Cyclic-Nucleotide- and HCN-Channel-Mediated Phototransduction in Intrinsically Photosensitive Retinal Ganglion Cells.

Non-image-forming vision in mammals is mediated primarily by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). In mouse M1-ipRGCs, by far the best-studied subtype, melanopsin activates PLCß4 (phospholipase C-ß4) to open TRPC6,7 channels, mechanistically similar to phototransduction in fly rhabdomeric (microvillous) photoreceptors. We report here that, surprisingly, mouse M4-ipRGCs rely on a different and hitherto undescribed melanopsin-driven, ciliary phototransduction mechanism involving cyclic nucleotide as the second messenger and HCN channels rather than CNG channels as the ion channel for phototransduction. Even more surprisingly, within an individual mouse M2-ipRGC, this HCN-channel-dependent, ciliary phototransduction pathway operates in parallel with the TRPC6,7-dependent rhabdomeric pathway. These findings reveal a complex heterogeneity in phototransduction among ipRGCs and, more importantly, break a general dogma about segregation of the two phototransduction motifs, likely with strong evolutionary implications.

A Population Representation of Absolute Light Intensity in the Mammalian Retina.

Environmental illumination spans many log units of intensity and is tracked for essential functions that include regulation of the circadian clock, arousal state, and hormone levels. Little is known about the neural representation of light intensity and how it covers the necessary range. This question became accessible with the discovery of mammalian photoreceptors that are required for intensity-driven functions, the M1 ipRGCs. The spike outputs of M1s are thought to uniformly track intensity over a wide range. We provide a different understanding: individual cells operate over a narrow range, but the population covers irradiances from moonlight to full daylight. The range of most M1s is limited by depolarization block, which is generally considered pathological but is produced intrinsically by these cells. The dynamics of block allow the population to code stimulus intensity with flexibility and efficiency. Moreover, although spikes are distorted by block, they are regularized during axonal propagation.

Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6.

Heterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is composed of the GTP-bound transducin α subunit (GαT·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis, leading to hyperpolarization of the photoreceptor cell. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GαT. The structure reveals two GαT·GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDEγ subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GαT·GTP subunits and PDE6 that supports an alternating-site catalytic mechanism.

Structural view of G protein-coupled receptor signaling in the retinal rod outer segment.

Visual phototransduction is the most extensively studied G protein-coupled receptor (GPCR) signaling pathway because of its quantifiable stimulus, non-redundancy of genes, and immense importance in vision. We summarize recent discoveries that have advanced our understanding of rod outer segment (ROS) morphology and the pathological basis of retinal diseases. We have combined recently published cryo-electron tomography (cryo-ET) data on the ROS with structural knowledge on individual proteins to define the precise spatial limitations under which phototransduction occurs. Although hypothetical, the reconstruction of the rod phototransduction system highlights the potential roles of phosphodiesterase 6 (PDE6) and guanylate cyclases (GCs) in maintaining the spacing between ROS discs, suggesting a plausible mechanism by which intrinsic optical signals are generated in the retina.

Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.

Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its α subunit (GαT). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the GαT helical domain (αHD) contacting the Gßγ subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gßγ during receptor-catalyzed nucleotide exchange.

Unusual phototransduction via cross-motif signaling from Gq to adenylyl cyclase in intrinsically photosensitive retinalganglion cells.

Nonimage-forming vision in mammals is mediated primarily by melanopsin (OPN4)-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). In mouse M1-ipRGCs, melanopsin predominantly activates, via Gαq,11,14, phospholipase C-ß4 to open transient receptor 6 (TRPC6) and TRPC7 channels. In M2- and M4-ipRGCs, however, a prominent phototransduction mechanism involves the opening of hyperpolarization- and cyclic nucleotide-gated channels via cyclic nucleotide, although the upstream steps remain uncertain. We report here experiments, primarily on M4-ipRGCs, with photo-uncaging of cyclic nucleotides and virally expressed CNGA2 channels to conclude that the second messenger is cyclic adenosine monophosphate (cAMP) - very surprising considering that cyclic guanosine monophosphate (cGMP) is used in almost all cyclic nucleotide-mediated phototransduction mechanisms across the animal kingdom. We further found that the upstream G protein is likewise Gq, which via its Gßγ subunits directly activates adenylyl cyclase (AC). Our findings are a demonstration in a native cell of a cross-motif GPCR signaling pathway from Gq directly to AC with a specific function.

Ultrafast spectra and kinetics of human green-cone visual pigment at room temperature.

Rhodopsin is the pigment that enables night vision, whereas cone opsins are the pigments responsible for color vision in bright-light conditions. Despite their importance for vision, cone opsins are poorly characterized at the molecular level compared to rhodopsin. Spectra and kinetics of the intermediate states of human green-cone visual pigment (mid-wavelength sensitive, or MWS opsin) were measured and compared with the intermediates and kinetics of bovine rhodopsin. All the major intermediates of the MWS opsin were recorded in the picosecond to millisecond time range. Several intermediates in MWS opsin appear to have characteristics similar to the intermediates of bovine rhodopsin; however, there are some marked differences. One of the most striking differences is in their kinetics, where the kinetics of the MWS opsin intermediates are slower compared to those of the bovine rhodopsin intermediates.

Coexistence within one cell of microvillous and ciliary phototransductions across M1- through M6-IpRGCs.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) serve as primary photoceptors by expressing the photopigment, melanopsin, and also as retinal relay neurons for rod and cone signals en route to the brain, in both cases for the purpose of non-image vision as well as aspects of image vision. So far, six subtypes of ipRGCs (M1 through M6) have been characterized. Regarding their phototransduction mechanisms, we have previously found that, unconventionally, rhabdomeric (microvillous) and ciliary signaling motifs co-exist within a given M1-, M2-, and M4-ipRGC, with the first mechanism involving PLCß4 and TRPC6,7 channels and the second involving cAMP and HCN channels. We have now examined M3-, M5-, and M6-cells and found that each cell likewise uses both signaling pathways for phototransduction, despite differences in the percentage representation by each pathway in a given ipRGC subtype for bright-flash responses (and saturated except for M6-cells). Generally, M3- and M5-cells show responses quite similar in kinetics to M2-responses, and M6-cell responses resemble broadly those of M1-cells although much lower in absolute sensitivity and amplitude. Therefore, similar to rod and cone subtypes in image vision, ipRGC subtypes possess the same phototransduction mechanism(s) even though they do not show microvilli or cilia morphologically.

Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function.

The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

Eyeless razor clam Sinonovacula constricta discriminates light spectra through opsins to guide Ca2+ and cAMP signaling pathways.

Phototransduction is based on opsins that drive distinct types of Gα cascades. Although nonvisual photosensitivity has long been known in marine bivalves, the underlying molecular basis and phototransduction mechanism are poorly understood. Here, we introduced the eyeless razor clam Sinonovacula constricta as a model to clarify this issue. First, we showed that S. constricta was highly diverse in opsin family members, with a significant expansion in xenopsins. Second, the expression of putative S. constricta opsins was highly temporal-spatio specific, indicating their potential roles in S. constricta development and its peripheral photosensitivity. Third, by cloning four S. constricta opsins with relatively higher expression (Sc_opsin1, 5, 7, and 12), we found that they exhibited different expression levels in response to different light environments. Moreover, we demonstrated that these opsins (excluding Sc_opsin7) couple with Gαq and Gαi cascades to mediate the light-dependent Ca2+ (Sc_opsin1 and 5) and cAMP (Sc_opsin12) signaling pathways. The results indicated that Sc_opsin1 and 5 belonged to Gq-opsins, Sc_opsin12 belonged to Gi-opsins, while Sc_opsin7 might act as a photo-isomerase. Furthermore, we found that the phototransduction function of S. constricta Gq-opsins was dependent on the lysine at the seventh transmembrane domain, and greatly influenced by the external light spectra in a complementary way. Thus, a synergistic photosensitive system mediated by opsins might exist in S. constricta to rapidly respond to the transient or subtle changes of the external light environment. Collectively, our findings provide valuable insights into the evolution of opsins in marine bivalves and their potential functions in nonvisual photosensitivity.

Probing the mechanism by which the retinal G protein transducin activates its biological effector PDE6.

Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (GαT). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant GαT subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of GαT and the inhibitor vardenafil occupying the active sites on the PDEα and PDEß subunits. We proposed GαT-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody GαT binds to PDE6 in a similar manner as observed when the antibody is present, does GαT activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 GαT-PDE6 complexes form with either recombinant or retinal GαT in the absence of the GαT antibody. We show that GαT binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables GαT to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting GαT-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of GαT for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.

Lipidome Unsaturation Affects the Morphology and Proteome of the Drosophila Eye.

Organisms respond to dietary and environmental challenges by altering the molecular composition of their glycerolipids and glycerophospholipids (GPLs), which may favorably adjust the physicochemical properties of lipid membranes. However, how lipidome changes affect the membrane proteome and, eventually, the physiology of specific organs is an open question. We addressed this issue in Drosophila melanogaster, which is not able to synthesize sterols and polyunsaturated fatty acids but can acquire them from food. We developed a series of semisynthetic foods to manipulate the length and unsaturation of fatty acid moieties in GPLs and singled out proteins whose abundance is specifically affected by membrane lipid unsaturation in the Drosophila eye. Unexpectedly, we identified a group of proteins that have muscle-related functions and increased their abundances under unsaturated eye lipidome conditions. In contrast, the abundance of two stress response proteins, Turandot A and Smg5, is decreased by lipid unsaturation. Our findings could guide the genetic dissection of homeostatic mechanisms that maintain visual function when the eye is exposed to environmental and dietary challenges.

Leading edge of the a-wave of the electroretinogram and sodium iodate-induced age-related macular degeneration: A model.

BACKGROUND: Iron-induced oxidative stress was thought to be the reason why the a-wave amplitude of the electroretinogram (ERG) dropped when iron ions were present. It is assumed that reactive oxygen species (ROS) are generated in the presence of iron ions, and this leads to a decrease in hyperpolarization of the photoreceptor. It is known that in age-related macular degeneration (AMD), sodium iodate can induce oxidative stress, apoptosis, and retinal damage, which mimic the effects of clinical AMD. Here, the reduction of the a-wave amplitude in mice with sodium iodate-induced age-related macular degeneration is explained. METHODS: The leading edge of the a-wave is divided into voltages developed by cones and rods. The same oxidative stress model is applied here since sodium iodate causes the creation of ROS in a manner similar to that caused by iron ions, with the exception that the retina is treated as a circuit of various resistances when computing the photoresponse. Moreover, sodium iodate also leads to apoptosis and, hence, may cause misalignment in cones (not in rods) during the initial stage of apoptosis in AMD. To include the effects of apoptosis and shortening in cones and rods, we have used a factor representing the fraction of total cones and rods that are alive. To include the effect of misalignment of cones on the reduction of the a-wave amplitude, we have used the Stiles-Crawford function to calculate the number of photoisomerizations occurring in a photoreceptor misaligned at an angle θ. The results are compared with experimental data. RESULTS: In sodium iodate-treated eyes, the ROS produced can attract calcium ions in the photoreceptor, which increases the calcium influx. In the case of the cones, the inclusion of the misalignment angle in the phototransduction process helps in determining the voltage and slope of the voltage vs. time graph.The smaller the fraction of active photoreceptors, the smaller the amplitude of the a-wave. The calcium influx, misaligned photoreceptors, and total photoreceptor loss all cause the amplitude of the a-wave to decrease, and at any time from the beginning of phototransduction cascade, the calcium influx causes the slope of the a-wave to increase. CONCLUSION: The reduction in the a-wave amplitude in the eyes of sodium iodate-treated mice is attributed to oxidative stress in both cones and rods and cone misalignment, which ultimately lead to apoptosis and vision loss in AMD.

The electroretinography to identify biomarkers of idiopathic hypersomnia and narcolepsy type 1.

Hypersomnia spectrum disorders are underdiagnosed and poorly treated due to their heterogeneity and absence of biomarkers. The electroretinography has been proposed as a proxy of central dysfunction and has proved to be valuable to differentiate certain psychiatric disorders. Hypersomnolence is a shared core feature in central hypersomnia and psychiatric disorders. We therefore aimed to identify biomarkers by studying the electroretinography profile in patients with narcolepsy type 1, idiopathic hypersomnia and in controls. Cone, rod and retinal ganglion cells electrical activity were recorded with flash-electroretinography in non-dilated eye of 31 patients with idiopathic hypersomnia (women 84%, 26.6 ± 5.9 years), 19 patients with narcolepsy type 1 (women 63%, 36.6 ± 12.7 years) and 43 controls (women 58%, 30.6 ± 9.3 years). Reduced cone a-wave amplitude (p = 0.039) and prolonged cone (p = 0.022) and rod b-wave (p = 0.009) latencies were observed in patients with narcolepsy type 1 as compared with controls, while prolonged photopic negative response-wave latency (retinal ganglion cells activity) was observed in patients with idiopathic hypersomnia as compared with controls (p = 0.033). The rod and cone b-wave latency clearly distinguished narcolepsy type 1 from idiopathic hypersomnia and controls (area under the curve > 0.70), and the photopic negative response-wave latency distinguished idiopathic hypersomnia and narcolepsy type 1 from controls with an area under the curve > 0.68. This first original study shows electroretinography anomalies observed in patients with hypersomnia. Narcolepsy type 1 is associated with impaired cone and rod responses, whereas idiopathic hypersomnia is associated with impaired retinal ganglion cells response, suggesting different phototransduction alterations in both hypersomnias. Although these results need to be confirmed with a larger sample size, the electroretinography may be a promising tool for clinicians to differentiate hypersomnia subtypes.

Dissecting and modeling photic and melanopsin effects to predict sleep disturbances induced by irregular light exposure in mice.

Artificial lighting, day-length changes, shift work, and transmeridian travel all lead to sleep-wake disturbances. The nychthemeral sleep-wake cycle (SWc) is known to be controlled by output from the central circadian clock in the suprachiasmatic nuclei (SCN), which is entrained to the light-dark cycle. Additionally, via intrinsically photosensitive retinal ganglion cells containing the photopigment melanopsin (Opn4), short-term light-dark alternations exert direct and acute influences on sleep and waking. However, the extent to which longer exposures typically experienced across the 24-h day exert such an effect has never been clarified or quantified, as disentangling sustained direct light effects (SDLE) from circadian effects is difficult. Recording sleep in mice lacking a circadian pacemaker, either through transgenesis (Syt10cre/creBmal1fl/- ) or SCN lesioning and/or melanopsin-based phototransduction (Opn4-/- ), we uncovered, contrary to prevailing assumptions, that the contribution of SDLE is as important as circadian-driven input in determining SWc amplitude. Specifically, SDLE were primarily mediated (>80%) through melanopsin, of which half were then relayed through the SCN, revealing an ancillary purpose for this structure, independent of its clock function in organizing SWc. Based on these findings, we designed a model to estimate the effect of atypical light-dark cycles on SWc. This model predicted SWc amplitude in mice exposed to simulated transequatorial or transmeridian paradigms. Taken together, we demonstrate this SDLE is a crucial mechanism influencing behavior on par with the circadian system. In a broader context, these findings mandate considering SDLE, in addition to circadian drive, for coping with health consequences of atypical light exposure in our society.

Transcriptomic Analysis Reveals That Excessive Thyroid Hormone Signaling Impairs Phototransduction and Mitochondrial Bioenergetics and Induces Cellular Stress in Mouse Cone Photoreceptors.

Thyroid hormone (TH) plays an essential role in cell proliferation, differentiation, and metabolism. Experimental and clinical studies have shown a potential association between TH signaling and retinal degeneration. The suppression of TH signaling protects cone photoreceptors in mouse models of retinal degeneration, whereas excessive TH signaling induces cone degeneration, manifested as reduced light response and a loss of cones. This work investigates the genes/transcriptomic alterations that might be involved in TH-induced cone degeneration in mice using single-cell RNA sequencing (scRNAseq) analysis. One-month-old C57BL/6 mice received triiodothyronine (T3, 20 µg/mL in drinking water) for 4 weeks as a model of hyperthyroidism/excessive TH signaling. At the end of the experiments, retinal cells were dissociated, and cell viability was analyzed before being subjected to scRNAseq. The resulting data were analyzed using the Seurat package and visualized using the Loupe browser. Among 155,866 single cells, we identified 14 cell clusters, representing various retinal cell types, with rod and cone clusters comprising 76% and 4.1% of the total cell population, respectively. Cone cluster transcriptomes demonstrated the most alterations after the T3 treatment, with 450 differentially expressed genes (DEGs), accounting for 38.5% of the total DEGs. Statistically significant changes in the expression of genes in the cone cluster revealed that phototransduction and oxidative phosphorylation were impaired after the T3 treatment, along with mitochondrial dysfunction. A pathway analysis also showed the activation of the sensory neuronal/photoreceptor stress pathways after the T3 treatment. Specifically, the eukaryotic initiation factor-2 signaling pathway and the cAMP response element-binding protein signaling pathway were upregulated. Thus, excessive TH signaling substantially affects cones at the transcriptomic level. The findings from this work provide an insight into how excessive TH signaling induces cone degeneration.

Eye proteome of Drosophila melanogaster.

Drosophila melanogaster is a popular model organism to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies, aging, light-induced damage, or dietary deficiencies. Large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which led to the discovery of key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins, including the absolute (molar) quantities of 43 proteins in the eye of adult male Drosophila reared on standard laboratory food. This work provides a generic and expandable resource for further genetic, pharmacological, and dietary studies.

Predicting and Manipulating Cone Responses to Naturalistic Inputs.

Primates explore their visual environment by making frequent saccades, discrete and ballistic eye movements that direct the fovea to specific regions of interest. Saccades produce large and rapid changes in input. The magnitude of these changes and the limited signaling range of visual neurons mean that effective encoding requires rapid adaptation. Here, we explore how macaque cone photoreceptors maintain sensitivity under these conditions. Adaptation makes cone responses to naturalistic stimuli highly nonlinear and dependent on stimulus history. Such responses cannot be explained by linear or linear-nonlinear models but are well explained by a biophysical model of phototransduction based on well-established biochemical interactions. The resulting model can predict cone responses to a broad range of stimuli and enables the design of stimuli that elicit specific (e.g., linear) cone photocurrents. These advances will provide a foundation for investigating the contributions of cone phototransduction and post-transduction processing to visual function.SIGNIFICANCE STATEMENT We know a great deal about adaptational mechanisms that adjust sensitivity to slow changes in visual inputs such as the rising or setting sun. We know much less about the rapid adaptational mechanisms that are essential for maintaining sensitivity as gaze shifts around a single visual scene. We characterize how phototransduction in cone photoreceptors adapts to rapid changes in input similar to those encountered during natural vision. We incorporate these measurements into a quantitative model that can predict cone responses across a broad range of stimuli. This model not only shows how cone phototransduction aids the encoding of natural inputs but also provides a tool to identify the role of the cone responses in shaping those of downstream visual neurons.

Absolute Quantification of Photoreceptor Outer Segment Proteins.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of 20 key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio among all 20 proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

Molecular insights into the maturation of phosphodiesterase 6 by the specialized chaperone complex of HSP90 with AIPL1.

Phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate phototransduction, and its maturation and function are known to critically depend on a specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Defects in PDE6 and AIPL1 underlie several severe retinal diseases, including retinitis pigmentosa and Leber congenital amaurosis. Here, we characterize the complex of AIPL1 with HSP90 and demonstrate its essential role in promoting the functional conformation of nascent PDE6. Our analysis suggests that AIPL1 preferentially binds to HSP90 in the closed state with a stoichiometry of 1:2, with the tetratricopeptide repeat domain and the tetratricopeptide repeat helix 7 extension of AIPL1 being the main contributors to the AIPL1/HSP90 interface. We demonstrate that mutations of these determinants markedly diminished both the affinity of AIPL1 for HSP90 and the ability of AIPL1 to cochaperone the maturation of PDE6 in a heterologous expression system. In addition, the FK506-binding protein (FKBP) domain of AIPL1 encloses a unique prenyl-binding site that anchors AIPL1 to posttranslational lipid modifications of PDE6. A mouse model with rod PDE6 lacking farnesylation of its PDE6A subunit revealed normal expression, trafficking, and signaling of the enzyme. Furthermore, AIPL1 was unexpectedly capable of inducing the maturation of unprenylated cone PDE6C, whereas mutant AIPL1 deficient in prenyl binding competently cochaperoned prenylated PDE6C. Thus, we conclude neither sequestration of the prenyl modifications is required for PDE6 maturation to proceed, nor is the FKBP-lipid interaction involved in the conformational switch of the enzyme into the functional state.