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Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors
Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors P D Calvert, SUNY Upstate Medical University, Syracuse, NY, USA V Y Arshavsky, Duke University, Durham, NC, USA ã 2010 Elsevier Ltd. All rights reserved.
Glossary Arrestin – A protein which binds to and inactivates photoactivated rhodopsin. Arrestin binding results in termination of transducin activation. Light adaptation – The ability of photoreceptors (and the visual system as a whole) to adapt the speed and sensitivity of light responses to ever-changing conditions of ambient illumination. Phosducin – A protein which interacts with the bg-subunit of transducin and reduces its membrane affinity. Recoverin – A regulatory protein which is thought to regulate the speed at which arrestin can bind to photoactivated rhodopsin. RGS9 – A protein responsible for returning activated transducin in its inactive form. Transducin – A G protein that mediates the visual signal between the photoactivated visual pigment, rhodopsin, and the downstream effector enzyme, cGMP phosphodiesterase. Transducin consists of two functional subunits, a and bg.
inactivated through its phosphorylation by rhodopsin kinase and arrestin binding, which blocks transducin activation. The rate of rhodopsin phosphorylation, and thus its active lifetime, is regulated by the Ca2+-binding protein, recoverin. Transducin (and accordingly PDE) activation is terminated upon the hydrolysis of GTP tightly bound to transducin a-subunit, a process markedly accelerated by the GTPase activating protein RGS9. Importantly, three of the above-mentioned proteins (transducin, arrestin, and recoverin) undergo massive light-driven translocation between the major subcellular compartments of photoreceptors (Figure 1). In rods, transducin moves out of the outer segment and accumulates primarily in the inner segment, arrestin moves in the opposite direction, and recoverin shifts from the outer segment toward the synapse. In cones, in light arrestin moves in the same direction, whereas transducin moves very little, if at all. Recoverin translocation has not yet been analyzed in cones. A similar phenomenon involving the G protein (Gq), arrestin, and the transient receptor potential-like (TRPL) channel takes place in rhabdomeric invertebrate photoreceptors.
Light Dependency of Protein Translocation Introduction Rod and cone photoreceptors are highly polarized cells which transduce information encoded by photons into electrical activity that can be processed by higher-order neurons. At the one end, photoreceptors have specialized ciliary organelles, outer segments, which are enriched in proteins directly involved in light detection and signal transduction. At the opposite end, synapses convey the information gathered by outer segments to downstream neurons. Vision begins when a molecule of rhodopsin in the outer segment becomes excited by light and activates a G protein, transducin. The transducin a-subunit stimulates its effector, cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE), which leads to the reduction in intracellular cGMP and to the electrical response mediated by the closure of the cGMP-gated cationic channels in the plasma membrane. The recovery of the photoresponse requires complete inactivation of all these molecular components. Photoexcited rhodopsin is
Quantitative experiments revealed that the translocations of arrestin and transducin in rods take place in bright light. The outer segments of the rod contain very little arrestin (estimated under 7% of its total cellular content) in the dark and under moderate illumination. In mouse rods, arrestin begins to move to outer segments when the light intensity reaches a critical threshold, exciting over 1000 rhodopsins per rod per second, which is within the upper limit at which mammalian rods can signal variations in light. Transducin translocation is also triggered at a threshold light intensity, although brighter, exciting 5000 rhodopsins per rod per second, an intensity that completely saturates rods. The time required for the completion of protein translocation in saturating light in rods is on the order of tens of minutes. Although no such quantitative measurements are available for cones, available data indicate that cone arrestin translocation also requires fairly bright light. The existence of cone transducin translocation in intact cells remains somewhat
577
578
Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors
Transducin Arrestin Recoverin Dark 80–90% <7% ∼12%
OS
IS
N ST Bright light 10–20% ∼80% <2%
Figure 1 Schematic illustration of transducin, arrestin, and recoverin distribution in dark- and light-adapted rods. The numbers on the left, color-coded to the corresponding translocating proteins, represent the percentage of the proteins found in the outer segments in the dark or following bright light illumination. The subcellular rod compartments are abbreviated on the right: OS – outer segment; IS – inner segment; N – nucleus; ST – synaptic terminal. Reproduced from Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568.
controversial. Most investigators do not see it at any light intensity, whereas one group reports small degree of translocation observed in extremely bright light. The most recent report argues that the inability of cone transducin to efficiently translocate in light reflects specific physicochemical properties of its individual subunits.
Hypotheses on the Functional Roles of Protein Translocation Photoreceptors adjust their sensitivity over a broad range of ambient light intensities, and protein translocation has been proposed to contribute to this process at the high end of adaptive light intensities. The reduction of transducin content in outer segments of rod in bright light correlates with a reduction in signal amplification in the rhodopsin–transducin–phosphodiesterase cascade, likely due to the reduction in transducin activation rate. This is likely to move the dynamic range over which rods
operate to higher light intensities. Although transducin translocation takes place in light that is saturating for rods, this range adjustment may be adaptive after the light is dimmed or extinguished. For example, such a mechanism could be useful as dusk approaches when vision is switching from being cone-dominant to rod-dominant. The fact that transducin translocation is triggered by light intensities that completely saturate rods makes it plausible to suggest that both phenomena, transducin translocation and response saturation, occur for essentially the same reason, the inability of rods to inactivate vast amounts of transducin beyond a certain light intensity. In this context, transducin translocation can be viewed as an elegant selfregulating mechanism triggered at the point where the rod exhausts other means of avoiding response saturation. Although not yet tested experimentally, arrestin translocation is thought to be adaptive as well. Its increased concentration in the outer segment could reduce the response amplitude and/or accelerate recovery. It could also allow rods and cones to prepare for inactivation of large amounts of photoexcited rhodopsin and its bleach products produced in bright light. Similarly, recoverin translocation from outer segments may increase the amount of rhodopsin kinase available to phosphorylate rhodopsin, thus further reducing light sensitivity. Light-driven protein translocation, particularly in rods, may also play a neuroprotective role. Rod saturation marks the transition from mesopic (mixed rod/cone) to cone-dominated photopic vision. Under these conditions, rods contribute little to vision and transducin translocation may prevent excessive energy consumption by rods by reducing the number of transducin molecules undergoing the cycle of activation/inactivation. This may, in turn, reduce the metabolic stress in the retina commonly believed to contribute to pathological processes. A reduced level of cellular signaling caused by transducin translocation may also reduce the chance of apoptotic death of the rod. At least in rodents, some forms of apoptosis are suggested to be caused by excessive signaling through the phototransduction cascade. The same argument may be applied to arrestin translocation, whose accumulation in the outer segment in bright light is likely to preamplify rhodopsin shutoff on a sunny day. Finally, when vision is dominated by cones, rods may perform more of their housekeeping functions, such as checking the integrity of proteins by the ubiquitin proteasome system located in the inner segments.
What Is the Mode of Protein Translocation: Active Transport or Diffusion? A major currently explored area in this field is whether phototransduction proteins translocate by diffusion or by
Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors
active transport via molecular motors. In principle, the mode of translocation of each protein could be different between the light- and dark-induced directions. Most investigators argue against the involvement of molecular motors. One major argument is that all of these proteins (or at least individual subunits in the case of transducin) are fairly soluble and any of their relocation by molecular motors may be negated by the subsequent diffusion throughout the entire cellular volume. Another argument is that, although protein translocation by motors is rapid, it could be easily saturated by the very large number of translocation protein molecules. On the other hand, intracellular diffusion of soluble proteins in rods is also sufficiently rapid to explain the observed protein translocation rates and, unlike molecular motors, could not be saturated by the amount of protein molecules undergoing lightinduced translocation. Yet, it should be noted that, based on the impediment of translocation by the cytoskeleton disrupting drugs, some believe that motor systems are involved, particularly in protein translocations in the dark-induced directions. However, diffusion alone cannot account for the phenomenon because it does not explain any disequilibrium of protein distributions with the free cytoplasmic volume of the rod or cone. Thus, while diffusion serves as the mode of protein movement, the observed patterns of light-dependent protein redistribution may be explained by light-dependent appearance or disappearance of specific protein-binding sites in individual subcellular compartments. The next two sections illustrate these ideas in regard to transducin and arrestin.
Specific Mechanisms of Protein Translocation Transducin Recent reports suggest that transducin translocation could be explained by the differences in the membrane affinities of its abg heterotrimer compared to the individual a- and bg-subunits. The heterotrimer is strongly membraneassociated due to the combined action of two lipid modifications: a farnesyl group on the g-subunit and an acyl group on the a-subunit. Thus, in the dark-adapted rod, transducin heterotrimer is predicted to be concentrated on the disk membranes of the outer segment. When transducin is activated in light, a-subunits bind GTP and dissociate from the bg-subunits (Figure 2). Both subunits become more soluble since each has only one lipid modification, allowing them to diffuse throughout the cytoplasm. Inevitably, GTP hydrolysis by the a-subunit would result in the restoration of the poorly soluble trimer. When this happens in the outer segment, transducin becomes re-attached to the disk membranes; but when it
579
Translocation GTP
GDP
GTP
GDP
Figure 2 The putative role of transducin subunit dissociation in its translocation. When transducin is activated by photoexcited rhodopsin (R*), its a- and bg-subunits become separated from one another and the membrane affinity of each is less than that of the heterotrimer. In this state, subunits may dissociate from photoreceptor disk membranes and translocate from the outer segment. The efficiency of translocation is dependent on the time during which transducin subunits stay apart before re-formation of the trimer. This time is determined by the rate of the GTP hydrolysis on the a-subunit and is dependent on the absolute amount of activated transducin. Reproduced from Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568.
happens in the inner segment, the trimer may adhere to the membranous structures there, causing its transient accumulation. The central assumption of this hypothesis, that transducin subunits move apart from one another, is supported by the difference in their translocation rates and by transducin translocation being facilitated by transgenic or pharmacological manipulations promoting transducin subunits to remain in the dissociated state. The translocation of transducin bg-subunit is further enhanced by the protein called phosducin. Phosducin reduces membrane association of the bg-subunit, which presumably allows it to more easily diffuse throughout the cytoplasm. Another important mechanistic feature of transducin translocation is its light-intensity threshold. This threshold reflects the fact that the cellular content of transducin significantly exceeds that of RGS9, a protein responsible for rapid inactivation of transducin. Light of the threshold intensity produces activated transducin in the amount exceeding the capacity of RGS9 to inactivate it. Consequently, a fraction of activated transducin stays in the active, GTP-bound form (with a- and bg-subunits dissociated) for a longer time, sufficient for dissociation from the disk membranes and diffusion through the photoreceptor cytoplasm. Accordingly, the knockout of RGS9 allows transducin translocation at a lower light intensity, whereas RGS9 overexpression shifts the threshold to brighter light.
580
Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors
Arrestin It was first suggested that arrestin is equilibrated throughout the rod cytoplasm in the dark and is trapped in the outer segment in light upon binding to photoexcited rhodopsin. However, recent observations argue that the dark-adapted distribution of arrestin does not match the distribution of the free cytoplasm volume, indicating that most arrestin is bound to sites located in the inner segment. One hypothesis is that these sites are formed by microtubules. It was further proposed that arrestin translocation is explained by a simple competition between constitutive, low-affinity microtubule sites in the inner segment and transient, high-affinity sites in the outer segment formed upon rhodopsin photoexcitation. However, quantitative measurements indicated that, at the minimal light intensity sufficient to trigger arrestin translocation, the number of translocated arrestin molecules exceeds the number of photoactivated rhodopsin molecules by 30-fold. Even less rhodopsin activation is required to trigger arrestin translocation in knockout mice lacking RGS9 where transducin activation persists longer than normally. Therefore, triggering arrestin translocation is not dependent on the absolute amount of rhodopsin excited by light, but is rather dependent on arrestin release from the inner segment sites by a yet-tobe-identified signaling mechanism downstream from phototransduction. Proteins’ Return in the Dark The rates of transducin and arrestin return to their darkadapted locations upon switching from light to dark are much slower than their movement in the light-induced direction, with most measurements indicating that it takes at least an hour. This may be slow enough to be within the capacity of molecular motors, and indeed transducin and arrestin return can be blocked by cytoskeleton disrupting drugs. The specificity of these treatments remains unknown and artifacts such as clogging the connecting cilium could not be ruled out. On the other hand, arrestin and transducin return could also be explained by a combination of diffusion, removal of the light-induced binding sites, and restoration of the dark-adapted sites. Current evidence is insufficient to discriminate between the potential roles of molecular motors and diffusion in transducin and arrestin return to their dark-adapted cellular distributions.
See also: Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods.
Further Reading Artemyev, N. O. (2008). Light-dependent compartmentalization of transducin in rod photoreceptors. Molecular Neurobiology 37: 44–51. Brann, M. R. and Cohen, L. V. (1987). Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina. Science 235: 585–587. Broekhuyse, R. M., Tolhuizen, E. F., Janssen, A. P., and Winkens, H. J. (1985). Light induced shift and binding of S-antigen in retinal rods. Current Eye Research 4: 613–618. Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N. Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568. Fain, G. L. (2006). Why photoreceptors die (and why they don’t). BioEssays 28: 344–354. Hanson, S. M., Francis, D. J., Vishnivetskiy, S. A., Klug, C. S., and Gurevich, V. V. (2006). Visual arrestin binding to microtubules involves a distinct conformational change. Journal of Biological Chemistry 281: 9765–9772. Kerov, V., Chen, D. S., Moussaif, M., et al. (2005). Transducin activation state controls its light-dependent translocation in rod photoreceptors. Journal of Biological Chemistry 280: 41069–41076. Nair, K. S., Hanson, S. M., Mendez, A., et al. (2005). Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein–protein interactions. Neuron 46: 555–567. Philp, N. J., Chang, W., and Long, K. (1987). Light-stimulated protein movement in rod photoreceptor cells of the rat retina. FEBS Letters 225: 127–132. Reidel, B., Goldmann, T., Giessl, A., and Wolfrum, U. (2008). The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton. Cell Motility and the Cytoskeleton 65: 785–800. Slepak, V. Z. and Hurley, J. B. (2008). Mechanism of light-induced translocation of arrestin and transducin in photoreceptors: Interaction-restricted diffusion. IUBMB Life 60: 2–9. Sokolov, M., Lyubarsky, A. L., Strissel, K. J., et al. (2002). Massive light-driven translocation of transducin between the two major compartments of rod cells: A novel mechanism of light adaptation. Neuron 34: 95–106. Strissel, K. J., Lishko, P. V., Trieu, L. H., et al. (2005). Recoverin undergoes light-dependent intracellular translocation in rod photoreceptors. Journal of Biological Chemistry 280: 29250–29255. Strissel, K. J., Sokolov, M., Trieu, L. H., and Arshavsky, V. Y. (2006). Arrestin translocation is induced at a critical threshold of visual signaling and is superstoichiometric to bleached rhodopsin. Journal of Neuroscience 26: 1146–1153. Whelan, J. P. and McGinnis, J. F. (1988). Light-dependent subcellular movement of photoreceptor proteins. Journal of Neuroscience Research 20: 263–270.
Glossary Arrestin – A protein which binds to and inactivates photoactivated rhodopsin. Arrestin binding results in termination of transducin activation. Light adaptation – The ability of photoreceptors (and the visual system as a whole) to adapt the speed and sensitivity of light responses to ever-changing conditions of ambient illumination. Phosducin – A protein which interacts with the bg-subunit of transducin and reduces its membrane affinity. Recoverin – A regulatory protein which is thought to regulate the speed at which arrestin can bind to photoactivated rhodopsin. RGS9 – A protein responsible for returning activated transducin in its inactive form. Transducin – A G protein that mediates the visual signal between the photoactivated visual pigment, rhodopsin, and the downstream effector enzyme, cGMP phosphodiesterase. Transducin consists of two functional subunits, a and bg.
inactivated through its phosphorylation by rhodopsin kinase and arrestin binding, which blocks transducin activation. The rate of rhodopsin phosphorylation, and thus its active lifetime, is regulated by the Ca2+-binding protein, recoverin. Transducin (and accordingly PDE) activation is terminated upon the hydrolysis of GTP tightly bound to transducin a-subunit, a process markedly accelerated by the GTPase activating protein RGS9. Importantly, three of the above-mentioned proteins (transducin, arrestin, and recoverin) undergo massive light-driven translocation between the major subcellular compartments of photoreceptors (Figure 1). In rods, transducin moves out of the outer segment and accumulates primarily in the inner segment, arrestin moves in the opposite direction, and recoverin shifts from the outer segment toward the synapse. In cones, in light arrestin moves in the same direction, whereas transducin moves very little, if at all. Recoverin translocation has not yet been analyzed in cones. A similar phenomenon involving the G protein (Gq), arrestin, and the transient receptor potential-like (TRPL) channel takes place in rhabdomeric invertebrate photoreceptors.
Light Dependency of Protein Translocation Introduction Rod and cone photoreceptors are highly polarized cells which transduce information encoded by photons into electrical activity that can be processed by higher-order neurons. At the one end, photoreceptors have specialized ciliary organelles, outer segments, which are enriched in proteins directly involved in light detection and signal transduction. At the opposite end, synapses convey the information gathered by outer segments to downstream neurons. Vision begins when a molecule of rhodopsin in the outer segment becomes excited by light and activates a G protein, transducin. The transducin a-subunit stimulates its effector, cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE), which leads to the reduction in intracellular cGMP and to the electrical response mediated by the closure of the cGMP-gated cationic channels in the plasma membrane. The recovery of the photoresponse requires complete inactivation of all these molecular components. Photoexcited rhodopsin is
Quantitative experiments revealed that the translocations of arrestin and transducin in rods take place in bright light. The outer segments of the rod contain very little arrestin (estimated under 7% of its total cellular content) in the dark and under moderate illumination. In mouse rods, arrestin begins to move to outer segments when the light intensity reaches a critical threshold, exciting over 1000 rhodopsins per rod per second, which is within the upper limit at which mammalian rods can signal variations in light. Transducin translocation is also triggered at a threshold light intensity, although brighter, exciting 5000 rhodopsins per rod per second, an intensity that completely saturates rods. The time required for the completion of protein translocation in saturating light in rods is on the order of tens of minutes. Although no such quantitative measurements are available for cones, available data indicate that cone arrestin translocation also requires fairly bright light. The existence of cone transducin translocation in intact cells remains somewhat
577
578
Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors
Transducin Arrestin Recoverin Dark 80–90% <7% ∼12%
OS
IS
N ST Bright light 10–20% ∼80% <2%
Figure 1 Schematic illustration of transducin, arrestin, and recoverin distribution in dark- and light-adapted rods. The numbers on the left, color-coded to the corresponding translocating proteins, represent the percentage of the proteins found in the outer segments in the dark or following bright light illumination. The subcellular rod compartments are abbreviated on the right: OS – outer segment; IS – inner segment; N – nucleus; ST – synaptic terminal. Reproduced from Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568.
controversial. Most investigators do not see it at any light intensity, whereas one group reports small degree of translocation observed in extremely bright light. The most recent report argues that the inability of cone transducin to efficiently translocate in light reflects specific physicochemical properties of its individual subunits.
Hypotheses on the Functional Roles of Protein Translocation Photoreceptors adjust their sensitivity over a broad range of ambient light intensities, and protein translocation has been proposed to contribute to this process at the high end of adaptive light intensities. The reduction of transducin content in outer segments of rod in bright light correlates with a reduction in signal amplification in the rhodopsin–transducin–phosphodiesterase cascade, likely due to the reduction in transducin activation rate. This is likely to move the dynamic range over which rods
operate to higher light intensities. Although transducin translocation takes place in light that is saturating for rods, this range adjustment may be adaptive after the light is dimmed or extinguished. For example, such a mechanism could be useful as dusk approaches when vision is switching from being cone-dominant to rod-dominant. The fact that transducin translocation is triggered by light intensities that completely saturate rods makes it plausible to suggest that both phenomena, transducin translocation and response saturation, occur for essentially the same reason, the inability of rods to inactivate vast amounts of transducin beyond a certain light intensity. In this context, transducin translocation can be viewed as an elegant selfregulating mechanism triggered at the point where the rod exhausts other means of avoiding response saturation. Although not yet tested experimentally, arrestin translocation is thought to be adaptive as well. Its increased concentration in the outer segment could reduce the response amplitude and/or accelerate recovery. It could also allow rods and cones to prepare for inactivation of large amounts of photoexcited rhodopsin and its bleach products produced in bright light. Similarly, recoverin translocation from outer segments may increase the amount of rhodopsin kinase available to phosphorylate rhodopsin, thus further reducing light sensitivity. Light-driven protein translocation, particularly in rods, may also play a neuroprotective role. Rod saturation marks the transition from mesopic (mixed rod/cone) to cone-dominated photopic vision. Under these conditions, rods contribute little to vision and transducin translocation may prevent excessive energy consumption by rods by reducing the number of transducin molecules undergoing the cycle of activation/inactivation. This may, in turn, reduce the metabolic stress in the retina commonly believed to contribute to pathological processes. A reduced level of cellular signaling caused by transducin translocation may also reduce the chance of apoptotic death of the rod. At least in rodents, some forms of apoptosis are suggested to be caused by excessive signaling through the phototransduction cascade. The same argument may be applied to arrestin translocation, whose accumulation in the outer segment in bright light is likely to preamplify rhodopsin shutoff on a sunny day. Finally, when vision is dominated by cones, rods may perform more of their housekeeping functions, such as checking the integrity of proteins by the ubiquitin proteasome system located in the inner segments.
What Is the Mode of Protein Translocation: Active Transport or Diffusion? A major currently explored area in this field is whether phototransduction proteins translocate by diffusion or by
Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors
active transport via molecular motors. In principle, the mode of translocation of each protein could be different between the light- and dark-induced directions. Most investigators argue against the involvement of molecular motors. One major argument is that all of these proteins (or at least individual subunits in the case of transducin) are fairly soluble and any of their relocation by molecular motors may be negated by the subsequent diffusion throughout the entire cellular volume. Another argument is that, although protein translocation by motors is rapid, it could be easily saturated by the very large number of translocation protein molecules. On the other hand, intracellular diffusion of soluble proteins in rods is also sufficiently rapid to explain the observed protein translocation rates and, unlike molecular motors, could not be saturated by the amount of protein molecules undergoing lightinduced translocation. Yet, it should be noted that, based on the impediment of translocation by the cytoskeleton disrupting drugs, some believe that motor systems are involved, particularly in protein translocations in the dark-induced directions. However, diffusion alone cannot account for the phenomenon because it does not explain any disequilibrium of protein distributions with the free cytoplasmic volume of the rod or cone. Thus, while diffusion serves as the mode of protein movement, the observed patterns of light-dependent protein redistribution may be explained by light-dependent appearance or disappearance of specific protein-binding sites in individual subcellular compartments. The next two sections illustrate these ideas in regard to transducin and arrestin.
Specific Mechanisms of Protein Translocation Transducin Recent reports suggest that transducin translocation could be explained by the differences in the membrane affinities of its abg heterotrimer compared to the individual a- and bg-subunits. The heterotrimer is strongly membraneassociated due to the combined action of two lipid modifications: a farnesyl group on the g-subunit and an acyl group on the a-subunit. Thus, in the dark-adapted rod, transducin heterotrimer is predicted to be concentrated on the disk membranes of the outer segment. When transducin is activated in light, a-subunits bind GTP and dissociate from the bg-subunits (Figure 2). Both subunits become more soluble since each has only one lipid modification, allowing them to diffuse throughout the cytoplasm. Inevitably, GTP hydrolysis by the a-subunit would result in the restoration of the poorly soluble trimer. When this happens in the outer segment, transducin becomes re-attached to the disk membranes; but when it
579
Translocation GTP
GDP
GTP
GDP
Figure 2 The putative role of transducin subunit dissociation in its translocation. When transducin is activated by photoexcited rhodopsin (R*), its a- and bg-subunits become separated from one another and the membrane affinity of each is less than that of the heterotrimer. In this state, subunits may dissociate from photoreceptor disk membranes and translocate from the outer segment. The efficiency of translocation is dependent on the time during which transducin subunits stay apart before re-formation of the trimer. This time is determined by the rate of the GTP hydrolysis on the a-subunit and is dependent on the absolute amount of activated transducin. Reproduced from Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568.
happens in the inner segment, the trimer may adhere to the membranous structures there, causing its transient accumulation. The central assumption of this hypothesis, that transducin subunits move apart from one another, is supported by the difference in their translocation rates and by transducin translocation being facilitated by transgenic or pharmacological manipulations promoting transducin subunits to remain in the dissociated state. The translocation of transducin bg-subunit is further enhanced by the protein called phosducin. Phosducin reduces membrane association of the bg-subunit, which presumably allows it to more easily diffuse throughout the cytoplasm. Another important mechanistic feature of transducin translocation is its light-intensity threshold. This threshold reflects the fact that the cellular content of transducin significantly exceeds that of RGS9, a protein responsible for rapid inactivation of transducin. Light of the threshold intensity produces activated transducin in the amount exceeding the capacity of RGS9 to inactivate it. Consequently, a fraction of activated transducin stays in the active, GTP-bound form (with a- and bg-subunits dissociated) for a longer time, sufficient for dissociation from the disk membranes and diffusion through the photoreceptor cytoplasm. Accordingly, the knockout of RGS9 allows transducin translocation at a lower light intensity, whereas RGS9 overexpression shifts the threshold to brighter light.
580
Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors
Arrestin It was first suggested that arrestin is equilibrated throughout the rod cytoplasm in the dark and is trapped in the outer segment in light upon binding to photoexcited rhodopsin. However, recent observations argue that the dark-adapted distribution of arrestin does not match the distribution of the free cytoplasm volume, indicating that most arrestin is bound to sites located in the inner segment. One hypothesis is that these sites are formed by microtubules. It was further proposed that arrestin translocation is explained by a simple competition between constitutive, low-affinity microtubule sites in the inner segment and transient, high-affinity sites in the outer segment formed upon rhodopsin photoexcitation. However, quantitative measurements indicated that, at the minimal light intensity sufficient to trigger arrestin translocation, the number of translocated arrestin molecules exceeds the number of photoactivated rhodopsin molecules by 30-fold. Even less rhodopsin activation is required to trigger arrestin translocation in knockout mice lacking RGS9 where transducin activation persists longer than normally. Therefore, triggering arrestin translocation is not dependent on the absolute amount of rhodopsin excited by light, but is rather dependent on arrestin release from the inner segment sites by a yet-tobe-identified signaling mechanism downstream from phototransduction. Proteins’ Return in the Dark The rates of transducin and arrestin return to their darkadapted locations upon switching from light to dark are much slower than their movement in the light-induced direction, with most measurements indicating that it takes at least an hour. This may be slow enough to be within the capacity of molecular motors, and indeed transducin and arrestin return can be blocked by cytoskeleton disrupting drugs. The specificity of these treatments remains unknown and artifacts such as clogging the connecting cilium could not be ruled out. On the other hand, arrestin and transducin return could also be explained by a combination of diffusion, removal of the light-induced binding sites, and restoration of the dark-adapted sites. Current evidence is insufficient to discriminate between the potential roles of molecular motors and diffusion in transducin and arrestin return to their dark-adapted cellular distributions.
See also: Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods.
Further Reading Artemyev, N. O. (2008). Light-dependent compartmentalization of transducin in rod photoreceptors. Molecular Neurobiology 37: 44–51. Brann, M. R. and Cohen, L. V. (1987). Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina. Science 235: 585–587. Broekhuyse, R. M., Tolhuizen, E. F., Janssen, A. P., and Winkens, H. J. (1985). Light induced shift and binding of S-antigen in retinal rods. Current Eye Research 4: 613–618. Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N. Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568. Fain, G. L. (2006). Why photoreceptors die (and why they don’t). BioEssays 28: 344–354. Hanson, S. M., Francis, D. J., Vishnivetskiy, S. A., Klug, C. S., and Gurevich, V. V. (2006). Visual arrestin binding to microtubules involves a distinct conformational change. Journal of Biological Chemistry 281: 9765–9772. Kerov, V., Chen, D. S., Moussaif, M., et al. (2005). Transducin activation state controls its light-dependent translocation in rod photoreceptors. Journal of Biological Chemistry 280: 41069–41076. Nair, K. S., Hanson, S. M., Mendez, A., et al. (2005). Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein–protein interactions. Neuron 46: 555–567. Philp, N. J., Chang, W., and Long, K. (1987). Light-stimulated protein movement in rod photoreceptor cells of the rat retina. FEBS Letters 225: 127–132. Reidel, B., Goldmann, T., Giessl, A., and Wolfrum, U. (2008). The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton. Cell Motility and the Cytoskeleton 65: 785–800. Slepak, V. Z. and Hurley, J. B. (2008). Mechanism of light-induced translocation of arrestin and transducin in photoreceptors: Interaction-restricted diffusion. IUBMB Life 60: 2–9. Sokolov, M., Lyubarsky, A. L., Strissel, K. J., et al. (2002). Massive light-driven translocation of transducin between the two major compartments of rod cells: A novel mechanism of light adaptation. Neuron 34: 95–106. Strissel, K. J., Lishko, P. V., Trieu, L. H., et al. (2005). Recoverin undergoes light-dependent intracellular translocation in rod photoreceptors. Journal of Biological Chemistry 280: 29250–29255. Strissel, K. J., Sokolov, M., Trieu, L. H., and Arshavsky, V. Y. (2006). Arrestin translocation is induced at a critical threshold of visual signaling and is superstoichiometric to bleached rhodopsin. Journal of Neuroscience 26: 1146–1153. Whelan, J. P. and McGinnis, J. F. (1988). Light-dependent subcellular movement of photoreceptor proteins. Journal of Neuroscience Research 20: 263–270.