) EGFP-fluorescence in living rods expressing EGFP-PDE6C. (B) EGFP-fluorescence (green), WGA-staining (red), and the EGFP/WGA overlay in transgenic PDE6C retina cryosection. Scale bar is 10 μm.

We first examined whether PDE6 contains specific OS targeting motifs besides the prenylated C-termini. A PDE5 chimera, 5-6ct, in which the C-terminal residues of bovine PDE5 (848-865) were replaced with the C-terminal residues of human PDE6C (817-858), was constructed and ectopically expressed in X. laevis rods (Fig. 1). In contrast to the OS localization of PDE6C, 5-6ct was largely mislocalized to the IS and the synaptic terminal (ST)(Fig. 3A,B). A small fraction of 5-6ct was targeted to the OS, where it displayed a peripheral striated distribution pattern reminiscent of the OS distribution of PDE6C (Fig. 3A,B). Immunoblot analysis revealed a single band of predicted size for the EGFP-fused 5-6ct protein and no signs of its proteolytic degradation (Fig. 3C). PDE6 is membrane-bound in isotonic buffers, but it can be extracted from the membrane by hypotonic buffers (Baehr et al. 1979), probably due to electrostatic repulsion with the negatively charged membrane phospholipids (Malinski & Wensel 1992). EGFP-PDE6C exhibited the expected membrane-binding profile (Fig. 3C). Very little PDE6C was solubilized in the isotonic retina extract, whereas the bulk of the protein was found in the subsequent hypotonic extract. Similarly to PDE6C, 5-6ct was membrane-bound in isotonic buffer, and it was solubilized in hypotonic buffer, indicating its association with intracellular membranes (Fig. 3C). These results suggest that the inability of 5-6ct to effectively localize to the OS is not due to protein degradation, deficient prenylation or altered membrane binding properties. Apparently, PDE6C contains OS localization signals not present in 5-6ct.

Figure 3. The lipid modifications of PDE6 are not sufficient for efficient OS localization.

Figure 3

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Chimera 5-6ct is largely mislocalized to the IS in transgenic rods. (A). EGFP-fluorescence in living 5-6ct rods. Top - cross-section of outer segments at the position marked by the green line in the bottom panel. The image indicates peripheral distribution of a small fraction of 5-6ct in the OS. (B) EGFP-fluorescence (green), WGA-staining (red), and the overlay of transgenic 5-6ct retina cryosection. Scale bar is 10 μm. (C) Isotonic (I) and hypotonic (H) extracts of transgenic retinas were analyzed by immunoblotting with anti-EGFP antibodies.

Inhibition of PDE6 by Pγ is not required for proper trafficking of the enzyme

Two features, besides prenylation, uniquely distinguish PDE6 and PDE5. One is the existence of the inhibitory Pγ subunit, and the second is the much higher kcat for cGMP hydrolysis when the inhibition by Pγ is relieved (Arshavsky & Burns 2012). To evaluate the potential role of these PDE6 features in its trafficking in rods, we generated transgenic X. laevis expressing PDE6C-4A mutant in which all the key Pγ C-terminus contact residues, Ile761Pro762Met763 and Phe782 (Barren et al. 2009, Zhang & Artemyev 2010), have been replaced by Ala residues. The atomic structure (Barren et al. 2009) and the published mutational analysis of the chimeric PDE5/6 catalytic domain (Zhang & Artemyev 2010) strongly predict that PDE6C-4A lacks the inhibitory interaction with the Pγ C-terminus. Live cell imaging as well as cryosection imaging revealed no differences in localization and distribution between PDE6C and PDE6C-4A (Fig. 4). This result indicates that the interaction of the PDE6 catalytic domain with the Pγ C-terminus is not essential for PDE6 translocation into OS.

Figure 4. Inhibition by Pγ is not required for PDE6 trafficking in rods.

Figure 4

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Normal localization of PDE6-4A mutant with disrupted inhibition by Pγ is observed by EGFP-fluorescence imaging in living rods (A) and by imaging of retina cryosections (B). EGFP-fluorescence – green, WGA-staining – red, and the overlay. Scale bar is 10 μm. (C) Membrane association profile of PDE6-4A is normal. Isotonic (I) and hypotonic (H) extracts of transgenic PDE6-4A retinas were analyzed by immunoblotting with anti-EGFP antibodies.

The OS targeting motif is localized within the N-terminal regulatory part of PDE6

To identify the OS localization motifs of PDE6, we first constructed two PDE5/PDE6C chimeras, 5-6-6ct and 6-5-6ct (Fig. 1). Chimera 5-6-6ct contained the C-terminal half from PDE6C including the PDE6 catalytic domain, whereas 6-5-6ct contained the PDE6C N-terminal GAF domains and the PDE6C C-terminus for isoprenylation. The subcellular distribution of 5-6-6ct in rods was very similar to that of 5-6ct. 5-6-6ct was predominantly localized to the IS and ST, with a minor presence in the OS in the typical striated pattern (Fig. 5A). In contrast, the presence of PDE6 GAF domains in 6-5-6ct to a large extent rescued correct OS targeting of the enzyme (Fig. 5B,C). Only a minor fraction of 6-5-6ct was seen in the rod inner compartments, indicating the GAF domains harbor important localization signals of PDE6 (Fig. 5B,C). Interestingly, the distribution pattern of 6-5-6ct was somewhat different from that of EGFP-PDE6C. 6-5-6ct rods were a mixture of rods with the striated pattern and rods with the diffused pattern (Fig. 5B). To explore the underlying cause for the diffused pattern, we examined the membrane-binding profile of 6-5-6ct. A greater fraction of 6-5-6ct was extracted from the membrane using isotonic buffer compared to EGFP-PDE6C (Fig. 3C), suggesting weaker membrane-binding of the chimera (Fig. 5D).

Figure 5. PDE6 targeting motif is localized within the N-terminal regulatory part.

Figure 5

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Mislocalization of chimera 5-6-6ct (A) and the rescue of the OS targeting for chimera 6-5-6ct (B, C) indicate the role of the N-terminal GAF domain(s) in PDE6 trafficking. (A, C –cryosection imaging, B - live cell imaging). Scale bar is 10 μm. (D) Membrane association profile of 6-5-6ct may explain the presence of diffusely fluorescent rods in (B). Isotonic (I) and hypotonic (H) extracts of transgenic 6-5-6ct retinas were analyzed by immunoblotting with anti-EGFP antibodies.

The GAFa domain is essential for proper photoreceptor localization of PDE6

Chimera 6ga-5-6ct containing the GAFa domain of PDE6C was produced to probe the role of the individual GAF domains in the transport of PDE6 (Figs. 1, 6). The presence of the GAFa domain combined with the prenylation site was sufficient for correct transport of 6ga-5-6ct to the OS (Fig. 6A,B). Similarly to the chimera 6-5-6ct, 6ga-5-6ct in rods displayed mixed striated/diffused distribution, and the fraction of the 6ga-5-6ct protein in the isotonic extract was increased (Fig. 6C). To confirm the presence of the PDE6 localization signal within the GAFa domain of PDE6, we tested whether this signal alone can target a soluble EGFP-GAFa fusion protein, 6ga, to the OS. Indeed, 6ga was predominantly and diffusely localized to the OS (Fig. 6D,E). The major fraction of 6ga was found in the hypotonic extract suggesting that the protein is not strongly associated with the membranes (Fig. 6F).

Figure 6. The GAFa domain is important for efficient OS localization of PDE6.

Figure 6

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Chimera 6ga-5-6ct containing the GAFa domain of PDE6C is efficiently targeted to the OS (A, B). The PDE6C GAFa domain targets soluble 6ga protein to the OS (D, E). (A, D - live cell imaging; B, E - cryosection imaging). Scale bar is 10 μm. Immunoblotting of 6ga-5-6ct (C) and 6ga (F) retina extracts (I - isotonic, H - hypotonic) with anti-EGFP antibodies.

Discussion

Organized and precise trafficking of newly synthesized lipidated phototransduction components from the IS to the OS is central to physiology and survival of photoreceptor cells (Pearring et al. 2013). In particular, cGMP hydrolysis by PDE6 in the OS balances its synthesis by GCs. Rapid retinal degeneration caused by elevation of cGMP ensues when PDE6 levels in the OS fall below critic threshold (Farber & Lolley 1974, Bowes et al. 1990, Pittler & Baehr 1991, Ramamurthy et al. 2004, Liu et al. 2004). PDE6 is a heterotetramer with the two catalytic subunits (PDE6AB in rods, PDE6C in cones) that are isoprenylated at the C-termini. The dual isoprenyl lipids provide for tight membrane association of PDE6. Thus, a directed vesicular transport mechanism is thought to underlie PDE6 transport to the OS (Karan et al. 2008, Christiansen & Ramamurthy 2012). The OS is a membrane-rich compartment filled with tightly stacked disc membranes. Hypothetically, the PDE6 lipid modifications might be sufficient for the protein targeting to the OS. Untargeted membrane-bound proteins may co-transport with the “default” trafficking pathway set forth for the rhodopsin delivery to the OS (Baker et al. 2008). However, this pathway, while operational in X. laevis rods, does not appear to carry untargeted proteins in much smaller mouse rods (Pearring et al. 2013). Furthermore, PDE6 and a subset of peripheral membrane proteins may utilize a carrier type different from rhodopsin-carrying vesicles (Hagstrom et al. 2012). Initially, PDE6 has been proposed to co-transport in GC1/GC2-bearing vesicles that are distinct from rhodopsin carriers (Karan et al. 2008, Karan et al. 2010). This model is based on the finding of downregulation of PDE6 in the GC1/GC2 double knockout mice (Baehr et al. 2007). Questioning the model, PDE6 was found to traffic normally to the OS in rd3−/− mouse rods lacking GC1/GC2 (Azadi et al. 2010). Finally, in mice lacking Tulp1, a protein involved in protein transport in photoreceptors, rhodopsin-bearing vesicles and GC1 were mislocalized, whereas the traffic of PDE6, transducin, and rhodopsin kinase to the OS was unimpeded (Hagstrom et al. 2012).

To explore the existence of sorting/targeting signals in PDE6, we generated transgenic X. laevis expressing chimeric PDE5 with the C-terminal isoprenylation motif of PDE6C (5-6ct). This transgenic model revealed that PDE6 contains specific OS targeting motifs required for efficient OS localization. Nonetheless, a small fraction of 5-6ct was present in the OS, where it was distributed in a striated peripheral pattern. We then examined the possibility that the unique inhibitory Pγ subunits are important for efficient localization of PDE6. In the Pγ-knockout mouse model, the levels of PDE6AB are reduced, and the residual PDE6 is catalytically inactive, leading to a rapid retinal degeneration (Tsang et al. 1996). Existing evidence suggests that PDE6 catalytic subunits must be correctly folded to interact with Pγ (Barren et al. 2009). Thus, inactivation of PDE6AB in Pγ−/− mice is conceivably a consequence of mistrafficking of the properly folded PDE6 and/or inappropriate PDE6 activity in the IS at the onset of PDE6 expression. Two regions of Pγ are primarily involved in the interaction with the PDE6 catalytic subunits. The Pγ polycationic region (~aa 24-45) interacts with the GAFa domain, whereas the Pγ C-terminus binds and inhibits the catalytic site (Barren et al. 2009, Artemyev et al. 1996, Muradov et al. 2002). To probe the role of the Pγ-inhibition of PDE6C for the enzyme trafficking, we disrupted the inhibitory interaction of the Pγ C-terminus with the catalytic site in the PDE6C-4A mutant. Previous biochemical studies suggested that this mutant is not inhibited by Pγ (Zhang & Artemyev 2010). Correct and robust localization of PDE6C-4A in the OS of transgenic X. laevis suggests that the Pγ-inhibition of PDE6C is not required in the protein transport.

Our analysis using expression of PDE5/PDE6 chimeras in rods of transgenic X. laevis revealed that efficient OS localization of PDE6 depends on the presence of the GAFa domain. Although the constructs also contained the sequence N-terminal to the PDE6GAFa domain, this sequence is not conserved among different PDE6 catalytic subunits and it is not likely to contain localization signals. The OS targeting of the PDE6C GAFa containing chimeras cannot be explained by their interaction (dimerization) with endogenous frog rod PDE6. Cone PDE6C is lacking the ability to form dimers with rod PDE6AB (Muradov et al. 2009, Muradov et al. 2010). Moreover, the GAFa localization signal was sufficient to target the EGFP-fusion protein 6ga to the OS. Interestingly, after delivery of 6ga to the OS, the protein appears to be retained in this compartment. Since 6ga is deficient of lipid modifications, its retention in the OS suggests lipid-independent interactions with the disc membrane and/or OS resident proteins. Apparently, these interactions are not strong and do not prevent 6ga extraction with isotonic buffer. It remains to be investigated whether the GAFa localization signal depends on the interaction of the PDE6 GAFa domain with the polycationic region of Pγ. The OS targeting of PDE6 seemingly parallels the trafficking of retinol dehydrogenase prRDH. prRDH, a peripheral membrane protein, is S-palmitoylated for membrane association, but a second rhodopsin-like V/IxPx sorting sequence at the very C-terminus is necessary for the efficient OS localization (Luo et al. 2004). One notable distinction is that in PDE6, the membrane-binding C-terminal lipid anchors are spatially separated from the N-terminal GAFa localization signal. Overall, our study supports the emerging view that membrane binding motifs are not sufficient for effective trafficking to the OS, which require a secondary sorting/targeting motif.

Acknowledgements

We would like to thank Dr. Sheila Baker (University of Iowa) for helpful comments on the manuscript. This work was supported by National Institutes of Health Grant EY-10843 to N.O.A.

Abbreviations

PDE6

photoreceptor phoshphodiesterase-6

PDE5

cGMP-binding, cGMP-specific phoshphodiesterase-5

GC

guanylyl cyclase

OS

outer segment

IS

inner segment

ST

synaptic terminal

CC

connecting cilium

GAF

domain named after proteins containing this domain (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA)

WGA

Wheat Germ Agglutinin

DTT

dithiothreitol

IPTG

isopropyl β-D-1-thiogalactopyranoside.

Footnotes

The authors declare no conflicts of interest.

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