Beatrice M. Tam

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

On-line monitoring and control of methanol concentration in shake-flask cultures of Pichia pastoris.

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol.

Dark Rearing Rescues P23H Rhodopsin-Induced Retinal Degeneration in a Transgenic Xenopus laevis Model of Retinitis Pigmentosa: A Chromophore-Dependent Mechanism Characterized by Production of N-Terminally Truncated Mutant Rhodopsin

To elucidate the molecular mechanisms underlying the light-sensitive retinal degeneration caused by the rhodopsin mutation P23H, which causes retinitis pigmentosa (RP) in humans, we expressed Xenopus laevis, bovine, human, and murine forms of P23H rhodopsin in transgenic X. laevis rod photoreceptors. All P23H rhodopsins caused aggressive retinal degeneration associated with low expression levels and retention of P23H rhodopsin in the endoplasmic reticulum (ER), suggesting involvement of protein misfolding and ER stress. However, light sensitivity varied dramatically between these RP models, with complete or partial rescue by dark rearing in the case of bovine and human P23H rhodopsin, and no rescue for X. laevis P23H rhodopsin. Rescue by dark rearing required an intact 11-cis-retinal chromophore binding site within the mutant protein and was associated with truncation of the P23H rhodopsin N terminus. This yielded an abundant nontoxic ∼27 kDa form that escaped the ER and was transported to the rod outer segment. The truncated protein was produced in the greatest quantities in dark-reared retinas expressing bovine P23H rhodopsin and was not observed with X. laevis P23H rhodopsin. These results are consistent with a mechanism involving enhanced protein folding in the presence of 11-cis-retinal chromophore, with ER exit assisted by proteolytic truncation of the N terminus. This study provides a molecular mechanism for light sensitivity observed in other transgenic models of RP and for phenotypic variation among RP patients.

Mislocalized Rhodopsin Does Not Require Activation to Cause Retinal Degeneration and Neurite Outgrowth in Xenopus laevis

Mutations in the C terminus of rhodopsin disrupt a rod outer segment localization signal, causing rhodopsin mislocalization and aggressive forms of retinitis pigmentosa (RP). Studies of cultured photoreceptors suggest that activated mislocalized rhodopsin can cause cell death via inappropriate G-protein-coupled signaling. To determine whether this pathway occurs in vivo, we developed a transgenic Xenopus laevis model of RP based on the class I rhodopsin mutation Q344Ter (Q350Ter in X. laevis). We used a second mutation, K296R, to block the ability of rhodopsin to bind chromophore and activate transducin. We compared the effects of expression of both mutants on X. laevis retinas alone and in combination. K296R did not significantly alter the cellular distribution of rhodopsin and did not induce retinal degeneration. Q350Ter caused rhodopsin mislocalization and induced an RP-like degeneration, including loss of rods and development of sprouts or neurites in some remaining rods, but did not affect the distribution of endogenous rhodopsin. The double mutant K296R/Q350Ter caused a similar degeneration and neurite outgrowth. In addition, we found no protective effects of dark rearing in these animals. Our results demonstrate that the degenerative effects of mislocalized rhodopsin are not mediated by the activated form of rhodopsin and therefore do not proceed via conventional G-protein-coupled signaling.

The Role of Rhodopsin Glycosylation in Protein Folding, Trafficking, and Light-Sensitive Retinal Degeneration

Several mutations in the N terminus of the G-protein-coupled receptor rhodopsin disrupt NXS/T consensus sequences for N-linked glycosylation (located at N2 and N15) and cause sector retinitis pigmentosa in which the inferior retina preferentially degenerates. Here we examined the role of rhodopsin glycosylation in biosynthesis, trafficking, and retinal degeneration (RD) using transgenic Xenopus laevis expressing glycosylation-defective human rhodopsin mutants. Although mutations T4K and T4N caused RD, N2S and T4V did not, demonstrating that glycosylation at N2 was not required for photoreceptor viability. In contrast, similar mutations eliminating glycosylation at N15 (N15S and T17M) caused rod death. Expression of T17M was more toxic than T4K to transgenic photoreceptors, further suggesting that glycosylation at N15 plays a more important physiological role than glycosylation at N2. Together, these results indicate that the structure of the rhodopsin N terminus must be maintained by an appropriate amino acid sequence surrounding N2 and may require a carbohydrate moiety at N15. The mutant rhodopsins were rendered less toxic in their dark inactive states, because RD was abolished or significantly reduced when transgenic tadpoles expressing T4K, T17M, and N2S/N15S were protected from light exposure. Regardless of their effect on rod viability, all of the mutants primarily localized to the outer segment and Golgi and showed little or no endoplasmic reticulum accumulation. Thus, glycosylation was not crucial for rhodopsin biosynthesis or trafficking. Interestingly, expression of similar bovine rhodopsin mutants did not cause rod cell death, possibly attributable to greater stability of bovine rhodopsin.

Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe.

Human serum transferrin N-lobe (hTF/2N) contains three conserved tryptophan residues, Trp(8), Trp(128) and Trp(264), located in three different environments. The present report addresses the different contributions of the three tryptophan residues to the UV-visible, fluorescence and NMR spectra of hTF/2N and the effect of the mutations at each tryptophan residue on the iron-binding properties of the protein. Trp(8) resides in a hydrophobic box containing a cluster of three phenylalanine side chains and is H bonded through the indole N to an adjacent water cluster lying between two beta-sheets containing Trp(8) and Lys(296) respectively. The fluorescence of Trp(8) may be quenched by the benzene rings. The apparent increase in the rate of iron release from the Trp(8)-->Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the second shell network. The partial quenching in the fluorescence of Trp(128) results from the nearby His(119) residue. Difference-fluorescence spectra reveal that any protein containing Trp(128) shows a blue shift upon binding metal ion, and the NMR signal of Trp(128) broadens out and disappears upon the binding of paramagnetic metals to the protein. These data imply that Trp(128) is a major fluorescent and NMR reporter group for metal binding, and possibly for cleft closure in hTF/2N. Trp(264) is located on the surface of the protein and does not connect to any functional residues. This explains the facts that Trp(264) is the major contributor to both the absorbance and fluorescence spectra, has a strong NMR signal and the mutation at Trp(264) has little effect on the iron-binding and release behaviours of the protein.

In Situ Visualization of Protein Interactions in Sensory Neurons: Glutamic Acid-Rich Proteins (GARPs) Play Differential Roles for Photoreceptor Outer Segment Scaffolding

Vertebrate photoreceptors initiate vision via a G-protein-mediated signaling cascade organized within a specialized cilium, the outer segment (OS). The membranous “stacked pancake” architecture of this organelle must be partially renewed daily to maintain cell function and viability; however, neither its static structure nor renewal process is well described in molecular terms. Glutamic acid-rich proteins (GARPs), including the cyclic nucleotide-gated cation channel (CNGB1) and GARP2 (a CNGB1 splice-variant), are proposed to contribute to OS organization in concert with peripherin/rds (P/rds), a retinal tetraspanin. We developed and applied an in situ fluorescence complementation approach that offers an unprecedented glimpse at the formation, trafficking, and localization of GARP-P/rds interactions in transgenic Xenopus laevis rod photoreceptors. Interactions for these (and other) proteins could be readily visualized using confocal microscopy. Nearly all associations, including CNGB1-P/rds interaction, were initiated within inner segments (ISs) before trafficking to OSs. In contrast, GARP2-P/rds interactions were only observed downstream, at or near sites of disk morphogenesis. These results suggest that GARP2-P/rds interaction participates directly in structuring disk stacks but CNGB1-P/rds interaction does not and instead serves mainly to localize plasma membrane ion channels. Altogether, the results lead us to propose that differential interaction of GARPs with P/rds may contribute to the broad phenotypic heterogeneity produced by inherited defects in P/rds. Analogous experiments applied to the synaptic protein RIBEYE suggest that monomers can oligomerize at the level of the IS before ribbon assembly and demonstrate the general applicability of this strategy for in situ analysis of protein interactions in sensory neurons.

The Dependence of Retinal Degeneration Caused by the Rhodopsin P23H Mutation on Light Exposure and Vitamin A Deprivation

PURPOSE To characterize the influence of light and vitamin A on retinal degeneration in an animal model of retinitis pigmentosa caused by the rhodopsin P23H mutation. METHODS Retinal degeneration was examined in transgenic Xenopus laevis expressing P23H rhodopsin, in which retinal degeneration is completely rescued by preventing light exposure. The sensitivity of this retinal degeneration to varying intensities, wavelengths, and durations of light exposure, and to vitamin A deprivation was characterized. RESULTS Green light was the most effective inducer of retinal degeneration in this model. Retinal degeneration was induced by prolonged exposure to green light and was prevented by filters that block short wavelengths. Reducing the duration of light exposure prevented retinal degeneration, even when the light intensity was proportionally increased. Vitamin A deprivation also induced retinal degeneration associated with defects in P23H rhodopsin biosynthesis. Vitamin A deprivation did not cause retinal degeneration in nontransgenic animals. CONCLUSIONS The mechanism of retinal degeneration in this animal model of RP involves the interaction of light with rhodopsin rather than with free chromophore or bleached rhodopsin. These results may explain the clinical benefits of vitamin A for patients with retinitis pigmentosa and may indicate that pharmacological chaperones are a viable approach to RP therapy. Results also suggest strategies for minimizing RD in patients through controlling light exposure duration or wavelengths.

Kinesin family 17 (osmotic avoidance abnormal-3) is dispensable for photoreceptor morphology and function

In Caenorhabditis elegans, homodimeric [kinesin family (KIF) 17, osmotic avoidance abnormal‐3 (OSM‐3)] and heterotrimeric (KIF3) kinesin‐2 motors are required to establish sensory cilia by intraflagellar transport (IFT) where KIF3 and KIF17 cooperate to build the axoneme core and KIF17 builds the distal segments. However, the function of KIF17 in vertebrates is unresolved. We expressed full‐length and motorless KIF17 constructs in mouse rod photoreceptors using adeno‐associated virus in Xenopus laevis rod photoreceptors using a transgene and in ciliated IMCD3 cells. We found that tagged KIF17 localized along the rod outer segment axoneme when expressed in mouse and X. laevis photoreceptors, whereas KIF3A was restricted to the proximal axoneme. Motorless KIF3A and KIF17 mutants caused photoreceptor degeneration, likely through dominant negative effects on IFT. KIF17 mutant lacking the motor domain translocated to nuclei after exposure of a C‐terminal nuclear localization signal. Germ‐line deletion of Kif17 in mouse did not affect photoreceptor function. A rod‐specific Kif3/Kif17 double knockout mouse demonstrated that KIF17 and KIF3 do not act synergistically and did not prevent rhodopsin trafficking to rod outer segments. In summary, the nematode model of KIF3/KIF17 cooperation apparently does not apply to mouse photoreceptors in which the photosensory cilium is built exclusively by KIF3.—Jiang, L., Tam, B. M., Ying, G., Wu, S., Hauswirth, W. W., Frederick, J. M., Moritz, O. L., Baehr, W. Kinesin family 17 (osmotic avoidance abnormal‐3) is dispensable for photoreceptor morphology and function. FASEB J. 29, 4866–4880 (2015). www.fasebj.org

Xenopus laevis red cone opsin and Prph2 promoters allow transgene expression in amphibian cones, or both rods and cones

We have cloned the promoter regions of two Xenopus laevis genes, Prph2 (also called RDS) and red cone opsin (RCO) using a polymerase chain reaction-based gene-walking method. The proteins coded by these genes are expressed exclusively in retinal photoreceptors. Although these promoter sequences are evolutionarily distant from previously described homologues, potentially informative similarities were noted that suggest conserved binding sites of the transcription factors Crx and Rx. The promoters were tested for function in transgenic X. laevis. RCO-driven expression was restricted to cones and pinealocytes, while the Prph2 promoter drove expression of a reporter green fluorescent protein transgene in both rod and cone photoreceptors, as well as low levels of expression in muscle tissue. This is the first description of transgene expression driven by a Prph2 promoter homologue from any species. In combination with the previously reported X. laevis opsin and arrestin promoters, these sequences will facilitate the development and analysis of X. laevis models of inherited retinal degeneration.