RESUMO
Rhodopsin is a G-protein-coupled receptor that detects light and initiates the intracellular signalling cascades that underpin vertebrate vision. Light sensitivity is achieved by covalent linkage to 11-cis retinal, which isomerizes upon photo-absorption. Serial femtosecond crystallography data collected from rhodopsin microcrystals grown in the lipidic cubic phase were used to solve the room-temperature structure of the receptor. Although the diffraction data showed high completeness and good consistency to 1.8â Å resolution, prominent electron-density features remained unaccounted for throughout the unit cell after model building and refinement. A deeper analysis of the diffraction intensities uncovered the presence of a lattice-translocation defect (LTD) within the crystals. The procedure followed to correct the diffraction intensities for this pathology enabled the building of an improved resting-state model. The correction was essential to both confidently model the structure of the unilluminated state and interpret the light-activated data collected after photo-excitation of the crystals. It is expected that similar cases of LTD will be observed in other serial crystallography experiments and that correction will be required in a variety of systems.
RESUMO
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.
Assuntos
Rodopsina , Visão Ocular , Animais , Sítios de Ligação/efeitos da radiação , Cristalografia , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Isomerismo , Fótons , Ligação Proteica/efeitos da radiação , Conformação Proteica/efeitos da radiação , Retinaldeído/química , Retinaldeído/metabolismo , Retinaldeído/efeitos da radiação , Rodopsina/química , Rodopsina/metabolismo , Rodopsina/efeitos da radiação , Fatores de Tempo , Visão Ocular/fisiologia , Visão Ocular/efeitos da radiaçãoRESUMO
Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump.
RESUMO
Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically fixed and stained wild-type mouse retina, including axons and synapses, with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analyzing disease processes like neurodegeneration at sub-200â nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptor synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam/scanning electron microscope tomography. The subcellular structures of synapses obtained with the X-ray technique matched the electron microscopy data, demonstrating that PXCT is a powerful scanning method for tissue volumes of more than 60 cells and sensitive enough for identification of regions as small as 200â nm, which remain available for further structural and biochemical investigations.
Assuntos
Retina , Tomografia , Animais , Imageamento Tridimensional , Camundongos , Microscopia Eletrônica , Sinapses , Tomografia Computadorizada por Raios XRESUMO
Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.
Assuntos
Flavobacteriaceae/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/efeitos da radiação , Sítios de Ligação , Cristalografia , Elétrons , Transporte de Íons , Isomerismo , Lasers , Prótons , Teoria Quântica , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Sódio/metabolismo , Análise Espectral , Eletricidade Estática , Fatores de TempoRESUMO
Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for classical single-crystal analysis even in cryogenic conditions. The application of serial crystallography for studying protein dynamics is mentioned.
Assuntos
Proteínas de Membrana/química , Rodopsina/química , Cristalização , Cristalografia/instrumentação , Cristalografia/métodos , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Conformação Proteica , Síncrotrons , Temperatura , ViscosidadeRESUMO
Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs.
Assuntos
Proteínas de Artrópodes/ultraestrutura , Rodopsina/ultraestrutura , Transdução de Sinais/efeitos da radiação , Aranhas , Animais , Proteínas de Artrópodes/isolamento & purificação , Proteínas de Artrópodes/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Luz , Simulação de Dinâmica Molecular , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Rodopsina/isolamento & purificação , Rodopsina/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The development of X-ray free-electron lasers (XFELs) has opened the possibility to investigate the ultrafast dynamics of biomacromolecules using X-ray diffraction. Whereas an increasing number of structures solved by means of serial femtosecond crystallography at XFELs is available, the effect of radiation damage on protein crystals during ultrafast exposures has remained an open question. We used a split-and-delay line based on diffractive X-ray optics at the Linac Coherent Light Source XFEL to investigate the time dependence of X-ray radiation damage to lysozyme crystals. For these tests, crystals were delivered to the X-ray beam using a fixed-target approach. The presented experiments provide probe signals at eight different delay times between 19 and 213 femtoseconds after a single pump event, thereby covering the time-scales relevant for femtosecond serial crystallography. Even though significant impact on the crystals was observed at long time scales after exposure with a single X-ray pulse, the collected diffraction data did not show significant signal reduction that could be assigned to beam damage on the crystals in the sampled time window and resolution range. This observation is in agreement with estimations of the applied radiation dose, which in our experiment was clearly below the values expected to cause damage on the femtosecond time scale. The experiments presented here demonstrate the feasibility of time-resolved pump-multiprobe X-ray diffraction experiments on protein crystals.
RESUMO
Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/efeitos da radiação , Retinaldeído/química , Retinaldeído/efeitos da radiação , Ácido Aspártico/química , Transporte de Íons , Isomerismo , Conformação Proteica , Bases de Schiff/química , Fatores de Tempo , Água/química , Raios XRESUMO
G protein coupled receptors (GPCRs) are a key family of membrane proteins in all eukaryotes and also very important drug targets for medical intervention. The extensively studied visual pigment rhodopsin is a prime example of a family A GPCR. Its chromophore ligand retinal is covalently linked to a lysine in helix seven forming a protonated Schiff base. Interestingly, this is the same situation in other-non-GPCR-retinal proteins, like the prototype light-driven microbial proton pump bacteriorhodopsin, albeit there is no (or only a very remote) phylogenetical link. Close to the retinal ligand, several water molecules help to organise a functionally important hydrogen bond network that undergoes significant changes during photo-activation. Such water-mediated networks are also critical for ligand binding of other GPCRs and they are becoming increasingly important in drug discovery. GPCRs also contain a partially conserved water mediated hydrogen bond network that stabilises the ground state of the receptor, and rearrangement of this network leads to the stabilization of the active state. Some water molecules have a specific role in this process to appropriately orient specific residues relative to the Schiff base, and to modulate the fine structure of the transmembrane bundle, particularly near the intracellular G protein binding site. While the atomic details of these mechanisms are still missing, the recent developments in free electron lasers (FELs) are enabling us to begin to observe the changes in waters and relevant side chains shortly after photo activation at an unprecedented level of spatial and temporal resolution.
Assuntos
Bacteriorodopsinas/química , Receptores Acoplados a Proteínas G/química , Água/química , Lasers , Ligantes , Processos FotoquímicosRESUMO
Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.
RESUMO
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
Assuntos
Bacteriorodopsinas/química , Cristalografia por Raios X/métodos , Lasers , Lipídeos/química , Cristalografia por Raios X/instrumentação , Estudos de Viabilidade , Conformação Proteica , Síncrotrons , Fatores de Tempo , Viscosidade , Espectroscopia por Absorção de Raios X/instrumentação , Espectroscopia por Absorção de Raios X/métodosRESUMO
We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution of 180 nm. As model for a biological system we use Chlamydomonas cells in buffer solution confined in a microcapillary. We use ptychographic X-ray computed tomography to image the entire specimen, including the 18 µm-diameter capillary, thereby providing directly an absolute mass density measurement of biological matter with an uncertainty of about 6%. The resulting maps have sufficient contrast to distinguish cells from the surrounding ice and several organelles of different densities inside the cells. Organelles are identified by comparison with a stained, resin-embedded specimen, which can be compared with established transmission electron microscopy results. For some identified organelles, the knowledge of their elemental composition reduces the uncertainty of their mass density measurement down to 1% with values consistent with previous measurements of dry weight concentrations in thin cellular sections by scanning transmission electron microscopy. With prospects of improving the spatial resolution in the near future, we expect that the capability of non-destructive three-dimensional mapping of mass density in biological samples close to their native state becomes a valuable method for measuring the packing of organic matter on the nanoscale.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Chlamydomonas reinhardtii/ultraestrutura , Imageamento Tridimensional/métodos , Organelas/ultraestrutura , Tomografia Computadorizada por Raios X/métodos , CongelamentoRESUMO
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.
Assuntos
Lasers/estatística & dados numéricos , Rodopsina/química , Difração de Raios X/métodos , Animais , Bovinos , CristalizaçãoRESUMO
In recent years, improved protein expression and crystallization strategies, as well as advanced synchrotron radiation sources and crystallographic tools considerably increased the number of crystal structures of integral membrane proteins from higher eukaryotes. However, seen as a proportion of the total number of candidate proteins, these achievements still appear meager, reflecting the huge effort that is often required to obtain high-level and functional expression of eukaryotic membrane proteins. Besides bacteria, yeast, insect, or mammalian cells are frequently used for heterologous expression, but despite considerable investments in time, labor, and money, there are numerous drawbacks to these systems. Are there other strategies that allow for an effective, large-scale production of functional membrane proteins? This chapter describes the expression of proteins in photoreceptor cells of transgenic Drosophila as an easily accessible, versatile alternative. We present step-by-step protocols starting from the cloning of the target gene into a suitable vector for fly eye expression and ending with the harvest of transgenic Drosophila and subsequent protein purification from the eye. Our examples span a number of eukaryotic membrane proteins from different classes-including receptors, transporters, channels, and enzymes-that were successfully expressed without further optimization. The protocols provided here are robust and straightforward to follow even without prior experience in Drosophila work.
Assuntos
Animais Geneticamente Modificados/genética , Clonagem Molecular/métodos , Drosophila melanogaster/genética , Proteínas de Membrana/genética , Células Fotorreceptoras/metabolismo , Animais , Ácido Aspártico Endopeptidases/análise , Ácido Aspártico Endopeptidases/genética , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/análise , Células Fotorreceptoras/citologiaRESUMO
Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4â Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.
RESUMO
Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.
RESUMO
The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.
Assuntos
Arrestina/genética , Receptor Quinase 1 Acoplada a Proteína G/genética , Mutação , Cegueira Noturna/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/genética , Animais , Arrestina/metabolismo , Bovinos , HDL-Colesterol/química , HDL-Colesterol/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Regulação da Expressão Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Cegueira Noturna/metabolismo , Cegueira Noturna/patologia , Opsinas/genética , Opsinas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Células Fotorreceptoras Retinianas Bastonetes/patologia , Rodopsina/metabolismo , Transdução de SinaisRESUMO
We present active-state structures of the G protein-coupled receptor (GPCRs) rhodopsin carrying the disease-causing mutation G90D. Mutations of G90 cause either retinitis pigmentosa (RP) or congenital stationary night blindness (CSNB), a milder, non-progressive form of RP. Our analysis shows that the CSNB-causing G90D mutation introduces a salt bridge with K296. The mutant thus interferes with the E113Q-K296 activation switch and the covalent binding of the inverse agonist 11-cis-retinal, two interactions that are crucial for the deactivation of rhodopsin. Other mutations, including G90V causing RP, cannot promote similar interactions. We discuss our findings in context of a model in which CSNB is caused by constitutive activation of the visual signalling cascade.
Assuntos
Oftalmopatias Hereditárias/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação de Sentido Incorreto , Miopia/genética , Cegueira Noturna/genética , Rodopsina/química , Arrestina/química , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Rodopsina/genética , Bases de Schiff , Homologia Estrutural de Proteína , Temperatura de TransiçãoRESUMO
BACKGROUND: Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. PRINCIPAL FINDINGS: We describe the homologous and heterologous expression of 10 different targets from the three major MP classes--G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2-0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter--both involved in synaptic transmission--showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. SIGNIFICANCE: We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.