Alvina Bragin

Mouse Cones Require an Arrestin for Normal Inactivation of Phototransduction

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

Co- and Posttranslational Translocation Mechanisms Direct Cystic Fibrosis Transmembrane Conductance Regulator N Terminus Transmembrane Assembly

Transmembrane topology of most eukaryotic polytopic proteins is established cotranslationally at the endoplasmic reticulum membrane through the action of alternating signal and stop transfer sequences. Here we demonstrate that the cystic fibrosis transmembrane conductance regulator (CFTR) achieves its N terminus topology through a variation of this mechanism that involves both co- and posttranslational translocation events. Using a series of defined chimeric and truncated proteins expressed in a reticulocyte lysate system, we have identified two topogenic determinants encoded within the first (TM1) and second (TM2) membrane-spanning segments of CFTR. Each sequence independently (i) directed endoplasmic reticulum targeting, (ii) translocated appropriate flanking residues, and (iii) achieved its proper membrane-spanning orientation. Signal sequence activity of TM1, however, was inefficient due to the presence of two charged residues, Glu92 and Lys95, located within its hydrophobic core. As a result, TM1 was able to direct correct topology for less than half of nascent CFTR chains. In contrast to TM1, TM2 signal sequence activity was both efficient and specific. Even in the absence of a functional TM1 signal sequence, TM2 was able to direct CFTR N terminus topology through a ribosome-dependent posttranslational mechanism. Mutating charged residues Glu92 and Lys95 to alanine improved TM1 signal sequence activity as well as the ability of TM1 to independently direct CFTR N terminus topology. Thus, a single functional signal sequence in either the first or second TM segment was sufficient for directing proper CFTR topology. These results identify two distinct and redundant translocation pathways for CFTR N terminus transmembrane assembly and support a model in which TM2 functions to ensure correct topology of CFTR chains that fail to translocate via TM1. This novel arrangement of topogenic information provides an alternative to conventional cotranslational pathways of polytopic protein biogenesis.

Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator.

Abnormal folding of mutant cystic fibrosis transmembrane conductance regulator (CFTR) and subsequent degradation in the endoplasmic reticulum is the basis for most cases of cystic fibrosis. Structural differences between wild-type (WT) and mutant proteins, however, remain unknown. Here we examine the intracellular trafficking, degradation, and transmembrane topology of two mutant CFTR proteins, G85E and G91R, each of which contains an additional charged residue within the first putative transmembrane helix (TM1). In microinjected Xenopus laevis oocytes, these mutations markedly disrupted CFTR plasma membrane chloride channel activity. G85E and G91R mutants (but not a conservative mutant, G91A) failed to acquire complex N-linked carbohydrates, and were rapidly degraded before reaching the Golgi complex thus exhibiting a trafficking phenotype similar to DeltaF508 CFTR. Topologic analysis revealed that neither G85E nor G91R mutations disrupted CFTR NH2 terminus transmembrane topology. Instead, WT as well as mutant TM1 spanned the membrane in the predicted C-trans (type II) orientation, and residues 85E and 91R were localized within or adjacent to the plane of the lipid bilayer. To understand how these charged residues might provide structural cues for ER degradation, we examined the stability of WT, G85E, and G91R CFTR proteins truncated at codons 188, 393, 589, or 836 (after TM2, TM6, the first nucleotide binding domain, or the R domain, respectively). These results indicated that G85E and G91R mutations affected CFTR folding, not by gross disruption of transmembrane assembly, but rather through insertion of a charged residue within the plane of the bilayer, which in turn influenced higher order tertiary structure.

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which ∼40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion.

The relaxation of fluorescence from diffraction‐limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo‐conversion was measured in solutions of varied viscosity (η), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point‐spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, Dap. The model incorporates two novel features that make it of broad utility. First, the model includes the no‐flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of Dap. Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between Dap and η−1 were obtained for the two proteins: for PAGFP, Daq= 89 ± 2.4 μm2 s−1 (mean ± 95% confidence interval), and for EGFP Daq= 91 ± 1.8 μm2 s−1. In CHO cells, the application of the model yielded Dap= 20 ± 3 μm2 s−1 (PAGFP) and 19 ± 2 μm2 s−1 (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked ‘bound fraction’ explanation of the deviation of the terminal fluorescence recovery from its pre‐bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488‐nm excitation, and the requirement for ∼1/200th the cumulative light energy to produce data of comparable signal/noise.

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis. Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, whereas CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium

A main requisite in the phagocytosis of ingested material is a coordinated series of maturation steps which lead to the degradation of ingested cargo. Photoreceptor outer segment (POS) renewal involves phagocytosis of the distal disk membranes by the retinal pigment epithelium (RPE). Previously, we identified melanoregulin (MREG) as an intracellular cargo-sorting protein required for the degradation of POS disks. Here, we provide evidence that MREG-dependent processing links both autophagic and phagocytic processes in LC3-associated phagocytosis (LAP). Ingested POS phagosomes are associated with endogenous LC3 and MREG. The LC3 association with POSs exhibited properties of LAP; it was independent of rapamycin pretreatment, but dependent on Atg5. Loss of MREG resulted in a decrease in the extent of LC3-POS association. Studies using DQ™-BSA suggest that loss of MREG does not compromise the association and fusion of LC3-positive phagosomes with lysosomes. Furthermore, the mechanism of MREG action is likely through a protein complex that includes LC3, as determined by colocalization and immunoprecipitation in both RPE cells and macrophages. We posit that MREG participates in coordinating the association of phagosomes with LC3 for content degradation with the loss of MREG leading to phagosome accumulation.

Phagocytosis-dependent ketogenesis in retinal pigment epithelium

Daily, the retinal pigment epithelium (RPE) ingests a bolus of lipid and protein in the form of phagocytized photoreceptor outer segments (OS). The RPE, like the liver, expresses enzymes required for fatty acid oxidation and ketogenesis. This suggests that these pathways play a role in the disposal of lipids from ingested OS, as well as providing a mechanism for recycling metabolic intermediates back to the outer retina. In this study, we examined whether OS phagocytosis was linked to ketogenesis. We found increased levels of β-hydroxybutyrate (β-HB) in the apical medium following ingestion of OS by human fetal RPE and ARPE19 cells cultured on Transwell inserts. No increase in ketogenesis was observed following ingestion of oxidized OS or latex beads. Our studies further defined the connection between OS phagocytosis and ketogenesis in wild-type mice and mice with defects in phagosome maturation using a mouse RPE explant model. In explant studies, the levels of β-HB released were temporally correlated with OS phagocytic burst after light onset. In the Mreg−/− mouse where phagosome maturation is delayed, there was a temporal shift in the release of β-HB. An even more pronounced shift in maximal β-HB production was observed in the Abca4−/− RPE, in which loss of the ATP-binding cassette A4 transporter results in defective phagosome processing and accumulation of lipid debris. These studies suggest that FAO and ketogenesis are key to supporting the metabolism of the RPE and preventing the accumulation of lipids that lead to oxidative stress and mitochondrial dysfunction.

Loss of melanoregulin (MREG) enhances cathepsin-D secretion by the retinal pigment epithelium.

Cathepsin-D (Cat-D) is a major proteolytic enzyme in phagocytic cells. In the retinal pigment epithelium (RPE), it is responsible for the daily degradation of photoreceptor outer segments (POSs) to maintain retinal homeostasis. Melanoregulin (MREG)-mediated loss of phagocytic capacity has been linked to diminished intracellular Cat-D activity. Here, we demonstrate that loss of MREG enhances the secretion of intermediate Cat-D (48 kDa), resulting in a net enhancement of extracellular Cat-D activity. These results suggest that MREG is required to maintain Cat-D homeostasis in the RPE and likely plays a protective role in retinal health. In this regard, in the Mreg dsu/dsu mouse, we observe increased basal laminin. Loss of the Mreg dsu allele is not lethal and therefore leads to slow age-dependent changes in the RPE. Thus, we propose that this model will allow us to study potential dysregulatory functions of Cat-D in retinal disease.