Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables 1

Characterization of Mutations Associated with Retinal Disease 2

Benjamin M. Scott,* Steven K. Chen,* Nihar Bhattacharyya,* Abdiwahab Y. Moalim,* Sergey 3

V. Plotnikov,* Elise Heon,† Sergio G. Peisajovich,* Belinda S.W. Chang *,‡,§ 4

5

*Department of Cell and Systems Biology, University of Toronto, ON, Canada 6

†Department of Ophthalmology, Hospital for Sick Children, Toronto, ON, Canada 7

‡Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, Canada 8

§Centre for the Analysis of Genome Evolution and Function, University of Toronto, ON, Canada 9

Author for correspondence: Belinda S.W. Chang belinda.chang@utoronto.ca 10

Keywords: Visual degenerative disease; retinitis pigmentosa; G protein-coupled receptor; disease 11

model; rhodopsin 12

13

Abstract 14

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in 15

eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number 16

of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but 17

can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult 18

for the visual pigment rhodopsin, a GPCR activated by light, for which hundreds of mutations 19

have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa (RP). In 20

this study, we successfully engineered, for the first time, activation by human rhodopsin of the 21

yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel 22

assay for rhodopsin light-dependent activation with studies of subcellular localization, and the 23

Genetics: Early Online, published on December 4, 2018 as 10.1534/genetics.118.301733

Copyright 2018.

upregulation of the unfolded protein response (UPR) in response to misfolded rhodopsin protein. 24

We use these assays to characterize a panel of rhodopsin mutations with known molecular 25

phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some 26

interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes 27

from patients with novel disease-linked mutations. We demonstrate that our engineered yeast 28

strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular 29

mechanisms underlying their pathogenicity. This approach may also be applied to better 30

understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as 31

a tool for investigating molecular mechanisms relevant to human disease. 32

33

Introduction 34

The diversity of biologically relevant signals that G protein-coupled receptors (GPCRs) 35

detect make them critical to how cells sense and respond to their environment. Missense 36

mutations within this large family of cell surface receptors can therefore have serious 37

physiological effects, and many human diseases have been linked with missense mutations in 38

GPCRs (Heng et al. 2013). Over 63,000 missense mutations have been identified in human 39

GPCRs via large scale human genome studies (Pandy-Szekeres et al. 2018), but the majority 40

have not been studied in detail, so the significance to human health remains unclear. Missense 41

mutations can disrupt GPCR function in many ways, ranging from ligand binding, to protein 42

stability, to changes in downstream signaling and interactions with negative regulators (Stoy and 43

Gurevich 2015). Consequently, disease can arise through many different molecular phenotypes 44

and not all mutations are disease causing. 45

To better understand how human missense mutations contribute to disease, the yeast S. 46

cerevisiae has emerged as a powerful tool for characterizing human protein function due to 47

conserved molecular pathways, rapid growth, and ease of genetic manipulation (Laurent et al. 48

2016). These benefits have facilitated yeast models of human disease (Outeiro and Lindquist 49

2003, Perocchi et al. 2008), and studies of pathogenic human mutations (Sun et al. 2016, Yang et 50

al. 2017). Synthetic biology approaches, where human protein function and interactions are 51

quantified by yeast-based gene circuits, have enabled high-throughput experiments at an 52

impressive scale (Starita et al. 2015, Woodsmith et al. 2017, Weile et al. 2017, Sokolina et al. 53

2017). With genetic engineering, human GPCRs can be functionally linked to the yeast mating 54

pathway, and mating-responsive reporter genes have allowed for detailed studies of GPCR 55

activation (Liu et al. 2016). As human GPCRs retain their natural preferences for ligands and G 56

proteins in yeast (Brown et al. 2000), this application of synthetic biology combines the high-57

throughput capabilities of yeast-based studies with the ability to rapidly characterize GPCR 58

function in a cellular context. This has facilitated screens of chemical libraries for novel GPCR 59

ligands (Campbell et al. 1999, Horswill et al. 2007), and screens of mutated GPCRs to 60

characterize specific protein domains or to engineer novel function (Armbruster et al. 2007, 61

Erlenbach et al. 2001, Liu et al. 2015). 62

Despite the power of yeast-based studies of human GPCRs, only a small proportion of 63

GPCRs have been functionally linked to the yeast mating pathway, and all have been ligand-64

activated (Liu et al. 2016). Unlike these GPCRs, rhodopsin exists as a covalent complex between 65

its light-sensitive chromophore 11-cis retinal and the seven-helix transmembrane opsin 66

apoprotein (Smith 2010). Light exposure isomerizes the chromophore, which induces a 67

conformational change in rhodopsin’s transmembrane helices, activating the associated 68

heterotrimeric G protein, transducin, and triggering the visual transduction cascade that 69

eventually results in a signal to the brain that light has been perceived (Smith 2010). Not 70

surprisingly, missense mutations in rhodopsin are often associated with retinal diseases in 71

humans (Athanasiou et al. 2018). Retinitis pigmentosa (RP) is a highly heterogeneous, 72

degenerative retinal disorder that results in vision impairment and in some cases eventually 73

blindness, affecting approximately 1 in 4000 people worldwide (Fahim et al. 2000). The 74

heterogeneous nature of this degenerative disease has contributed to the difficulties in developing 75

effective prognoses and treatment. Missense mutations in rhodopsin have been associated with 76

20-30% of autosomal dominant RP and 1% of autosomal recessive RP cases, making rhodopsin 77

one of the most important RP genes (Fahim et al. 2000). Over 150 rhodopsin missense mutations 78

have been associated with disease (Stenson et al. 2014), and an additional >200 uncharacterized 79

mutations in rhodopsin exons are listed in the Genome Aggregation Database (Lek et al. 2016). 80

Without a tool to rapidly assess rhodopsin function, this increasing availability of genetic 81

information has yet to lead to a better understanding of pathogenicity of mutations associated 82

with RP and other inherited visual diseases. Determining the impact of missense mutations on 83

the ability of rhodopsin to respond to light currently relies on in vitro biochemical assays that are 84

labor intensive, requiring mammalian cell culture to produce one mutant protein at a time, 85

followed by immunofluorescence or immunoaffinity purification (Reeves et al. 1996, Sung et al. 86

1991). These technical challenges are compounded by the diverse molecular phenotypes of 87

rhodopsin mutations, ranging from constitutively active, to improper subcellular localization, to 88

disrupted post-translational modifications (Athanasiou et al. 2018). To efficiently characterize 89

the wide variety of patient-derived mutations, a rapid method that reliably recapitulates light-90

dependent signaling of rhodopsin is needed. 91

Here, we use synthetic biology approaches to engineer rhodopsin coupling to the yeast 92

mating pathway, demonstrating for the first time successful rhodopsin light-activated signal 93

transduction that can be rapidly quantified using a fluorescent reporter gene of mating pathway 94

activation. We compared our novel yeast-based assay to more established mammalian cell-based 95

methods, using a panel of previously-studied rhodopsin mutations. We found that measurements 96

of rhodopsin activation in yeast resemble in vitro results using rhodopsin purified from 97

mammalian cells, with some exceptions. We also found that a yeast-based reporter of the 98

unfolded protein response (UPR) produced results consistent with previous studies in 99

mammalian cells quantifying the effects of rhodopsin pathogenic mutants on cellular stress. 100

Finally, we used our combined approaches in yeast to investigate recently identified rhodopsin 101

mutations in patients with retinal disease, and were able to propose pathogenic classifications 102

that are supported by mammalian cell and clinical data. 103

Materials and Methods 104

Yeast Strain Engineering. The parent yeast strain for all strain engineering was CB008, 105

genotype W303 MATa, far1Δ, his3, trp1, leu2, ura3 (Table S1 contains all strain genotypes). All 106

gene knock-outs and knock-ins were conducted using homologous recombination of selectable 107

markers. pFUS1-mCherry was integrated at the MFA2 locus using plasmid pJW609 containing 108

the KanR marker. pFUS1 was defined as the 1636 bp immediately upstream of the Fus1 start 109

codon, the mCherry sequence used is from (Keppler-Ross et al. 2008), and ~1 kb homology 110

regions were used. STE2 and SST2 were targeted for deletion using TRP1 and HygB selectable 111

markers respectively, each with 180 bp of flanking homology regions identical to the sequences 112

flanking the ORF. The 5 C-terminal amino acids of Gpa1 (KIGII) was replaced with a Gpa1-Gαt 113

(transducin) chimera, containing the C-terminal amino acids from mammalian Gαt (DCGLF), 114

using plasmid pBS600 designed for this study (Figure S1), containing selectable marker LEU2, 115

and a sequence homologous to the 800 bp 3’ to natural Gpa1 gene. The C. albicans Adh 116

terminator was used downstream of both the pFUS1-mCherry and Gpa1-Gαt gene cassettes. 117

Strains were confirmed by PCR and flow cytometry. 118

Rhodopsin Mutation Selection and Patient Phenotyping. Rhodopsin mutations were selected 119

from across phenotypic classes, as reported in a recent comprehensive review (Athanasiou et al. 120

2018), and with at least one of the following assays previously published: transducin activation, 121

localization in mammalian cells, or spectroscopy indicating chromophore binding. The patient 122

cases were selected from an internal database and the phenotype information was collected 123

retrospectively. Other than basic demographic and genetic information, we collected information 124

about visual acuity (VA), color vision, Goldmann visual fields (GVF), electroretinography 125

(ERG) and imaging. Imaging included fundus photography (VisucamNM/FA - Carl Zeiss Meditec, 126

Dublin, California, USA and Optos), Optical coherence tomography (OCT, Cirrus from Carl 127

Zeiss Meditec, Dublin, California, USA). Genetic testing was done using gene panels based 128

sequencing by CLIA approved laboratories. This study was approved by the Human Research 129

Ethics Board of the hospital for Sick Children and met the Tenets of the declaration of Helsinki. 130

Cloning and Mutagenesis. The human rhodopsin sequence (RefSeq NP_000530.1) was 131

amplified using Pfu polymerase (Thermo) from plasmid pJET HuRh (Morrow et al. 2017), using 132

primers to insert flanking AarI restriction sites. Following AarI digestion, the rhodopsin 133

sequence was ligated to the yeast centromere plasmids pRS316 and pRS313 which each 134

contained the TDH3 promoter (pTDH3; alternatively called pGPD). For mammalian expression, 135

rhodopsin mutations were introduced into the wild-type bovine rhodopsin sequence in the p1D4 136

vector for immunoaffinity purification or the pGFP vector for SK-N-SH immunofluorescence 137

microscopy (Figure S2). Mutagenesis was conducted via PCR following the QuikChange site-138

directed mutagenesis protocol (Agilent) and using PfuUltra II Fusion HS DNA Polymerase 139

(Agilent). Mutagenesis primers were designed with 20 or 21 nucleotides identical to human or 140

bovine rhodopsin, flanking the mutant nucleotide(s). 141

Yeast Plasmid Transformation. Yeast strain BS017 or yJW1200 were transformed with 142

individual plasmids by standard lithium-acetate method and plated on selective media (SD-URA 143

or SD-HIS respectively). 144

Rhodopsin Purification. Culturing and transfection of HEK293T cells was performed as 145

previously described (Bhattacharyya et al. 2017). Briefly, p1D4 vector containing a rhodopsin 146

gene was transfected with Lipofectamine 2000 (Invitrogen) and cells were harvested after 48 147

hours. Rhodopsin was regenerated with 11-cis retinal for 2 hours before solubilization in 1% 148

dodecylmaltoside (Anatrace) and immunoaffinity purified with 1D4 monoclonal antibody 149

(Molday and MacKenzie 1983). The ultraviolet-visible absorption spectra of the rhodopsin 150

proteins were recorded using a Cary 4000 double beam spectrophotometer (Agilent). Pigments 151

were light bleached with a Fiber-Lite MI-150 high intensity illuminator (Dolan-Jenner) for 60 152

seconds at 20ºC. Dark-light difference spectra were calculated by subtracting the light-bleached 153

absorbance spectra from the dark spectra. 154

Yeast Light Activation Assay. Yeast strain BS017 transformed with a human rhodopsin mutant 155

gene in the pRS316 pTDH3 vector was incubated overnight in SD-URA media in a 30oC shaker. 156

The same strain transformed with a plasmid not containing the rhodopsin sequence (Vector) was 157

used as a negative control. Cells were diluted to OD600 0.05 in fresh media containing 5 μM 9-cis 158

retinal (Sigma-Aldrich) and incubated for 2 hours protected from light in a 30°C shaker. 9-cis 159

retinal is a common alternative to the natural chromophore 11-cis retinal, and gives comparable 160

in vitro results (Opefi et al. 2013). LightSafe 50 mL centrifuge tubes (Sigma-Aldrich) were used 161

for 5 mL cultures, and 96-well deep well blocks (VWR) wrapped in aluminum foil for 600 μL 162

cultures. Indicated cultures were then exposed to light using a Fiber-Lite MI-150 high intensity 163

illuminator (Dolan-Jenner) set to full intensity for 15 minutes at room temperature. After 100 μL 164

samples were taken for analysis, an additional 5 μM 9-cis retinal was added to indicated culture, 165

both light exposed and cultures kept in the dark, and placed back in the 30oC shaker. Light 166

exposure followed by retinal addition was conducted every hour for a total of six hours following 167

the first light exposure. Cells were then treated with the protein synthesis inhibitor 168

cycloheximide, to a final concentration of 10 μg/mL. The mCherry signal of at least 6,000 cells 169

was measured for each sample with a Miltenyi Biotec MACSQuant VYB. The mean mCherry 170

fluorescence was determined using FlowJo. After subtracting the mCherry fluorescence signal of 171

the Vector control, fluorescence values were normalized to the wild-type rhodopsin control used 172

in the same experiment, to allow comparisons between experiments performed on different days. 173

Immunofluorescence Microscopy. SK-N-SH neuroblastoma (ATCC HTB-11) cells were 174

grown and cultured in full media (DMEM (Life technologies), 10% FBS (Invitrogen), and 175

Penicillin-Streptomycin (Invitrogen)) at 37º in 5% CO2 and seeded into 24-well plates with 176

coverslips (Sarstedt) while under 5 passages. Once cells reached approximately 75% confluence, 177

they were transfected with 645 ng of pGFP plasmid containing the appropriate bovine rhodopsin 178

gene, using Lipofectamine 2000 (Invitrogen) protocols. After 24 hours, half the wells were 179

incubated with WGA in HBSS for 10 minutes at 37º to label the plasma membrane. All cells 180

were then rinsed with PBS and fixed with 2% paraformaldehyde in PBS. To label cells with the 181

endoplasmic reticulum marker antibody anti-calreticulin (1:400, Abcam), cells were washed and 182

permeabilized in PBS containing 1% bovine serum albumin (Sigma) and 0.1% saponin (PBS-183

BS). Anti-calreticulin was diluted in PBS-BS and incubated for 1hr at room temp. After washing 184

with PBS-BS, secondary antibody (Cy3-conjugated goat anti-rabbit IgGt, 1:200, Jackson 185

Immunoresearch) was diluted in PBS-BS and added to the wells for 1 hour. Nuclei were stained 186

with Hoechst (1:1000 in PBS, Hoechst type 33258 Invitrogen) for 10 minutes. Cells were 187

mounted with ProlLong Gold Antifade (Thermofisher), a coverslip as applied, and allowed to 188

cure for 24 hours in the dark prior to imaging on Leica TCSSP8 confocal microscope. ImageJ 189

was used to construct Z-stacks, maximum projection images and scale bars. 190

Yeast Microscopy. Yeast strain BS017 expressing human rhodopsin C-terminally tagged with 191

GFP were grown to log phase in selective media, then plated on glass-bottomed dishes (Greiner 192

Bio-one) treated with 1 mg/mL concanavalin A (Sigma-Aldrich). The centromere plasmid 193

pRS316 pTDH3 was used, and the GFP sequence used is from (Moser et al. 2013). Restriction 194

enzyme sites introduced a short amino acid linker (GGERGS) between the final rhodopsin 195

residue and first GFP residue. Images of the cells adhered to the dishes were acquired using a 196

Leica TCS SP8 confocal microscope with a 100X 1.4NA objective and a hybrid detector (Leica). 197

GFP fluorescence was analyzed using a custom Fiji-MATLAB pipeline (File S1, File S2), which 198

was similar to analyses performed to quantify fluorescence peaks in mammalian cells (Cheng et 199

al. 1999). On Fiji (Schindelin et al. 2012), yeast cell maps were generated by first removing 200

high-frequency noise using ROF Denoise (theta=25) and preparing for thresholding using 201

Enhance Contrast (0.3 saturation with Normalization). Manual thresholding, filling holes, and 202

finally selecting of cells using the watershed plugin generated final cell maps. Cell membrane 203

maps were defined in MATLAB as the 7 outer pixels (~0.7 µm) of each cell map. Fluorescent 204

patches at the cell membrane were defined in MATLAB as pixels with fluorescence intensity at 205

least 2.0 standard deviations above the edge mean. Cells with patchy membrane expression of 206

rhodopsin were defined in MATLAB as cells with at least 10% of their cell membrane 207

containing membrane patches. Bootstrapped standard errors were generated in MATLAB by 208

using the Statistics and Machine Learning Toolbox functions (Mathworks). 209

UPR Activation Assay. Yeast strain yJW1200 was generously provided by the Weissman lab, 210

University of California, San Francisco. This strain contains a 4x repeat of the unfolded protein 211

response element upstream of GFP, and the constitutive TEF2 promoter upstream of RFP 212

(Jonikas et al. 2009). yJW1200 transformed with a human rhodopsin mutant gene in the pRS313 213

pTDH3 vector was incubated overnight in 3 mL SD-HIS media in a 30oC shaker. The same 214

strain transformed with a plasmid not containing the rhodopsin sequence (Vector) was used as a 215

negative control. Cells were diluted to OD600 0.2 in 600 μL fresh media and incubated for 4 216

hours in a 30°C shaker. Cells were then treated with the protein synthesis inhibitor 217

cycloheximide, to a final concentration of 10 μg/mL. The GFP and RFP signal of at least 10,000 218

cells was measured for each sample with a Miltenyi Biotec MACSQuant VYB. The mean GFP 219

and RFP fluorescence was determined using FlowJo. Fluorescence values were normalized to the 220

wild-type rhodopsin control used in the same experiment, to allow comparisons between 221

experiments performed on different days. 222

Statistical Analyses and Graphs. Statistical analyses were performed using Prism (GraphPad), 223

using one-way ANOVA for UPR comparisons to wild-type rhodopsin, and for yeast light and 224

dark activation comparisons to wild-type rhodopsin. Student’s t-test was used to compare the 225

results of one mutant or condition to one other mutant or condition. Graphs were generated using 226

Prism or Excel (Microsoft). 227

Data Availability Statement. Strains and plasmids are available upon request. All 228

supplementary figures, tables, and files have been uploaded to figshare. 229

230

Results 231

Human Rhodopsin Functionally Couples to the Yeast Mating Pathway and Signal 232

Transduction is Dependent on Light 233

We have engineered, for the first time, a vertebrate rhodopsin that can successfully 234

couple to the yeast mating pathway. This was accomplished by first knocking out the genes 235

encoding Far1, to prevent cell cycle arrest, and the GTPase-activating protein Sst2, a negative 236

regulator of the mating pathway (Brown et al. 2000). The endogenous mating pathway GPCR 237

Ste2 was also knocked out, to prevent unnecessary interactions with downstream mating 238

pathway proteins. Next, a chimeric G alpha protein and the fluorescent protein mCherry under 239

the regulation of the mating-responsive FUS1 promoter (pFUS1) were inserted into the yeast 240

genome (Figure 1, Table S1). As rhodopsin is known to only interact with G alpha proteins 241

containing the same five amino acid C-terminal sequence (transducin in rod photoreceptors and 242

Gαi1 in engineered systems (Sun et al. 2015, Maeda et al. 2014)), only one Gpa1 chimera (Gpa1-243

Gαt) was required for our study. To ensure functional coupling between Gpa1-Gαt and human 244

rhodopsin, we first expressed a known constitutively active rhodopsin mutant, E113Q M257Y 245

(Han et al. 1998). Consistent and high levels of mCherry fluorescence were observed, regardless 246

of the presence of retinal chromophore (Figure S3), indicative of productive rhodopsin 247

expression and the ability to activate the yeast mating pathway. 248

Wild-type human rhodopsin was then expressed using the same strain, and when 249

incubated with retinal, induced the expression of mCherry only in response to light (Figure 2). 5 250

μM 9-cis retinal in culture media was sufficient to elicit this light-dependent response, a 251

concentration also used for the heterologous expression of rhodopsin using mammalian cells 252

(Opefi et al. 2013). The 5- to 10-fold increase in mCherry fluorescence in response to GPCR 253

activation was comparable to previous reported activation of the natural mating pathway GPCR 254

Ste2 (Ishii et al. 2008, Kompella et al. 2017). Increasing the retinal concentration did not 255

increase activation of the mating pathway, indicating rhodopsin molecules were saturated with 256

chromophore (Figure S4). However, mating pathway response was enhanced when retinal was 257

added after each hourly light exposure, to account for the lack of retinal recycling enzymes in 258

yeast. 259

260

Magnitude of Light-Activated Signal Transduction in Yeast Comparable to Assays of 261

Rhodopsin Expressed in Mammalian Cells 262

After establishing light-dependent activation of rhodopsin in yeast, we next sought to 263

compare these new yeast-based methods to traditional in vitro methods utilizing protein purified 264

from mammalian cells. Previously characterized rhodopsin mutations P23H, M39R, and G51A 265

were specifically chosen to establish a gradient of phenotypic severity. P23H is the most 266

common RP-associated rhodopsin mutation in North America (Dryja et al. 1990, Mendes et al. 267

2005), and has been characterized in a number of cell and animal models. P23H rhodopsin 268

consistently displays poor stability (Krebs et al. 2010, Chen et al. 2014), aggregation in the ER 269

(Chiang et al. 2012b), and disrupted transducin activation (Opefi et al. 2013, Chen et al. 2014), 270

leading to severe retinal degeneration (Athanasiou et al. 2017, LaVail et al. 2018, Cideciyan et 271

al. 1998). The less severe M39R mutation, which is also associated with RP, has been studied 272

using both bovine and human rhodopsin genes displaying a more severe cytosolic aggregation 273

phenotype in the human gene background (Ramon et al. 2014, Davies et al. 2012). As M39R 274

rhodopsin is more productively expressed by mammalian cells than P23H rhodopsin, and a 275

proportion remains able to form a light-responsive complex with retinal, it was selected as an 276

intermediate RP-associated rhodopsin mutation (Davies et al. 2012, Ramon et al. 2014). G51A is 277

the most common nonsynonymous rhodopsin mutation in humans (Lek et al. 2016), displays a 278

less severe phenotype in vitro and in patients (Bosch et al. 2003, Cideciyan et al. 1998), and may 279

be an asymptomatic variant (Athanasiou et al. 2018). These three rhodopsin mutants were each 280

expressed using the mCherry reporter yeast strain. Following exposure to light, the relative 281

mCherry fluorescence was observed as follows: P23H < M39R < G51A = WT (Figure 3A). 282

Next, we compared rhodopsin activation in yeast to traditional in vitro methods for 283

determining rhodopsin function in response to light. The same three mutants were purified 284

following heterologous expression in mammalian cells, then regenerated with retinal. By 285

recording the absorption spectra before and after exposure to light, difference spectra showing 286

the response of rhodopsin to light could be measured. This method has been used extensively to 287

characterize missense mutations suspected to cause inherited retinal disease, as a measure of the 288

ability of rhodopsin to properly fold and respond to light (Sung et al. 1991, Opefi et al. 2013). 289

The relative response to light displayed a similar range of function as the yeast-expressed 290

mutants (Figure 3B). This suggested that not only is the function of yeast-expressed rhodopsin 291

similarly impacted by pathogenic mutations, but the relative activation of the mating pathway in 292

yeast is comparable to the severity of the mutant as measured in vitro using rhodopsin purified 293

from mammalian cells. 294

295

Pathogenic Mutations Known to Disrupt Rhodopsin Stability or G Protein Coupling 296

Prevent Light-Activated Signal Transduction in Yeast 297

After establishing yeast as a platform for quantifying light-dependent rhodopsin 298

activation, we investigated a larger panel of rhodopsin mutations to understand how the wide 299

range of known functional phenotypes translates to a response in yeast. Efforts have been made 300

to classify mutations based on these phenotypes, which range from completely inactive to 301

constitutively active (Figure S5, Table S2), as discussed in detail in a recent review (Athanasiou 302

et al. 2018). However, as this new yeast assay examines rhodopsin signaling in an engineered 303

cellular system, it was unknown how results would compare to traditional in vitro methods using 304

purified mammalian cell-expressed rhodopsin. We first focused on pathogenic mutations known 305

to disrupt rhodopsin folding and stability, as we hypothesized these loss-of-function mutations 306

would be easier to distinguish from wild-type. These mutations were placed into three groups 307

based on previous characterization: if the mutation intrinsically disrupts rhodopsin stability; if 308

the mutation indirectly affects stability by disrupting a post-translational modification 309

(glycosylation) motif; or if the mutation disrupts G protein coupling and leads to constitutive 310

endocytosis (Class 3). 311

When expressed in yeast and exposed to light, rhodopsin function was significantly 312

disrupted by each of the mutations known to result in misfolding or instability, with the 313

exception of D190N (Figure 4A). D190N had previously been shown to be a less severe RP-314

linked mutation (Tsui et al. 2008, Liu et al. 2013, Fishman et al. 1992), and ERG data from 315

patients matches our observation that this missense mutation does not completely disrupt 316

rhodopsin function (Sancho-Pelluz et al. 2012). The G89D and L125R mutations also had a 317

reduced but measurable response to light when expressed in yeast, which fits with previous 318

trends observed (Kaushal and Khorana 1994, Bosch et al. 2003). Interestingly, L125R lead to 319

signaling in the dark as well, equivalent to the mutant’s light-dependent activation, which has not 320

previously been reported. 321

Mutations in the N-terminal cap of rhodopsin (V20G, P23H, and Q28H) have been 322

functionally characterized in detail, and are known to poorly activate transducin in response to 323

light (Opefi et al. 2013). Similarly, mutations M39R, N55K, G106W, C110Y, G114D and 324

P171L have each been shown to disrupt or prevent productive formation of a opsin-retinal 325

complex (Davies et al. 2012, Ramon et al. 2014, Sung et al. 1991, Sung et al. 1993, Hwa et al. 326

1999, Andres et al. 2003), which matched our observation that light-activated signal transduction 327

was significantly impaired in yeast. 328

Mutations T4K, N15S, and T17M prevent glycosylation at residues N2 and N15, 329

resulting in a severe reduction in rhodopsin stability (Kaushal et al. 1994, Opefi et al. 2013). The 330

NXS/T glycosylation consensus sequence is recognized across eukaryotes (Lam et al. 2013), and 331

a previous study indicated that yeast-expressed bovine rhodopsin was glycosylated 332

(Mollaaghababa et al. 1996). The reduced stability of unglycosylated rhodopsin is known to 333

prevent productive transducin activation in vitro (Opefi et al. 2013), and comparable reductions 334

in signaling were observed in our yeast assay. A general trend of T4K (40% wild-type activity) 335

being less severe than N15S (4%) and T17M (15%) was observed, similar to previous studies 336

which indicated glycosylation is more important on N15 than it is on N2 (Kaushal et al. 1994, 337

Tam and Moritz 2009, Opefi et al. 2013). 338

Of the rhodopsin mutations we studied with impaired signaling, R135G is unique as it 339

does not cause misfolding and it does not prevent the formation of a stable complex with retinal, 340

when using rhodopsin purified from mammalian cells (Sung et al. 1993). R135G mutates the 341

highly conserved E/DRY motif, where R135 is the arginine residue in this motif and is crucial 342

for G protein coupling (Acharya and Karnik 1996, Rovati et al. 2007). In addition, when 343

heterologously expressed in mammalian cells, R135 mutations cause rhodopsin to be 344

hyperphosphorylated, leading to aggregation with visual arrestin and constitutively undergoing 345

endocytosis (Chuang et al. 2004). With two unique molecular mechanisms contributing to 346

pathogenicity, R135 mutants have been placed in their own “Class 3” category (Athanasiou et al. 347

2018, Chuang et al. 2004). The observed absence of signaling in yeast was in line with the 348

reported in vitro transducin activation defect using mammalian cell-derived R135G rhodopsin 349

(Min et al. 1993, Acharya and Karnik 1996). However, as our light-activated signal transduction 350

assay could not distinguish between disrupted G protein coupling versus aberrant endocytosis, 351

this assay alone could not determine the molecular mechanism behind the lack of R135G 352

signaling in yeast. 353

354

Pathogenic Mutations that Enhance or Do Not Disrupt Transducin Activation Respond 355

Similarly in Yeast 356

Based on the wild-type-like activity of G51A we observed in yeast, and the reduced or 357

inactive response of misfolded and unstable mutants, we hypothesized that rhodopsin mutations 358

which maintain or increase light-dependent activation in vitro may behave similarly in yeast. 359

Constitutively active rhodopsin mutants are also associated with disease, causing congenital 360

stationary night blindness (CSNB) and RP (Park 2014). We specifically selected a panel of 361

rhodopsin mutations to characterize in yeast where activity was known to vary greatly, from 362

asymptomatic, to constitutively active, to increased signaling in the dark. 363

Across this diversity of function, yeast-expressed rhodopsin again behaved comparably to 364

rhodopsin purified from mammalian cells, with both wild-type-like signals and increased 365

signaling observed depending on the mutation (Figure 4B). We grouped mutations known to 366

increase downstream transducin activation in vitro, although this increased activity can occur in 367

the light, dark, or in both states (Park 2014). The M44T mutation showed a significantly higher 368

response than wild-type, at over 1.6-fold wild-type, which matches in vitro transducin activation 369

data for M44T (Andres et al. 2003). T94I trended higher than wild-type, and is also believed to 370

cause CSNB due to constitutive activation (119% WT signaling in vitro) (Gross et al. 2003), but 371

the increase we observed versus wild-type (115% WT signaling in yeast) was not statistically 372

significant. V137M has been reported to activate transducin 1.25-fold greater than wild-type 373

(Andres et al. 2003), but we did not observe an increase in rhodopsin activity. The V137M 374

mutation is known to have highly variable clinical phenotypes (Ayuso et al. 1996), and it has 375

been suggested to be an asymptomatic variant (Rakoczy et al. 2011). 376

Of the rhodopsin mutations with known increased activity that we studied, S186W is 377

unique as it is believed to cause autosomal dominant RP due to increased signaling in the dark 378

(Liu et al. 2013). This is a result of reduced thermal stability of the inactive dark state, where 379

spontaneous thermal isomerization of the chromophore leads to signaling in the dark (Liu et al. 380

2013), a phenomenon called “dark noise” (Luo et al. 2011). The increased signaling that we 381

observed in the dark for S186W, equivalent to 30% of activated wild-type rhodopsin, fit with this 382

proposed mechanism of RP pathogenesis. E150K was also found to have significantly elevated 383

signaling in the dark, when expressed in yeast. This data is in line with a mouse model of 384

E150K, which was found to have elevated photoreceptor signaling in the dark, also believed to 385

be due to reduced thermal stability of the dark state (Zhang et al. 2013). The E150K mutation is 386

associated with autosomal recessive RP (arRP) and was previously shown to have a 1.3-fold 387

increased activation of transducin after light exposure (Zhang et al. 2013). An identical value 388

was observed using our yeast-based assay but was not statistically significant. 389

To determine if dark state signaling in yeast was retinal-dependent, we investigated 390

signaling without the addition of retinal for selected mutants (Figure S6). Elevated dark state 391

signaling appeared to be retinal-dependent for only M44T, L125R, E150K, and S186W. This fits 392

with the proposed mechanism of thermal isomerization of retinal contributing to dark noise for 393

the E150K and S186 mutants (Zhang et al. 2013, Liu et al. 2013), while providing new insight 394

on the M44T and L125R mutants. 395

Some rhodopsin mutations may cause disease by preventing the formation of rhodopsin 396

homodimers (Ploier et al. 2016), but their pathogenicity is debated due to their relatively high 397

frequency in sequenced human genomes (>1:80,000) (Athanasiou et al. 2018). The F45L and 398

V209M mutations were found to activate the mating pathway at wild-type levels when exposed 399

to light, matching published in vitro transducin activation assays (Ploier et al. 2016). F220L was 400

found to have a 1.5-fold greater response, which was unexpectedly higher than the wild-type-like 401

value reported for the F220C mutation (Ploier et al. 2016). Similarly, although V104I is 402

considered asymptomatic, light activation of the mating pathway was approximately 1.3-fold 403

greater than wild-type. This mutation does not segregate with RP in genetic studies (Macke et al. 404

1993), but transducin activation assays have not previously been performed, so it is unclear how 405

our results in yeast relate to a potential human phenotype. 406

Mutations in the C-terminus of rhodopsin disrupt trafficking to the rod outer segment 407

(ROS), but do not affect trafficking in other mammalian cell types and do not affect transducin 408

activation in vitro (Sung et al. 1994), therefore we did not expect their function to differ from 409

wild-type rhodopsin in yeast. Interestingly, V345M significantly affected light-activated 410

signaling in yeast, despite the mutation occurring in the C-terminus which is not believed to be 411

required for G protein activation. A study of transducin activation using V345M rhodopsin 412

purified from mammalian cells has not been performed to compare to our yeast results. 413

There were two examples where light-activated signaling in yeast differed from reported 414

in vitro results using rhodopsin purified from mammalian cells. The A292E mutation is 415

associated with CSNB, and has constitutive activation in vitro in the absence of retinal, but 416

reduced light-dependent transducin activation when retinal is supplied (Gross et al. 2003). In our 417

yeast signaling assay, a 2.1-fold increase in light-activated signaling versus wild-type was 418

observed, greater than any other mutation we studied. A292E signaling in the dark with retinal 419

added was not significantly increased, and was equivalent to not adding retinal. G90D, another 420

constitutively active mutant associated with CSNB (Rao et al. 1994), activated the mating 421

pathway at only 44% of wild-type’s response. This result was similar to one transducin 422

activation study using G90D (59% of wild-type) (Zvyaga et al. 1996), while others have shown 423

wild-type-like responses to light but constitutive activation in the absence of retinal, similar to 424

A292E (Gross et al. 2003, Rao et al. 1994). 425

426

Light-Activated Signal Transduction in Yeast Correlates with Published Assays of 427

Rhodopsin Function 428

Of the 33 mutations and controls studied, 23 had previously reported measurements of 429

light-dependent activation of transducin in vitro, or measurements of photoreceptor activity by 430

ERG (Table S2). When plotted together, our yeast-based measurements closely matched this 431

available data on rhodopsin signaling, approaching a 1:1 ratio (Figure S7). This held true for a 432

diverse array of phenotypes, ranging from pathogenic due to inactivation, or pathogenic due to 433

constitutive activation, to asymptomatic. A292E was found to be an outlier from this trend. The 434

rate of light-dependent transducin activation has been reported at ~80% of wild-type, although 435

this mutant has constitutive activation in the absence of retinal in vitro (Gross et al. 2003). As 436

signaling in yeast occurs in a cellular context, versus traditional in vitro assays that use purified 437

protein, the increase in light-activated signaling we observed for A292E may have been a 438

combination of signaling both with and without retinal bound, or may represent a unique 439

signaling state in yeast. Overall, however, the general trend strongly supports the use of yeast to 440

quantify rhodopsin mediated G protein signaling in response to light, as the yeast-based assay 441

was comparable to more laborious assays for characterizing patient derived mutations. 442

443

Subcellular Localization of Rhodopsin is Comparable Between Yeast and Mammalian 444

Cells 445

As the majority of disease linked rhodopsin mutations cause the receptor to misfold 446

(Athanasiou et al. 2018), comparing the subcellular localization of rhodopsin using mammalian 447

cells is a common technique to study protein trafficking and ER retention, and is predictive of 448

pathogenicity (Sung et al. 1991, Behnen et al. 2018). To establish phenotypes in mammalian 449

cells to compare to, we again used the rhodopsin mutations P23H, M39R, and G51A to create a 450

gradient of phenotypic severity. Rhodopsin mutants were expressed in SK-N-SH neuroblastoma 451

cells with a C-terminal GFP tag that has previously been shown to not affect rhodopsin stability 452

in vitro or in vivo (Moritz et al. 2001) 453

The P23H mutation has been characterized in a number of cell models, consistently 454

showing poor plasma membrane expression regardless of cell type (Sung et al. 1991, Chiang et 455

al. 2012b, Chiang et al. 2015). When expressed in SK-N-SH neuroblastoma cells, 456

immunohistochemistry revealed that P23H rhodopsin did not localize to the plasma membrane, 457

forming aggregates in the cytosol and colocalized with an ER-specific marker (Figure 5A, Figure 458

S8). M39R rhodopsin displayed a nearly wild-type phenotype, colocalizing with a plasma 459

membrane marker but with some evidence of mutant rhodopsin retained within the cell (Figure 460

S8), similar to a previous study (Davies et al. 2012). G51A displayed robust wild-type-like 461

localization on the plasma membrane, consistent with a recent report (Behnen et al. 2018). This 462

comparative range of subcellular localization matched what we previously observed in assays of 463

rhodopsin function for these three mutations. 464

We next sought to determine if the same rhodopsin mutants expressed in yeast trafficked 465

to the plasma membrane in a similar manner. The same yeast strain used to functionally couple 466

human rhodopsin to the mating pathway was used to express select rhodopsin mutants with GFP 467

fused to the C-terminus. Similar techniques have been used to investigate productive expression 468

of other human GPCRs in yeast, by observing expression on the plasma membrane or the 469

presence of aggregates (O'Malley et al. 2009). Mirroring our mammalian cell-based 470

observations, P23H and M39R were poorly distributed across the yeast plasma membrane, with 471

highly localized “patchy” expression, while G51A localized consistently to the membrane 472

(Figure 5A). The peri-nuclear ring observed is similar to the localization of other human GPCRs 473

expressed in yeast, indicating the ER membrane (O'Malley et al. 2009, Hashi et al. 2017). 474

Although P23H was observed to form aggregates in the ER of our mammalian cells, this was not 475

observed in yeast, however M39R did appear to form aggregates in the cytosol of yeast. 476

We expanded this microscopy-based analysis to additional rhodopsin mutants expressed 477

in yeast (Figure S9). Image analysis software was used to quantify rhodopsin distribution on the 478

plasma membrane of each yeast cell, identifying localized regions or “patches” where rhodopsin 479

appeared to aggregate (Figure 5B). Wild-type rhodopsin was observed to have a low number of 480

cells displaying a “patchy” phenotype, suggesting even distribution across the plasma membrane. 481

Some mutants associated with misfolding or reduced stability (i.e. N15S, M39R, L125R) 482

exhibited incomplete distribution on the plasma membrane, characterized by a “patchy” 483

phenotype. Mutations in the C-terminus of rhodopsin (Q344ter, V345M, P347L) disrupt the 484

VXPX motif, which is crucial for trafficking rhodopsin in photoreceptors (Wang and Deretic 485

2014). However, mutations within this motif do not affect rhodopsin localization in mammalian 486

cells that are not photoreceptors (Sung et al. 1991, Sung et al. 1993), and were not found to 487

affect rhodopsin localization in yeast. In general, these findings indicated that rhodopsin 488

maintains its subcellular localization when expressed in yeast, which is likely dependent on 489

rhodopsin folding and stability, just as it is with heterologous expression in mammalian cells. 490

491

The Yeast Unfolded Protein Response is Upregulated by Misfolded Rhodopsin Mutants 492

After discovering that the subcellular localization of rhodopsin and changes in responses 493

to light were preserved in yeast, we investigated additional molecular pathways known to be 494

affected by pathogenic rhodopsin mutations. Rhodopsin mutations P23H and T17M have been 495

shown to activate the UPR in mammalian systems, indicative of severe misfolding (Lin et al. 496

2007, Kunte et al. 2012). P23H has been shown to preferentially activate the IRE1 UPR pathway 497

in mammalian cells (Chiang et al. 2015), a pathway also present in yeast. Similar to mammalian 498

IRE1, yeast IRE1 serves as a sensor of misfolded protein in the ER, which activates the 499

transcription factor HAC1 (Kimata and Kohno 2011). Yeast strains and plasmids have been 500

devised utilizing a HAC1-responsive promoter to express reporter genes, which have been used 501

to predict productive expression of other human GPCRs in yeast, where greater UPR activation 502

was associated with GPCR misfolding and aggregation (O'Malley et al. 2009). Due to the 503

conserved pathway, and established results with other GPCRs, we hypothesized that the severity 504

of misfolded rhodopsin could be quantified using a yeast-based sensor of UPR upregulation. The 505

strain designed by Jonikas et al. (2009) has an additional gene cassette constitutively expressing 506

RFP, which can be used to correct for changes in global protein expression (Jonikas et al. 2009). 507

We used this strain to study the 33 selected rhodopsin mutations, plus controls, to quantify their 508

effect on UPR upregulation. 509

Expressed in the UPR-reporter strain, P23H and T17M recapitulated the expected 510

elevated UPR activation in yeast, in addition to several other mutations known to disrupt 511

rhodopsin stability (Figure 6). T4K and N15S, which like T17M disrupt glycosylation, also 512

upregulated the UPR, suggesting a crucial stabilizing nature of these posttranslational 513

modifications. C110Y prevents the formation of a critical disulphide bond in rhodopsin, severely 514

impacting stability and function (Hwa et al. 1999), and the observed increased UPR in yeast 515

matches in silico predictions that this mutant is highly unstable (Rakoczy et al. 2011). Elsewhere 516

in transmembrane helix three, the G114D and R135G mutations similarly increased the UPR. In 517

mammalian cells, R135G is hyperphosporylated and aggregates in endosomes (Chuang et al. 518

2004), but accumulation in the ER or activation of the mammalian UPR has not been reported. 519

That R135G activated the yeast UPR suggests that this mutant may be misfolded in yeast. 520

The constitutively active mutant A292E showed an upregulation in the UPR which has 521

not previously been reported, which may be due to the replacement of a small uncharged residue 522

with a large negatively charged residue proximal to the retinal binding pocket. A reduced UPR 523

for mutations M44T, E150K, and V345M compared to wild-type was observed, but the 524

physiological relevance is unclear. Interestingly, culturing in selective media alone was sufficient 525

to upregulate the UPR, revealed by the difference between the Vector control and the 526

untransformed strain grown in rich media. A log2 GFP/RFP ratio of ~1.0 was previously shown 527

to indicate moderate UPR upregulation (Jonikas et al. 2009), which was observed for the Vector 528

control prior to normalizing the data. Expressing wild-type rhodopsin gave a similar value, 529

which suggested baseline UPR upregulation was due to growth in selective media and not the 530

overexpression of rhodopsin. 531

532

Yeast Assays of Rhodopsin Mutations V81Δ, A164E, A164V Found in Retinal Disease 533

Patients 534

Having established a suite of new yeast-based techniques to investigate rhodopsin 535

functional and molecular phenotypes, we applied these approaches to study three rhodopsin 536

mutations found in degenerative retinal disease patients diagnosed with retinitis pigmentosa. One 537

of the rhodopsin mutations, V81Δ, is a new mutation that has not been previously reported (Case 538

1), and two of the other mutations are previously reported but have had little (A164V, Case 2), or 539

no experimental characterization with respect to those rhodopsin mutations (A164E, Case 3). 540

Missense mutations A164E and A164V have been previously linked to autosomal dominant RP 541

(Fuchs et al. 1994, Hwa et al. 1997), but the molecular mechanism underlying the disruption of 542

rhodopsin function at this site remains to be elucidated. Studies of A164V suggest that helical 543

packing may be an issue (Hwa et al. 1997, Stojanovic et al. 2003), but the effects of introducing 544

a charged residue at this site have not yet been investigated. Mutations at the same residue in 545

rhodopsin can have highly heterogenous phenotypes (Bosch et al. 2003), so we sought to 546

compare Al64E to A164V in greater detail. We identified a new V81Δ mutation in a patient 547

(Case 1) with early-onset autosomal dominant RP (Table S3). This mutation completely removes 548

the V81 codon from the rhodopsin DNA sequence, resulting in a deleted amino acid in the 549

central part of the second transmembrane domain (Figure 7A). Such an amino acid deletion 550

could disrupt alpha helix formation and stability in the membrane, and is likely to lead to a 551

severe molecular phenotype for V81Δ rhodopsin. 552

We characterized V81Δ, A164E, and A164V using the yeast-based methods for 553

investigating rhodopsin molecular phenotype and function, with experiments conducted in the 554

same manner as previously described (Figure 7B-E). Plasma membrane localization of all three 555

mutants were poor, with 28-40% of yeast cells displaying patchy expression of rhodopsin. Light 556

activated signal transduction in yeast was completely abolished for each, similar to the other 557

mutants we studied known to be unstable or misfold. UPR activation was elevated for all three 558

mutants, with A164E higher than any other rhodopsin mutant we studied. Together, our results 559

suggest that all three of these mutants have a severe phenotype in yeast, based on decreased 560

function, subcellular localization, and UPR activation, where we may expect a difference in 561

severity between the A164 mutants based on UPR activation. 562

563

Yeast Assays Comparable to Mammalian Cell Data for Rhodopsin Mutations V81Δ, 564

A164E, A164V 565

We compared our yeast-based assays of V81Δ, A164E, and A164V to expression in 566

mammalian cells. A164V colocalized with the plasma membrane marker, suggesting a less 567

severe phenotype in mammalian cells, which contrasted with the yeast data (Figure 8A). 568

However, A164E and V81Δ were completely retained inside mammalian cells, colocalizing with 569

the ER marker, indicating they were severely misfolded. Following immunoaffinity purification 570

from mammalian cells, the three RP-associated mutations also showed a range in their response 571

to light (Figure 8B). Heterologous expression of V81Δ produced no functional protein, 572

demonstrating a very severe phenotype. A164E, while not as severe phenotype as V81Δ, 573

expressed poorly and produced limited functional protein. A164V expression did produce a high 574

amount of functional protein, consistent with another in vitro study of this mutation (Stojanovic 575

et al. 2003). The sum of this cellular and functional data suggested the same functional trend we 576

first predicted using yeast-based methods, although there was more evidence of functional 577

A164V protein in mammalian cells. 578

579

Patient Clinical Data Supports Functional Trend Predicted by Yeast and Mammalian Cells 580

Next, we looked at patient clinical data. The comparative severity in vitro was found to 581

follow the same trend as available patient phenotype information (Table S3). The most severe 582

phenotype was exhibited by the patient with the V81Δ mutation (Case 1). This patient first had 583

symptoms around 10 years of age, with difficulty adapting to a dim lit environment (nyctalopia). 584

This slowly progressed, and at 39 years she has moderate visual acuity loss (20/50), mildly 585

abnormal color vision, constriction of the visual field to the central 5 degrees. At age 26 years 586

electroretinography already documented severe reduction of rod and cone function. The 587

phenotype of the patient with the A164V mutation (Case 2) is milder than that of the patient with 588

the A164E mutation. Although Case 2 had symptoms of nyctalopia since childhood, the 589

progression of his disease was extremely slow. At age 64 years his electroretinogram was 590

recordable and only mildly abnormal. His central visual acuity at 67 years was 20/40 and despite 591

a paracentral scotoma (area of decreased vision), he maintained a peripheral field. In contrast the 592

patient with the A164E mutation (Case 3) has a good central visual acuity and normal color 593

vision at age 53 years. However, her paracentral scotoma were more severe and progressed to 594

form an annular scotoma at the age of 53 years. Unlike Case 1 (V81Δ), she also preserved some 595

good peripheral field of vision at the age of 45 years, and her ERG was only moderately 596

abnormal. 597

The V81Δ patient is the youngest of the three with a highly reduced retinal function, 598

while the A164V patient is the eldest of the three with the best retinal function (Figure 9). A164E 599

again appeared intermediate to both. These results show that the overall trend of clinical severity 600

was accurately predicted by combining both sets of yeast-derived and mammalian cell-derived 601

data. The yeast methods provided additional information on UPR upregulation which also 602

supported the difference in severity between A164E and A164V mutations, while being less 603

labor intensive than mammalian microscopy and expression methods. 604

605

Discussion 606

Yeast Provide New Methods to Investigate Rhodopsin Structure and Function 607

In this study, we have engineered the yeast S. cerevisiae to characterize both known and 608

novel pathogenic mutations of the visual pigment rhodopsin. In comparing our new assays to 609

traditional mammalian cell-based approaches, we demonstrate that the molecular phenotypes of 610

this light-activated human GPCR are similar in yeast, and that these phenotypes reflect patient 611

clinical data. There are a number of advantages and differences when compared to traditional 612

techniques, as these yeast-based rhodopsin assays are performed in a cellular context, which 613

provides a new perspective on signal transduction pathways, subcellular localization, and UPR 614

upregulation. 615

A direct measurement of downstream pathway activation in response to light was 616

achieved by functionally coupling rhodopsin to the yeast mating pathway. This required 617

productive in vivo activation of a G protein, mimicking the initial step that occurs in human 618

photoreceptors, even if downstream signaling differs. By using yeast, many rhodopsin mutations 619

could be studied in the same experiment, with multiple replicates, without laborious purification 620

steps which are traditionally required for in vitro transducin activation assays (Reeves et al. 621

1996). Not only did yeast-expressed rhodopsin maintain its ability to respond to light, but the 622

magnitude of signal pathway activation was comparable to many previous studies of mutations 623

expressed in mammalian systems. We were also able to observe rhodopsin mutations that 624

resulted in the activation of dark state rhodopsin, which has previously required sensitive 625

spectroscopic assays using immunoaffinity purified rhodopsin (Liu et al. 2013), or gene knock-in 626

animal models (Zhang et al. 2013). This included providing new data on L125R signaling in the 627

dark, a mutant poorly expressed in mammalian cells which has made previous characterization of 628

function challenging (Stojanovic et al. 2003). Mutations at site L125 have been shown to reduce 629

the thermal stability of rhodopsin (Andres et al. 2001), which could lead to dark noise through 630

thermal isomerization of the bound chromophore (Luo et al. 2011). This mechanism was 631

supported by our finding that L125R dark noise was retinal-dependent in yeast, which could be a 632

result of thermal isomerization. Thus, yeast may serve as a platform for studying difficult to 633

express rhodopsin proteins, allowing for the rapid quantification of downstream G protein 634

activation under various conditions. 635

Mutations known to disrupt rhodopsin folding or stability tended to have incomplete 636

localization on the yeast plasma membrane, similar to mammalian cell expressed rhodopsin. 637

Although there was significant heterogeneity between the investigated mutants, this observation 638

suggests that the biochemical properties of rhodopsin were conserved in yeast, despite known 639

differences in glycosylation patterns (Mollaaghababa et al. 1996). We quantified membrane 640

localization using a novel automated image analysis procedure, which may be useful for studies 641

of other GPCRs and associated pathogenic mutations when expressed in yeast. However, not all 642

human GPCRs are productively expressed in yeast (O'Malley et al. 2009), so these methods 643

should first be validated with the wild-type receptor. 644

The majority of disease-linked rhodopsin mutations that have been identified cause the 645

protein to misfold, which can activate the UPR in the ER, and eventually lead to photoreceptor 646

cell death (Athanasiou et al. 2018). Modulating the UPR has been investigated as a potential 647

treatment for RP (Tam et al. 2010, Chiang et al. 2012a, Parfitt et al. 2014), but the effect on UPR 648

upregulation had only been determined for three rhodopsin mutations (Lin et al. 2007, Kunte et 649

al. 2012, Marsili et al. 2015). Determining UPR upregulation by mutant rhodopsin has 650

previously required microscopy, immunoblot, and qPCR methods (Kunte et al. 2012, Marsili et 651

al. 2015). We took advantage of an engineered yeast strain that possesses a reporter linked to the 652

IRE1 UPR pathway, which is the only one of three UPR pathways that is conserved between 653

mammals and yeast (Kimata and Kohno 2011). This yeast-based reporter of UPR activity 654

enables the use of flow cytometry, a more simple and high-throughput method, and offers the 655

advantage of measuring UPR upregulation directly in live cells. We found that upregulation of 656

this pathway was associated with certain rhodopsin mutants known to misfold or with reduced 657

stability, which may be predictive of the molecular mechanism contributing to retinal 658

degeneration. 659

However, yeast do not contain the PERK and ATF6 UPR pathways found in mammalian 660

cells (Kimata and Kohno 2011). Thus, although the yeast-based methods provide insight into 661

UPR upregulation, these studies would need to be combined with mammalian cells to better 662

understand how all UPR pathways may be affected by rhodopsin and other GPCR mutations. 663

This may also explain why nine of the fifteen mutants that are believed to misfold did not cause 664

an increase UPR activation in yeast, suggesting significant heterogeneity between rhodopsin 665

mutations. Determining a missense mutation’s contribution to UPR upregulation is highly 666

relevant to pharmacogenomics, as an inability for rhodopsin to respond to light may not 667

necessarily indicate the pathogenic mutation can be rescued by UPR modulation. 668

Overall, many known rhodopsin phenotypes were recapitulated in yeast, a requirement 669

for any assay of human gene function seeking to determine the clinical relevance of patient 670

derived missense mutations (Amendola et al. 2016). There are also important differences to 671

consider when comparing our yeast-based assays to mammalian cell-based assays. Importantly, 672

when a lack of signaling is observed in yeast it is difficult to separate rhodopsin mutants that 673

misfold, from mutants that fold properly but do not productively activate the downstream 674

pathway. Although our quantified microscopy data supported the notion that rhodopsin mutants 675

with inconsistent plasma membrane expression in yeast are also mutants known to be unstable or 676

misfolded, unique aspects of yeast cellular machinery or post-translational processing may 677

influence these mutants. Results from the R135G mutation highlight these differences, where this 678

mutant activated the UPR in yeast, but does not aggregate in the ER of mammalian cells 679

(Chuang et al. 2004). 680

The signaling phenotypes of the G90D and A292E mutants in yeast also differed from the 681

reported constitutive activity in vitro when using purified protein (Gross et al. 2003). That 682

A292E signaled higher and G90D lower than expected, suggests that constitutive activity in 683

yeast is dependent on cellular conditions which would not be revealed in an in vitro assay using 684

purified protein, such as a renewing supply of both rhodopsin and G protein. It is interesting to 685

note, however, that A292E has the highest constitutive activity reported of a CSNB-associated 686

mutation in vitro (Gross et al. 2003), which fits the trend observed in yeast. That light-dependent 687

signaling in yeast was affected by the V104I, F220L, and V345M mutations was unexpected, 688

which provides interesting new data for these previously uncharacterized mutants that should be 689

followed up in mammalian cell-based assays. Thus, these yeast-based methods provide 690

complimentary but independent data to traditional mammalian-cell and in vitro biochemical 691

techniques, offering a unique perspective on rhodopsin structure and function in a cellular 692

context. 693

694

Characterizing Novel Rhodopsin Mutations with Yeast 695

Determining mutant function rapidly and accurately has become increasingly important 696

with the rise of whole genome sequencing, and the ever expanding rise in gene mutations with an 697

unknown impact on human health. Rhodopsin mutations linked to inherited retinal disease have 698

been used as examples of how many gene mutations discovered in patients are rarely 699

characterized, and that the molecular basis for pathology is poorly understood (Chiang and Gorin 700

2016, Davies 2014). Animal models and traditional in vitro assays provide detailed information, 701

but they have not kept pace with the hundreds (>350) of rhodopsin mutations identified to date. 702

This is true of many other genetic diseases, but new methods of functional characterization are 703

helping to address this (Starita et al. 2017), including using yeast-based assays (Sun et al. 2016, 704

Yang et al. 2017). 705

We investigated the use of yeast to characterize novel and understudied pathogenic 706

mutations, and compared to clinical data for patients with varying severity of RP. Our yeast-707

based approaches predicted severe phenotypes for the V81Δ and A164 mutations, which 708

included determinations of their light-activation, subcellular localization, and UPR upregulation. 709

By combining yeast and mammalian cell-based assays, the relative severity of these mutations 710

was revealed, as compared with clinical phenotypes measuring decline in visual function in 711

patients. The V81Δ rhodopsin mutation showed the most severe phenotype in our combined 712

assays and the most extensive visual deterioration clinically, in contrast to A164V, which had the 713

mildest phenotype both clinically and experimentally, and A164E, which was found to be 714

intermediate. 715

The difference in severity for the two missense mutations found at site 164 highlights the 716

heterogenic nature of retinitis pigmentosa, and the importance of characterizing individual 717

disease phenotypes. These results indicate that yeast-based approaches could be useful not only 718

for investigating the molecular basis of retinal disease, but also for better prediction of mutation 719

pathogenicity, to help improve the accuracy of prognoses for patients associated with specific 720

mutations in rhodopsin. Integrating the results of both UPR and light-activation assays (Figure 721

S10) may also help determine the molecular mechanism of disease, to differentiate between 722

mutations that cause severe misfolding, versus disruptions in retinal binding or stability that 723

prevent activation but do not cause cell stress. 724

Recent successes in gene therapy for inherited retinal disease means that mutation 725

classification is of utmost importance, to determine if such therapy is required (U.S. Food and 726

Drug Administration 2017). This issue is particularly important to address for degenerative 727

diseases, such as inherited retinal disease, where early intervention is crucial. A method to 728

determine the functional consequences of mutations throughout rhodopsin, rapidly and 729

accurately, would therefore be highly beneficial. 730

The methods presented here could also extend to functionally characterizing mutations of 731

many other GPCRs. Indeed, although over 30 human GPCRs have been functionally linked to 732

the yeast mating pathway, no previous yeast-based study has focused on direct functional 733

characterization of human GPCR mutations. As discussed in a recent review of GPCR 734

pharmacogenomics, characterizing GPCR mutations could lead to a better understanding of 735

disease and drug responses in patients (Hauser et al. 2018). Missense mutations that modulate 736

interactions with downstream signaling and regulatory proteins are known to play a role in this, 737

so assays which accurately reflect GPCR function and interactions in a cellular context, such as 738

the yeast assays presented here, will be key to understanding the impact of GPCR mutations on 739

human health. 740

741

Figures 742

743

Figure 1 Representation of the Engineered Mating Pathway. Rhodopsin activation was 744

functionally coupled to the expression of a fluorescent reporter protein, mCherry, utilizing the 745

mating-responsive promoter pFUS1. Modifications to the mating pathway included the knockout 746

of negative regulator Sst2, the gene encoding Far1 which halts cell growth in the wild-type 747

mating pathway, and the endogenous mating pathway GPCR Ste2. The chimeric G alpha protein 748

(Gpa1-Gαt) contains the 5 C-terminal amino acids of the G alpha subunit of human transducin 749

(Gαt). 750

751

Figure 2 Light-Dependent Activation of the Mating Pathway. Human rhodopsin was found to 752

activate the mating pathway only in response to light, requiring the presence of retinal 753

chromophore. Adding retinal after each hourly light exposure improved the overall response 754

approximately 1.6-fold. Incubating with the same concentration of retinal but keeping the culture 755

in the dark did not result in mating pathway activation. “Vector” denotes yeast transformed with 756

a plasmid not containing the rhodopsin gene. Data points represent results of four individual 757

colonies, each in a 5 mL culture. Error bars represent standard deviation. 758

759

Figure 3 Characterization of Rhodopsin Light-Dependent Function. (A) Response to light 760

from yeast-expressed rhodopsin mutants, indicating a similar magnitude of response as the 761

mammalian cell-expressed protein. “WT” denotes wild-type human rhodopsin. Yeast data points 762

represent results of nine individual colonies, each in a 600 μL culture, minus the mCherry 763

fluorescence of the same strain transformed with empty plasmid control (Vector), and 764

normalized to wild-type. * P < 0.05 vs WT or between indicated mutants. (B) Difference spectra 765

of mammalian cell-expressed rhodopsin mutants in response to light. The peak at 500 nm 766

indicates a light-dependent response. 767

768

Figure 4 The Light-Activated Signal Transduction of Pathogenic Rhodopsin Mutants in 769

Yeast. (A) Missense mutations that intrinsically disrupt rhodopsin stability, or that indirectly 770

affect stability by disrupting a post-translational modification (PTM) motif. Class 3 denotes a 771

unique category of mutations at site R135, which disrupt G protein coupling and lead to 772

constitutive endocytosis in mammalian cells. (B) Pathogenic and asymptomatic variants that 773

increase or do not disrupt rhodopsin activation by light. “WT” denotes wild-type human 774

rhodopsin. “Vector” denotes yeast transformed with a plasmid not containing the rhodopsin 775

gene. “arRP” denotes autosomal recessive retinitis pigmentosa. Data points represent results of 776

nine individual colonies, each in a 600 μL culture, minus the mCherry fluorescence of Vector, 777

and normalized to wild-type. Error bars represent the 95% CI, * P < 0.05 vs WT. 778

779

780

781

782

783

Figure 5 Representative Subcellular Localization of Rhodopsin Mutants (A) Comparative 784

subcellular localization of rhodopsin mutants in SK-N-SH and yeast cells. “PM merge” indicates 785

fluorescence of a plasma membrane marker, merged with GFP-tagged rhodopsin. Images 786

represent maximal projections, with scale bars representing 30 μm and 5 μm respectively. (B) 787

Quantified localization of GFP-tagged rhodopsin mutants on the yeast plasma membrane. Yeast 788

cells with patchy membrane expression of rhodopsin had at least 10% of their cell membrane 789

containing separated patches of rhodopsin. Error bars represent the bootstrapped standard error. 790

791

Figure 6 UPR Activation in Yeast Relative to Wild-Type Rhodopsin. GFP expression is a 792

reporter of UPR upregulation, while RFP is constitutively expressed to help correct for changes 793

in global protein expression. “WT” denotes wild-type human rhodopsin. “Vector” denotes yeast 794

transformed with a plasmid not containing the rhodopsin gene. “Strain” denotes the yJW1200 795

strain not transformed with plasmid and grown in rich media. “arRP” denotes autosomal 796

recessive retinitis pigmentosa. Data points represent results of nine individual colonies, each in a 797

600 μL culture, normalized to wild-type. Boxes extend from the 25th to 75th percentile, the line 798

across the box represents the median value. Bars represent the min and max recorded values. * P 799

< 0.05 vs WT. 800

801

802

Figure 7 Characterization of Novel Rhodopsin Mutants using Yeast. (A) Crystal structure of 803

rhodopsin, highlighting residues V81 and A164 in red. The other rhodopsin mutants 804

characterized in this study are highlighted in cyan, and the approximate location of the 805

membrane is indicated by the dotted line (1U19.pdb). Quantified phenotype and functional 806

assays of V81Δ, A164E, and A164V in yeast (B) Representative subcellular localization, scale 807

bars are 5 μm, (C) quantified consistency of rhodopsin localization on the yeast plasma 808

membrane, (D) light-activated signal transduction, and (E) UPR activation. “WT” denotes wild-809

type human rhodopsin. The number of biological replicates and error bars are identical to 810

previous figures. * P < 0.05 vs WT in all panels unless otherwise indicated. 811

812

Figure 8 Characterization of Novel Rhodopsin Mutants using Mammalian Cell Expression. 813

(A) Comparative subcellular localization of rhodopsin mutants in SK-N-SH cells. “PM” 814

indicates fluorescence of a plasma membrane marker, “ER” indicates fluorescence of a ER-815

specific marker. Scale bars are 30 μm. (B) Difference spectra of mammalian cell-expressed 816

rhodopsin mutants in response to light. The peak at 500 nm indicates a light-dependent response. 817

818

819

820

Figure 9 Clinical Assessment of Patients with Rhodopsin Mutations V81Δ, A164E, and 821

A164V. (A) Goldmann visual fields of the right eye at two time points. Normal fields would 822

reach the gray dotted line. The solid blue line outlines the actual field. The hatched areas are 823

scotoma, i.e. areas of loss in sensitivity. Darker areas refer to denser scotoma. (B) Structural 824

retinal phenotype of the right eye from cases carrying the A164E and V81Δ mutations. Optical 825

coherence tomography (OCT) above showing the different retinal layers. Brackets show area of 826

preserved outer retina; A164E > V81Δ. Unlike for A164E, the OCT of V81Δ shows disturbed 827

lamination of the retina with degenerative cysts, reflecting more advanced disease. The retinal 828

photograph below centered on the posterior pole. Photograph on the right is taken with a wider 829

field camera. ON: optic nerve. The dotted white line indicated the foveal area at the center of the 830

macula. Double white arrow indicates vessel attenuation, while single arrow shows typical 831

pigmentary deposits (few in these cases). The width of the central visual field corresponds to the 832

area of preserved outer retina on the OCT. 833

834

835

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