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HIGHLIGHTED ARTICLE| INVESTIGATION
Coupling of Human Rhodopsin to a Yeast SignalingPathway Enables
Characterization of Mutations
Associated with Retinal DiseaseBenjamin M. Scott,* Steven K.
Chen,* Nihar Bhattacharyya,* Abdiwahab Y. Moalim,*
Sergey V. Plotnikov,* Elise Heon,† Sergio G. Peisajovich,* and
Belinda S. W. Chang*,‡,§,1
*Department of Cell and Systems Biology, ‡Department of Ecology
and Evolutionary Biology, and §Centre for the Analysis ofGenome
Evolution and Function, University of Toronto, Ontario M5S 3G5,
Canada and †Department of Ophthalmology, Hospital
for Sick Children, Toronto, Ontario M5G 1X8, Canada
ORCID ID: 0000-0002-6525-4429 (B.S.C.)
ABSTRACT G protein-coupled receptors (GPCRs) are crucial sensors
of extracellular signals in eukaryotes, with multiple GPCRmutations
linked to human diseases. With the growing number of sequenced
human genomes, determining the pathogenicity of amutation is
challenging, but can be aided by a direct measurement of
GPCR-mediated signaling. This is particularly difficult for
thevisual pigment rhodopsin—a GPCR activated by light—for which
hundreds of mutations have been linked to inherited
degenerativeretinal diseases such as retinitis pigmentosa. In this
study, we successfully engineered, for the first time, activation
by human rhodopsinof the yeast mating pathway, resulting in
signaling via a fluorescent reporter. We combine this novel assay
for rhodopsin light-dependent activation with studies of
subcellular localization, and the upregulation of the unfolded
protein response in response tomisfolded rhodopsin protein. We use
these assays to characterize a panel of rhodopsin mutations with
known molecular phenotypes,finding that rhodopsin maintains a
similar molecular phenotype in yeast, with some interesting
differences. Furthermore, we compareour assays in yeast with
clinical phenotypes from patients with novel disease-linked
mutations. We demonstrate that our engineeredyeast strain can be
useful in rhodopsin mutant classification, and in helping to
determine the molecular mechanisms underlying theirpathogenicity.
This approach may also be applied to better understand the clinical
relevance of other human GPCR mutations,furthering the use of yeast
as a tool for investigating molecular mechanisms relevant to human
disease.
KEYWORDS Visual degenerative disease; retinitis pigmentosa; G
protein-coupled receptor; disease model; rhodopsin
THEdiversity of biologically relevant signals
thatGprotein-coupled receptors (GPCRs) detect make them critical
tohow cells sense and respond to their environment.
Missensemutationswithin this large family of cell surface receptors
cantherefore have serious physiological effects, andmany
humandiseases have been linked with missense mutations in
GPCRs(Heng et al. 2013). Over 63,000 missense mutations havebeen
identified in human GPCRs via large-scale human ge-nome studies
(Pándy-Szekeres et al. 2018), but the majorityhave not been studied
in detail, so the significance to human
health remains unclear. Missense mutations can disruptGPCR
function in many ways, ranging from ligand binding,to protein
stability, to changes in downstream signaling andinteractions with
negative regulators (Stoy and Gurevich2015). Consequently, disease
can arise through many differ-ent molecular phenotypes, and not all
mutations are diseasecausing.
To better understand how human missense mutationscontribute to
disease, the yeast Saccharomyces cerevisiae hasemerged as a
powerful tool for characterizing human pro-tein function due to
conserved molecular pathways, rapidgrowth, and ease of genetic
manipulation (Laurent et al.2016). These benefits have facilitated
yeast models of humandisease (Outeiro and Lindquist 2003; Perocchi
et al. 2008)and studies of pathogenic human mutations (Sun et al.
2016;Yang et al. 2017). Synthetic biology approaches, where hu-man
protein function and interactions are quantified by
Copyright © 2019 by the Genetics Society of Americadoi:
https://doi.org/10.1534/genetics.118.301733Manuscript received June
13, 2018; accepted for publication November 29, 2018;published
Early Online December 04, 2018.Supplemental material available at
Figshare: https://doi.org/10.25386/genetics.7414109.1Corresponding
author: University of Toronto, 25 Harbord St., Toronto, ON M5S3G5,
Canada. E-mail: [email protected]
Genetics, Vol. 211, 597–615 February 2019 597
http://orcid.org/0000-0002-6525-4429https://doi.org/10.1534/genetics.118.301733https://doi.org/10.25386/genetics.7414109https://doi.org/10.25386/genetics.7414109mailto:[email protected]
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yeast-based gene circuits, have enabled high-throughput
ex-periments at an impressive scale (Starita et al. 2015;
Sokolinaet al. 2017; Weile et al. 2017; Woodsmith et al. 2017).
Withgenetic engineering, human GPCRs can be functionally linkedto
the yeast mating pathway, and mating-responsive reportergenes have
allowed for detailed studies of GPCR activation(Liu et al. 2016).
As human GPCRs retain their natural prefer-ences for ligands and G
proteins in yeast (Brown et al. 2000),this application of synthetic
biology combines the high-throughput capabilities of yeast-based
studies with the abilityto rapidly characterize GPCR function in a
cellular context.This has facilitated screens of chemical libraries
for novel GPCRligands (Campbell et al. 1999; Horswill et al. 2007),
andscreens of mutated GPCRs to characterize specific protein
do-mains or to engineer novel function (Erlenbach et al.
2001;Armbruster et al. 2007; Liu et al. 2015).
Despite the power of yeast-based studies of humanGPCRs,only a
small proportion of GPCRs have been functionallylinked to the yeast
mating pathway, and all have beenligand-activated (Liu et al.
2016). Unlike these GPCRs,rhodopsin exists as a covalent complex
between its light-sensitive chromophore 11-cis retinal and the
seven-helixtransmembrane opsin apoprotein (Smith 2010). Light
ex-posure isomerizes the chromophore, which induces a
con-formational change in rhodopsin’s transmembrane
helices,activating the associated heterotrimeric G protein,
transdu-cin, and triggering the visual transduction cascade that
even-tually results in a signal to the brain that light has
beenperceived (Smith 2010). Not surprisingly, missense muta-tions
in rhodopsin are often associated with retinal diseasesin humans
(Athanasiou et al. 2018). Retinitis pigmentosa(RP) is a highly
heterogeneous, degenerative retinal disorderthat results in vision
impairment and in some cases even-tually blindness, affecting �1 in
4000 people worldwide(Fahim et al. 2000). The heterogeneous nature
of this de-generative disease has contributed to the difficulties
indeveloping effective prognoses and treatment. Missense mu-tations
in rhodopsin have been associated with 20–30% ofautosomal dominant
RP and 1% of autosomal recessive RPcases, making rhodopsin one of
the most important RP genes(Fahim et al. 2000). Over 150 rhodopsin
missense mutationshave been associated with disease (Stenson et al.
2014), andan additional .200 uncharacterized mutations in
rhodopsinexons are listed in the Genome Aggregation Database (Leket
al. 2016).
Without a tool to rapidly assess rhodopsin function,
thisincreasing availability of genetic information has yet to lead
toa better understanding of pathogenicity of mutations
associ-atedwith RP and other inherited visual diseases.
Determiningthe impactofmissensemutationson theability of rhodopsin
torespond to light currently relies on in vitro biochemical
assaysthat are labor intensive, requiring mammalian cell culture
toproduce one mutant protein at a time, followed by
immuno-fluorescence or immunoaffinity purification (Sung et al.
1991;Reeves et al. 1996). These technical challenges are
com-pounded by the diverse molecular phenotypes of rhodopsin
mutations, ranging from constitutively active, to
impropersubcellular localization, to disrupted post-translational
mod-ifications (Athanasiou et al. 2018). To efficiently
character-ize the wide variety of patient-derived mutations, a
rapidmethod that reliably recapitulates light-dependent signalingof
rhodopsin is needed.
Here, we use synthetic biology approaches to engineerrhodopsin
coupling to the yeast mating pathway, demonstrat-ing for the first
time successful rhodopsin light-activatedsignal transduction that
can be rapidly quantified using afluorescent reporter gene of
mating pathway activation. Wecompared our novel yeast-based assay
to more establishedmammalian cell-based methods, using a panel of
previouslystudied rhodopsinmutations.We found thatmeasurements
ofrhodopsin activation in yeast resemble in vitro results
usingrhodopsin purified from mammalian cells, with some
excep-tions. We also found that a yeast-based reporter of the
un-folded protein response (UPR) produced results consistentwith
previous studies in mammalian cells quantifying theeffects of
rhodopsin pathogenic mutants on cellular stress.Finally, we used
our combined approaches in yeast to inves-tigate recently
identified rhodopsin mutations in patientswith retinal disease, and
were able to propose pathogenicclassifications that are supported
by mammalian cell andclinical data.
Materials and Methods
Yeast strain engineering
The parent yeast strain for all strain engineering was
CB008,genotype W303 MATa, far1D, his3, trp1, leu2, ura3
(Supple-mental Material, Table S1 contains all strain genotypes).
Allgene knock-outs and knock-ins were conducted using homol-ogous
recombination of selectable markers. pFUS1-mCherrywas integrated at
the MFA2 locus using plasmid pJW609containing the KanR marker.
pFUS1 was defined as the1636 bp immediately upstream of the Fus1
start codon, themCherry sequence used is from Keppler-Ross et al.
(2008),and �1 kb homology regions were used. STE2 and SST2were
targeted for deletion using TRP1 and HygB selectablemarkers
respectively, each with 180 bp of flanking homologyregions
identical to the sequences flanking the ORF. The fiveC-terminal
amino acids of Gpa1 (KIGII) was replaced with aGpa1-Gat
(transducin) chimera, containing the C-terminalamino acids from
mammalian Gat (DCGLF), using plasmidpBS600 designed for this study
(Figure S1), containing se-lectable marker LEU2, and a sequence
homologous to the800 bp 39 to the natural Gpa1 gene. The C.
albicans Adhterminator was used downstream of both the
pFUS1-mCherryand Gpa1-Gat gene cassettes. Strains were confirmed by
PCRand flow cytometry.
Rhodopsin mutation selection and patient phenotyping
Rhodopsin mutations were selected from across pheno-typic
classes, as reported in a recent comprehensive review
598 B. M. Scott et al.
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(Athanasiou et al. 2018), and with at least one of the
follow-ing assays previously published: transducin activation,
lo-calization in mammalian cells, or spectroscopy
indicatingchromophore binding. The patient cases were selected
froman internal database and the phenotype information was
col-lected retrospectively. Other than basic demographic and
ge-netic information, we collected information about visualacuity
(VA), color vision, Goldmann visual fields (GVF),
elec-troretinography (ERG), and imaging. Imaging included fun-dus
photography (VisucamNM/FA; Carl Zeiss Meditec, Dublin,CA and Optos)
and optical coherence tomography (OCT, Cir-rus from Carl Zeiss
Meditec). Genetic testing was done usinggene panels based
sequencing by CLIA-approved laborato-ries. This study was approved
by the Human Research EthicsBoard of the hospital for Sick Children
and met the Tenets ofthe declaration of Helsinki.
Cloning and mutagenesis
The human rhodopsin sequence (RefSeq NP_000530.1) wasamplified
using Pfu polymerase (Thermo) from plasmid pJETHuRh (Morrow et al.
2017), using primers to insert flankingAarI restriction sites.
Following AarI digestion, the rhodopsinsequence was ligated to the
yeast centromere plasmidspRS316 and pRS313, which each contained
the TDH3 pro-moter (pTDH3; alternatively called pGPD). For
mammalianexpression, rhodopsin mutations were introduced into
thewild-type bovine rhodopsin sequence in the p1D4 vector
forimmunoaffinity purification or the pGFP vector for
SK-N-SHimmunofluorescence microscopy (Figure S2). Mutagenesiswas
conducted via PCR following the QuikChange site-di-rected
mutagenesis protocol (Agilent) and using PfuUltra IIFusion HS DNA
Polymerase (Agilent). Mutagenesis primerswere designed with 20 or
21 nucleotides identical to humanor bovine rhodopsin flanking the
mutant nucleotide(s).
Yeast plasmid transformation
Yeast strain BS017 or yJW1200 were transformed with indi-vidual
plasmids by a standard lithium-acetate method andplated on
selective media (SD-URA or SD-HIS, respectively).
Rhodopsin purification
Culturingand transfectionofHEK293Tcellswasperformedaspreviously
described (Bhattacharyya et al. 2017). Briefly,p1D4 vector
containing a rhodopsin gene was transfectedwith Lipofectamine 2000
(Invitrogen) and cells were har-vested after 48 hours. Rhodopsin
was regenerated with11-cis retinal for 2 hours before
solubilization in 1% dodecylmaltoside (Anatrace) and immunoaffinity
purified with 1D4monoclonal antibody (Molday and MacKenzie 1983).
Theultraviolet-visible absorption spectra of the rhodopsin
pro-teins were recorded using a Cary 4000 double beam
spectro-photometer (Agilent). Pigments were light bleached with
aFiber-Lite MI-150 high intensity illuminator (Dolan-Jenner)for 60
sec at 20�. Dark-light difference spectra were calcu-lated by
subtracting the light-bleached absorbance spectrafrom the dark
spectra.
Yeast light activation assay
Yeast strain BS017 transformed with a human rhodopsinmutant gene
in the pRS316 pTDH3 vector was incubatedovernight in SD-URA media
in a 30� shaker. The same straintransformed with a plasmid not
containing the rhodopsinsequence (Vector) was used as a negative
control. Cells werediluted to OD600 0.05 in fresh media containing
5 mM 9-cisretinal (Sigma-Aldrich) and incubated for 2 hours,
protectedfrom light, in a 30� shaker; 9-cis retinal is a common
alterna-tive to the natural chromophore 11-cis retinal, and
givescomparable in vitro results (Opefi et al. 2013).
LightSafe50-ml centrifuge tubes (Sigma-Aldrich) were used for 5
mlcultures, and 96-well deep well blocks (VWR) wrapped inaluminum
foil for 600 ml cultures. Indicated cultures werethen exposed to
light using a Fiber-Lite MI-150 high intensityilluminator
(Dolan-Jenner) set to full intensity for 15 min atroom temperature.
After 100 ml samples were taken for anal-ysis, an additional 5 mM
9-cis retinal was added to indicatedcultures—to both light exposed
and cultures kept in thedark—and placed back in the 30� shaker.
Light exposure fol-lowed by retinal addition was conducted every
hour for atotal of 6 hours following the first light exposure.
Cells werethen treated with the protein synthesis inhibitor
cyclohexi-mide, to a final concentration of 10 mg/ml. The
mCherryfluorescence of at least 6000 cells was measured for
eachsample with a Miltenyi Biotec MACSQuant VYB. The meanmCherry
fluorescence was determined using FlowJo. Aftersubtracting the
mCherry fluorescence signal of the Vector con-trol, fluorescence
values were normalized to the wild-typerhodopsin control used in
the same experiment, to allow com-parisons between experiments
performed on different days.
Immunofluorescence microscopy
SK-N-SH neuroblastoma (ATCC HTB-11) cells were grownand cultured
in full media [DMEM (Life Technologies), 10%FBS (Invitrogen), and
1% Penicillin-Streptomycin (Invi-trogen)] at 37� in 5% CO2 and
seeded into 24-well plateswith coverslips (Sarstedt) while under
five passages. Oncecells reached �75% confluence, they were
transfected with645 ng of pGFP plasmid containing the appropriate
bovinerhodopsin gene, using Lipofectamine 2000 (Invitrogen)
pro-tocols. After 24 hours, half the wells were incubated withWheat
germ Agglutinin (Invitrogen) in HBSS for 10 min at37� to label the
plasma membrane. All cells were then rinsedwith PBS and fixed with
2% paraformaldehyde in PBS. Tolabel cells with the endoplasmic
reticulum marker antibodyanti-calreticulin (1:400; Abcam), cells
were washed andpermeabilized in PBS containing 1% bovine serum
albu-min (Sigma) and 0.1% saponin (PBS-BS). Anti-calreticulinwas
diluted in PBS-BS and incubated for 1 hours at roomtemperature.
After washing with PBS-BS, secondary anti-body (Cy3-conjugated goat
anti-rabbit IgG, 1:200; JacksonImmunoresearch) was diluted in
PBS-BS and added to thewells for 1 hours. Nuclei were stained with
Hoechst (1:1000in PBS, Hoechst type 33258 Invitrogen) for 10 min.
Cells
Rhodopsin-Coupled Yeast Signaling 599
http://www.yeastgenome.org/locus/S000003424/overview
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were mounted with ProLong Gold Antifade (Thermo), a cov-erslip
was applied, and allowed to cure for 24 hours in thedark prior to
imaging on Leica TCS SP8 confocal microscope.ImageJ was used to
construct Z-stacks, maximum projectionimages, and scale bars.
Yeast microscopy
Yeast strain BS017 expressing human rhodopsin C-terminallytagged
with GFP were grown to log phase in selective media,then plated on
glass-bottomed dishes (Greiner Bio-One)treated with 1 mg/ml
concanavalin A (Sigma-Aldrich). Thecentromere plasmid pRS316 pTDH3
was used, and the GFPsequence used is from Moser et al. (2013). A
short aminoacid linker (GGERGS) was introduced between the
finalrhodopsin residue and first GFP residue. Images of the
cellsadhered to the dishes were acquired using a Leica TCS
SP8confocal microscope with a 1003 1.4NA objective and ahybrid
detector (Leica). GFP fluorescence was analyzed us-ing a custom
Fiji-MATLAB pipeline (File S1 and File S2),which was similar to
analyses performed to quantify fluo-rescence peaks in mammalian
cells (Cheng et al. 1999). OnFiji using batch mode (Schindelin et
al. 2012), yeast cellmaps were generated by first removing
high-frequencynoise using ROF Denoise (theta = 25) and preparing
forthresholding using Enhance Contrast (0.3 saturation
withNormalization). Manual thresholding, filling holes, and
fi-nally selecting of cells using the watershed plugin
generatedfinal cell maps. Cell membrane maps were defined in
MAT-LAB as the seven outer pixels (�0.7 mm) of each cell
map.Fluorescent patches at the cell membrane were defined inMATLAB
as pixels with fluorescence intensity at least 2.0 SDabove the edge
mean. Cells with patchy membrane expres-sion of rhodopsin were
defined in MATLAB as cells with atleast 10% of their cell membrane
containing membranepatches. Bootstrapped SE were generated in
MATLAB byusing the Statistics and Machine Learning Toolbox
functions(Mathworks).
UPR activation assay
Yeast strain yJW1200 was generously provided by theWeissman
laboratory, University of California, San Francisco.This strain
contains a 43 repeat of the unfolded protein re-sponse element
upstream of GFP, and the constitutive TEF2promoter upstream of RFP
(Jonikas et al. 2009). yJW1200transformed with a human rhodopsin
mutant gene in thepRS313 pTDH3 vector was incubated overnight in 3
mlSD-HISmedia in a 30� shaker. The same strain transformedwitha
plasmid not containing the rhodopsin sequence (Vector) wasused as a
negative control. Cells were diluted to OD600 0.2 in600 ml fresh
media and incubated for 4 hours in a 30� shaker.Cells were then
treated with the protein synthesis inhibitor cy-cloheximide, to
afinal concentration of 10 mg/ml. TheGFP andRFP signal of at least
10,000 cells was measured for eachsample with a Miltenyi Biotec
MACSQuant VYB. The meanGFP and RFP fluorescence was determined
using FlowJo.Fluorescence values were normalized to the
wild-type
rhodopsin control used in the same experiment, to allow
com-parisons between experiments performed on different days.
Statistical analyses and graphs
Statistical analyses were performed using Prism (GraphPad),using
one-way ANOVA for UPR comparisons to wild-typerhodopsin, and for
yeast light anddarkactivation comparisonsto wild-type rhodopsin.
Student’s t-test was used to comparethe results of one mutant or
condition to one other mutantor condition. Graphs were generated
using Prism or Excel(Microsoft).
Data availability statement
Strains and plasmids are available upon request. All
supple-mental figures, tables, and files have been uploaded to
fig-share. Supplemental material available at Figshare:
https://doi.org/10.25386/genetics.7414109.
Results
Human rhodopsin functionally couples to the yeastmating pathway
and signal transduction is dependenton light
Wehave engineered, for the first time, a vertebrate
rhodopsinthat can successfully couple to the yeast mating pathway.
Thiswas accomplished by first knocking out the genes encodingFar1,
to prevent cell cycle arrest, and the GTPase-activatingprotein
Sst2, a negative regulator of the mating pathway(Brown et al.
2000). The endogenous mating pathwayGPCR Ste2 was also knocked out,
to prevent unnecessaryinteractions with downstream mating pathway
proteins.Next, a chimeric G alpha protein and the fluorescent
proteinmCherry under the regulation of the mating-responsiveFUS1
promoter (pFUS1) were inserted into the yeast genome(Figure 1 and
Table S1). As rhodopsin is known to interactonly with G alpha
proteins containing the same five aminoacid C-terminal sequence
[transducin in rod photoreceptorsand Gai1 in engineered systems
(Maeda et al. 2014; Sun et al.2015)], only one Gpa1 chimera
(Gpa1-Gat) was required forour study. To ensure functional coupling
between Gpa1-Gatand human rhodopsin, we first expressed a known
constitu-tively active rhodopsin mutant, E113Q M257Y (Han et
al.1998). Consistent and high levels of mCherry fluorescencewere
observed, regardless of the presence of retinal chromo-phore
(Figure S3), indicative of productive rhodopsin expres-sion and the
ability to activate the yeast mating pathway.
Wild-type human rhodopsin was then expressed using thesame
strain, and, when incubated with retinal, induced theexpression of
mCherry only in response to light (Figure 2);5 mM 9-cis retinal in
culture media was sufficient to elicit thislight-dependent
response—a concentration also used for theheterologous expression
of rhodopsin using mammalian cells(Opefi et al. 2013). The 5- to
10-fold increase in mCherryfluorescence in response to GPCR
activation was comparableto previous reported activation of the
natural mating path-way GPCR Ste2 (Ishii et al. 2008; Kompella et
al. 2017).
600 B. M. Scott et al.
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Increasing the retinal concentration did not increase
activa-tion of the mating pathway, indicating rhodopsin
moleculeswere saturated with chromophore (Figure S4).
However,mating pathway response was enhanced when retinal wasadded
after each hourly light exposure, to account for thelack of retinal
recycling enzymes in yeast.
Magnitude of light-activated signal transduction inyeast
comparable to assays of rhodopsin expressed inmammalian cells
After establishing light-dependent activation of rhodopsin
inyeast, we next sought to compare these new yeast-basedmethods to
traditional in vitro methods utilizing protein pu-rified from
mammalian cells. Previously characterized rho-dopsin mutations
P23H, M39R, and G51A were specificallychosen to establish a
gradient of phenotypic severity. P23His the most common
RP-associated rhodopsin mutation inNorth America (Dryja et al.
1990; Mendes et al. 2005), andhas been characterized in a number of
cell and animal mod-els. P23H rhodopsin consistently displays poor
stability(Krebs et al. 2010; Chen et al. 2014), aggregation in the
ER(Chiang et al. 2012b), and disrupted transducin activation(Opefi
et al. 2013; Chen et al. 2014), leading to severe
retinaldegeneration (Cideciyan et al. 1998; Athanasiou et al.
2017;LaVail et al. 2018). The less severe M39R mutation, which
isalso associated with RP, has been studied using both bovineand
human rhodopsin genes displaying a more severe cyto-solic
aggregation phenotype in the human gene background(Davies et al.
2012; Ramon et al. 2014). As M39R rhodopsinis expressed more
productively by mammalian cells thanP23H rhodopsin, and a
proportion remains able to forma light-responsive complex with
retinal, it was selected
as an intermediate RP-associated rhodopsin mutation (Davieset
al. 2012; Ramon et al. 2014). G51A is the most commonnonsynonymous
rhodopsin mutation in humans (Lek et al.2016), displays a less
severe phenotype in vitro and in pa-tients (Cideciyan et al. 1998;
Bosch et al. 2003), and may bean asymptomatic variant (Athanasiou
et al. 2018). Thesethree rhodopsin mutants were each expressed
using themCherry reporter yeast strain. Following exposure to
light,the relative mCherry fluorescence was observed as
follows:P23H , M39R , G51A = WT (Figure 3A).
Next, we compared rhodopsin activation in yeast to tradi-tional
in vitromethods for determining rhodopsin function inresponse to
light. The same three mutants were purified fol-lowing heterologous
expression in mammalian cells, thenregenerated with retinal. By
recording the absorption spectrabefore and after exposure to light,
difference spectra showingthe response of rhodopsin to light could
be measured. Thismethod has been used extensively to characterize
missensemutations suspected to cause inherited retinal disease, as
ameasure of the ability of rhodopsin to properly fold and re-spond
to light (Sung et al. 1991; Opefi et al. 2013). Therelative
response to light displayed a similar range of functionas the
yeast-expressed mutants (Figure 3B). This suggestednot only that
the function of yeast-expressed rhodopsin wassimilarly impacted by
pathogenic mutations, but also that therelative activation of the
mating pathway in yeast is compa-rable to the severity of the
mutant as measured in vitro usingrhodopsin purified from mammalian
cells.
Pathogenic mutations known to disrupt rhodopsinstability or G
protein coupling prevent light-activatedsignal transduction in
yeast
After establishing yeast as a platform for quantifying
light-dependent rhodopsin activation, we investigated a largerpanel
of rhodopsin mutations to understand how the widerangeofknown
functionalphenotypes translates toa responsein yeast.
Effortshavebeenmade to classifymutationsbasedonthese phenotypes,
which range from completely inactive toconstitutively active
(Figure S5 and Table S2), as discussed indetail in a recent review
(Athanasiou et al. 2018). However, asthis new yeast assay examines
rhodopsin signaling in an engi-neered cellular system, it was
unknown how results wouldcompare to traditional in vitro methods
using purified mam-malian cell-expressed rhodopsin. We first
focused on patho-genic mutations known to disrupt rhodopsin folding
andstability, as we hypothesized these loss-of-function
mutationswould be easier to distinguish from wild type. These
muta-tions were placed into three groups based on previous
char-acterization: mutations intrinsically disrupting
rhodopsinstability; mutations indirectly affecting stability by
disruptinga post-translational modification (glycosylation) motif;
ormutations disrupting G protein coupling, leading to constitu-tive
endocytosis (Class 3).
When expressed in yeast and exposed to light, rhodopsinfunction
was significantly disrupted by each of the mutationsknown to result
inmisfolding or instability, with the exception
Figure 1 Representation of the engineered mating pathway.
Rhodopsinactivation was functionally coupled to the expression of a
fluorescentreporter protein, mCherry, utilizing the
mating-responsive promoterpFUS1. Modifications to the mating
pathway included the knockout ofnegative regulator Sst2, the gene
encoding Far1, which halts cell growthin the wild-type mating
pathway, and the endogenous mating pathwayGPCR Ste2. The chimeric G
alpha protein (Gpa1-Gat) contains the fiveC-terminal amino acids of
the G alpha subunit of human transducin (Gat).
Rhodopsin-Coupled Yeast Signaling 601
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of D190N (Figure 4A). D190N had previously been shown tobe a
less severe RP-linked mutation (Fishman et al. 1992;Tsui et al.
2008; Liu et al. 2013), and ERG data from patientsmatches our
observation that this missense mutation doesnot completely disrupt
rhodopsin function (Sancho-Pelluzet al. 2012). The G89D and L125R
mutations also had a re-duced but measurable response to light when
expressed inyeast, which fits with previous trends observed
(Kaushal andKhorana 1994; Bosch et al. 2003). Interestingly, L125R
leadto signaling in the dark as well, equivalent to the
mutant’slight-dependent activation, which has not previously
beenreported.
Mutations in the N-terminal cap of rhodopsin (V20G,P23H, and
Q28H) have been functionally characterized indetail, and are known
to poorly activate transducin in re-sponse to light (Opefi et al.
2013). Similarly,mutationsM39R,N55K, G106W, C110Y, G114D, and P171L
have each beenshown to disrupt or prevent productive formation of a
opsin-retinal complex (Davies et al. 2012; Ramon et al. 2014;
Sunget al. 1991, 1993; Hwa et al. 1999; Andrés et al. 2003),
whichmatched our observation that light-activated signal
transduc-tion was significantly impaired in yeast.
Mutations T4K, N15S, and T17M prevent glycosylation atresidues
N2 and N15, resulting in a severe reduction inrhodopsin stability
(Kaushal et al. 1994; Opefi et al. 2013).The NXS/T glycosylation
consensus sequence is recognizedacross eukaryotes (Lam et al.
2013), and a previous studyindicated that yeast-expressed bovine
rhodopsin was glyco-sylated (Mollaaghababa et al. 1996). The
reduced stability ofunglycosylated rhodopsin is known to prevent
productivetransducin activation in vitro (Opefi et al. 2013), and
compa-rable reductions in signaling were observed in our yeast
assay. A general trend of T4K (40% wild-type activity) beingless
severe than N15S (4%) and T17M (15%) was observed,similar to
previous studies, which indicated glycosylation ismore important on
N15 than it is on N2 (Kaushal et al. 1994;Tam and Moritz 2009;
Opefi et al. 2013).
Of the rhodopsin mutations we studied with impairedsignaling,
R135G is unique as it does not cause misfoldingand it does not
prevent the formation of a stable complexwithretinal, when using
rhodopsin purified frommammalian cells(Sung et al. 1993). R135G
mutates the highly conservedE/DRYmotif, where R135 is the arginine
residue in this motifand is crucial for G protein coupling (Acharya
and Karnik1996; Rovati et al. 2007). In addition, when
heterologouslyexpressed in mammalian cells, R135 mutations cause
rho-dopsin to be hyper-phosphorylated, leading to aggregationwith
visual arrestin and constitutively undergoing endocyto-sis (Chuang
et al. 2004). With two unique molecular mech-anisms contributing to
pathogenicity, R135 mutants havebeen placed in their own “Class 3”
category (Chuang et al.2004; Athanasiou et al. 2018). The observed
absence of sig-naling in yeast was in line with the reported in
vitro trans-ducin activation defect using mammalian cell-derived
R135Grhodopsin (Min et al. 1993; Acharya and Karnik 1996).
How-ever, as our light-activated signal transduction assay couldnot
distinguish between disrupted G protein coupling vs. ab-errant
endocytosis, this assay alone could not determine themolecular
mechanism behind the lack of R135G signaling inyeast.
Pathogenic mutations that enhance or do not disrupttransducin
activation respond similarly in yeast
Based on the wild-type-like activity of G51A we observed
inyeast, and the reduced or inactive response of misfolded
andunstablemutants, we hypothesized that rhodopsinmutationsthat
maintain or increase light-dependent activation in vitromay behave
similarly in yeast. Constitutively active rhodop-sin mutants are
also associated with disease, causing congen-ital stationary night
blindness (CSNB) and RP (Park 2014).We specifically selected a
panel of rhodopsin mutationsto characterize in yeast where activity
was known to varygreatly, from asymptomatic, to constitutively
active, to in-creased signaling in the dark.
Across thisdiversity of function, yeast-expressed rhodopsinagain
behaved comparably to rhodopsin purified from mam-malian cells,
with both wild-type-like signals and increasedsignaling observed
depending on the mutation (Figure 4B).We grouped mutations known to
increase downstream trans-ducin activation in vitro, although this
increased activity canoccur in the light, dark, or in both states
(Park 2014). TheM44T mutation showed a significantly higher
response thanwild-type, at over 1.6-fold wild type, which matches
in vitrotransducin activation data for M44T (Andrés et al.
2003).T94I trended higher than wild type, and is also believedto
cause CSNB due to constitutive activation (119% WT sig-naling in
vitro) (Gross et al. 2003), but the increase we ob-served vs. wild
type (115% WT signaling in yeast) was not
Figure 2 Light-dependent activation of the mating pathway.
Humanrhodopsin was found to activate the mating pathway only in
responseto light, requiring the presence of retinal chromophore.
Adding retinalafter each hourly light exposure improved the overall
response �1.6-fold.Incubating with the same concentration of
retinal but keeping the culturein the dark did not result in mating
pathway activation. Vector: Yeasttransformed with a plasmid not
containing the rhodopsin gene. Datapoints represent results of four
individual colonies, each in a 5 ml culture.Error bars represent
SD.
602 B. M. Scott et al.
-
statistically significant. V137M has been reported to
activatetransducin 1.25-fold greater than wild type (Andrés et
al.2003), but we did not observe an increase in rhodopsin
ac-tivity. The V137M mutation is known to have highly
variableclinical phenotypes (Ayuso et al. 1996), and it has been
sug-gested to be an asymptomatic variant (Rakoczy et al. 2011).
Of the rhodopsin mutations with known increased activitythat we
studied, S186W is unique as it is believed to causeautosomal
dominant RP due to increased signaling in the dark(Liu et al.
2013). This is a result of reduced thermal stability ofthe inactive
dark state, where spontaneous thermal isomeriza-tion of the
chromophore leads to signaling in the dark (Liu et al.2013), a
phenomenon called “dark noise” (Luo et al. 2011).The increased
signaling that we observed in the dark forS186W, equivalent to 30%
of activated wild-type rhodopsin,fit with this proposed mechanism
of RP pathogenesis. E150Kwas also found to have significantly
elevated signaling in thedark, when expressed in yeast. This data
is in linewith amousemodel of E150K, which was found to have
elevated photore-ceptor signaling in the dark, also believed to be
due to reducedthermal stability of the dark state (Zhang et al.
2013). TheE150K mutation is associated with autosomal recessive
RP(arRP) and was previously shown to have a 1.3-fold
increasedactivation of transducin after light exposure (Zhang et
al.2013). An identical value was observed using our
yeast-basedassay but was not statistically significant.
To determine if dark state signaling in yeast was
retinal-dependent, we investigated signaling without the addition
ofretinal for selected mutants (Figure S6). Elevated dark
statesignaling appeared to be retinal-dependent for only
M44T,L125R, E150K, and S186W. This fits with the proposedmechanism
of thermal isomerization of retinal contributingto dark noise for
the E150K andS186mutants (Liu et al. 2013;
Zhang et al. 2013), while providing new insight on the M44Tand
L125R mutants.
Some rhodopsinmutationsmay cause disease bypreventingthe
formation of rhodopsin homodimers (Ploier et al. 2016),but their
pathogenicity is debated due to their relativelyhigh frequency in
sequenced human genomes (.1:80,000)(Athanasiou et al. 2018). The
F45L and V209M mutationswere found to activate the mating pathway
at wild-type lev-els when exposed to light, matching published in
vitro trans-ducin activation assays (Ploier et al. 2016). F220L was
foundto have a 1.5-fold greater response, which was
unexpectedlyhigher than the wild-type-like value reported for the
F220Cmutation (Ploier et al. 2016). Similarly, although V104I
isconsidered asymptomatic, light activation of themating path-way
was �1.3-fold greater than wild-type. This mutationdoes not
segregate with RP in genetic studies (Macke et al.1993), but
transducin activation assays have not previouslybeen performed, so
it is unclear how our results in yeast re-late to a potential human
phenotype.
Mutations in the C-terminus of rhodopsin disrupt traffick-ing to
the rod outer segment (ROS), but do not affect traf-ficking in
other mammalian cell types and do not affecttransducin activation
in vitro (Sung et al. 1994), thereforewe did not expect their
function to differ from wild-typerhodopsin in yeast. Interestingly,
V345M significantly af-fected light-activated signaling in yeast,
despite the mutationoccurring in the C-terminus, which is not
believed to be re-quired for G protein activation. A study of
transducin activa-tion using V345M rhodopsin purified from
mammalian cellshas not been performed to compare to our yeast
results.
There were two examples where light-activated signalingin yeast
differed from reported in vitro results using rhodopsinpurified
from mammalian cells. The A292E mutation is
Figure 3 Characterization of rhodopsin light-dependent function.
(A) Response to light from yeast-expressed rhodopsin mutants,
indicating a similarmagnitude of response as the mammalian
cell-expressed protein. WT: Wild-type human rhodopsin. Yeast data
points represent results of nine individualcolonies, each in a 600
ml culture, minus the mCherry fluorescence of the same strain
transformed with empty plasmid control (Vector), and normalizedto
wild-type. * P , 0.05 vs. WT or between indicated mutants. (B)
Difference spectra of mammalian cell-expressed rhodopsin mutants in
response tolight. The peak at 500 nm indicates a light-dependent
response.
Rhodopsin-Coupled Yeast Signaling 603
-
associated with CSNB, and has constitutive activation in vitroin
the absence of retinal, but reduced light-dependent trans-ducin
activation when retinal is supplied (Gross et al. 2003).In our
yeast signaling assay, a 2.1-fold increase in light-activated
signaling vs. wild type was observed, greater thanany other
mutation we studied. A292E signaling in the darkwith retinal added
was not significantly increased, and wasequivalent to not adding
retinal. G90D, another constitutivelyactive mutant associated with
CSNB (Rao et al. 1994), acti-vated the mating pathway at only 44%
of wild-type response.
This result was similar to one transducin activation studyusing
G90D (59% of wild type) (Zvyaga et al. 1996), whileothers have
shown wild-type-like responses to light but con-stitutive
activation in the absence of retinal, similar to A292E(Rao et al.
1994; Gross et al. 2003).
Light-activated signal transduction in yeast correlateswith
published assays of rhodopsin function
Of the 33 mutations and controls studied, 23 had previ-ously
reported measurements of light-dependent activation of
Figure 4 The light-activated signal transduction of pathogenic
rhodopsin mutants in yeast. (A) Missense mutations that
intrinsically disrupt rhodopsinstability, or that indirectly affect
stability by disrupting a post-translational modification (PTM)
motif. Class 3 denotes a unique category of mutations atsite R135,
which disrupt G protein coupling and lead to constitutive
endocytosis in mammalian cells. (B) Pathogenic and asymptomatic
variants thatincrease or do not disrupt rhodopsin activation by
light. WT: Wild-type human rhodopsin; Vector: yeast transformed
with a plasmid not containing therhodopsin gene; arRP: autosomal
recessive RP. Data points represent results of nine individual
colonies, each in a 600 ml culture, minus the mCherryfluorescence
of Vector, and normalized to wild-type. Error bars represent the
95% CI, * P , 0.05 vs. WT.
604 B. M. Scott et al.
-
transducin in vitro, or measurements of photoreceptor activ-ity
by ERG (Table S2). When plotted together, our yeast-based
measurements closely matched this available data onrhodopsin
signaling, approaching a 1:1 ratio (Figure S7).This held true for a
diverse array of phenotypes, ranging frompathogenic due to
inactivation, or pathogenic due to consti-tutive activation, to
asymptomatic. A292Ewas found to be anoutlier from this trend. The
rate of light-dependent transdu-cin activation has been reported at
�80% of wild type, al-though this mutant has constitutive
activation in the absenceof retinal in vitro (Gross et al. 2003).
As signaling in yeastoccurs in a cellular context, vs. traditional
in vitro assays thatuse purified protein, the increase in
light-activated signalingwe observed for A292E may have been a
combination ofsignaling both with and without retinal bound, or may
rep-resent a unique signaling state in yeast. Overall, however,
thegeneral trend strongly supports the use of yeast to
quantifyrhodopsin-mediated G protein signaling in response to
light,as the yeast-based assay was comparable to more
laboriousassays for characterizing patient derived mutations.
Subcellular localization of rhodopsin is comparablebetween yeast
and mammalian cells
As the majority of disease-linked rhodopsin mutations causethe
receptor to misfold (Athanasiou et al. 2018), comparingthe
subcellular localization of rhodopsin using mammaliancells is a
common technique to study protein trafficking andER retention, and
is predictive of pathogenicity (Sung et al.1991; Behnen et al.
2018). To establish phenotypes in mam-malian cells to compare to,
we again used the rhodopsinmutations P23H, M39R, and G51A to create
a gradient ofphenotypic severity. Rhodopsin mutants were expressed
inSK-N-SH neuroblastoma cells with a C-terminal GFP tag thathas
previously been shown to not affect rhodopsin stabilityin vitro or
in vivo (Moritz et al. 2001)
The P23Hmutation has been characterized in a number ofcell
models, consistently showing poor plasma membraneexpression
regardless of cell type (Sung et al. 1991; Chianget al. 2012b,
2015). When expressed in SK-N-SH neuro-blastoma cells,
immunocytochemistry revealed that P23Hrhodopsin did not localize to
the plasma membrane, formingaggregates in the cytosol and
colocalized with an ER-specificmarker (Figure 5A and Figure S8).
M39R rhodopsin dis-played a nearly wild-type phenotype,
colocalizing with aplasma membrane marker but with some evidence of
mutantrhodopsin retained within the cell (Figure S8), similar to
aprevious study (Davies et al. 2012). G51A displayed
robustwild-type-like localization on the plasma membrane,
consis-tent with a recent report (Behnen et al. 2018). This
compar-ative range of subcellular localization matched what we
hadpreviously observed in assays of rhodopsin function for
thesethree mutations.
We next sought to determine if the same rhodopsin mu-tants
expressed in yeast trafficked to the plasmamembrane ina similar
manner. The same yeast strain used to functionallycouple human
rhodopsin to the mating pathway was used to
express select rhodopsin mutants with GFP fused to
theC-terminus. Similar techniques have been used to inves-tigate
productive expression of other human GPCRs inyeast, by observing
expression on the plasma membraneor the presence of aggregates
(O’Malley et al. 2009). Mir-roring our mammalian cell-based
observations, P23H andM39R were poorly distributed across the yeast
plasmamembrane, with highly localized “patchy” expression,while
G51A localized consistently to the membrane (Fig-ure 5A). The
peri-nuclear ring observed is similar to thelocalization of other
human GPCRs expressed in yeast,indicating the ER membrane (O’Malley
et al. 2009; Hashiet al. 2018). Although P23Hwas observed to form
aggregatesin the ER of our mammalian cells, this was not observed
inyeast; however, M39R did appear to form aggregates in thecytosol
of yeast.
We expanded this microscopy-based analysis to
additionalrhodopsin mutants expressed in yeast (Figure S9).
Imageanalysis software was used to quantify rhodopsin
distributionon the plasma membrane of each yeast cell,
identifyinglocalized regions or “patches” where rhodopsin appeared
toaggregate (Figure 5B). Wild-type rhodopsin was observed tohave a
low number of cells displaying a “patchy” phenotype,suggesting even
distribution across the plasma membrane.Somemutants
associatedwithmisfolding or reduced stability(i.e., N15S, M39R,
L125R) exhibited incomplete distributionon the plasma membrane,
characterized by a “patchy” phe-notype. Mutations in the C-terminus
of rhodopsin (Q344ter,V345M, P347L) disrupt the VXPX motif, which
is crucial fortrafficking rhodopsin in photoreceptors (Wang and
Deretic2014). However, mutations within this motif do not
affectrhodopsin localization in mammalian cells that are not
pho-toreceptors (Sung et al. 1991, 1993), and were not found
toaffect rhodopsin localization in yeast. In general, these
find-ings indicated that rhodopsin maintains its subcellular
local-ization when expressed in yeast, which is likely dependent
onrhodopsin folding and stability, just as it is with
heterologousexpression in mammalian cells.
The yeast unfolded protein response is upregulated bymisfolded
rhodopsin mutants
Afterdiscovering that the subcellular localizationof
rhodopsinand changes in responses to light were preserved in yeast,
weinvestigated additional molecular pathways known to beaffected by
pathogenic rhodopsin mutations. Rhodopsin mu-tations P23H and T17M
have been shown to activate the UPRin mammalian systems, indicative
of severe misfolding (Linet al. 2007; Kunte et al. 2012). P23H has
been shown topreferentially activate the IRE1 UPR pathway in
mammaliancells (Chiang et al. 2015)—a pathway also present in
yeast.Similar to mammalian IRE1, yeast IRE1 serves as a sensor
ofmisfolded protein in the ER, which activates the
transcriptionfactor HAC1 (Kimata and Kohno 2011). Yeast strains
andplasmids have been devised utilizing a HAC1-responsive pro-moter
to express reporter genes, which have been used topredict
productive expression of other human GPCRs in yeast,
Rhodopsin-Coupled Yeast Signaling 605
http://www.yeastgenome.org/locus/S000001121/overviewhttp://www.yeastgenome.org/locus/S000001863/overviewhttp://www.yeastgenome.org/locus/S000001863/overview
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where greater UPR activation was associated with GPCRmisfolding
and aggregation (O’Malley et al. 2009). Dueto the conserved
pathway, and knowing results established
with other GPCRs, we hypothesized that the severity ofmisfolded
rhodopsin could be quantified using a yeast-based sensor of UPR
upregulation. The strain designedby Jonikas et al. (2009) has an
additional gene cassetteconstitutively expressing RFP, which can be
used to correctfor changes in global protein expression (Jonikas et
al.2009). We used this strain to study the 33 selected rho-dopsin
mutations, plus controls, to quantify their effect onUPR
upregulation.
Expressed in the UPR-reporter strain, P23H and T17Mrecapitulated
the expected elevated UPR activation in yeast,in addition to
several other mutations known to disruptrhodopsin stability (Figure
6). T4K and N15S, which likeT17M disrupt glycosylation, also
upregulated the UPR, sug-gesting a crucial stabilizing nature of
these posttranslationalmodifications. C110Y prevents the formation
of a criticaldisulfide bond in rhodopsin, severely impacting
stabilityand function (Hwa et al. 1999), and the observed
increasedUPR in yeast matches in silico predictions that this
mutant ishighly unstable (Rakoczy et al. 2011). Elsewhere in
trans-membrane helix three, the G114D and R135G mutationssimilarly
increased the UPR. In mammalian cells, R135G ishyper-phosporylated
and aggregates in endosomes (Chuanget al. 2004), but accumulation
in the ER or activation of themammalian UPR has not been reported.
That R135G acti-vated the yeast UPR suggests that this mutant may
be mis-folded in yeast.
The constitutively active mutant A292E showed an upre-gulation
in the UPR that has not previously been reported,which may be due
to the replacement of a small unchargedresidue with a large
negatively charged residue proximal tothe retinal binding pocket. A
reduced UPR for mutationsM44T, E150K, and V345M compared to wild
type was ob-served, but the physiological relevance is unclear.
Interest-ingly, culturing in selective media alone was sufficient
toupregulate the UPR, revealed by the difference between thevector
control and the untransformed strain grown in richmedium. A log2
GFP/RFP ratio of�1.0 was previously shownto indicate moderate UPR
upregulation (Jonikas et al. 2009),which was observed for the
Vector control prior to normaliz-ing the data. Expressing wild-type
rhodopsin gave a similarvalue, which suggested baseline UPR
upregulation was dueto growth in selective media and not to the
overexpression ofrhodopsin.
Yeast assays of rhodopsin mutations V81D, A164E, andA164V found
in retinal disease patients
Having established a suite of new yeast-based techniques
toinvestigate rhodopsin functional and molecular phenotypes,we
applied these approaches to study three rhodopsin muta-tions found
in degenerative retinal disease patients diagnosedwith RP. One of
the rhodopsin mutations, V81D, is a newmutation that has not been
previously reported (Case 1),and two of the other mutations are
previously reported buthave had little (A164V, Case 2), or no
(A164E, Case 3) ex-perimental characterization with respect to
those rhodopsin
Figure 5 Representative subcellular localization of rhodopsin
mutants (A)Comparative subcellular localization of rhodopsin
mutants in SK-N-SHand yeast cells. PM merge: Fluorescence of a
plasma membrane marker,merged with GFP-tagged rhodopsin. Images
represent maximal projec-tions, with scale bars representing 30 mm
and 5 mm respectively. (B)Quantified localization of GFP-tagged
rhodopsin mutants on the yeastplasma membrane. Yeast cells with
patchy membrane expression of rho-dopsin had at least 10% of their
cell membrane containing separatedpatches of rhodopsin. Error bars
represent the bootstrapped SE.
606 B. M. Scott et al.
-
mutations. Missense mutations A164E and A164V have
beenpreviously linked to autosomal dominant RP (Fuchs et al.1994;
Hwa et al. 1997), but the molecular mechanism un-derlying the
disruption of rhodopsin function at this site re-mains to be
elucidated. Studies of A164V suggest that helicalpacking may be an
issue (Hwa et al. 1997; Stojanovic et al.2003), but the effects of
introducing a charged residue at thissite have not yet been
investigated. Mutations at the sameresidue in rhodopsin can have
highly heterogenous pheno-types (Bosch et al. 2003), so we sought
to compare Al64E toA164V in greater detail. We identified a new
V81Dmutationin a patient (Case 1)with early-onset autosomal
dominant RP(Table S3). This mutation completely removes the V81
codonfrom the rhodopsin DNA sequence, resulting in a deletedamino
acid in the central part of the second transmembranedomain (Figure
7A). Such an amino acid deletion could dis-rupt alpha helix
formation and stability in the membrane,and is likely to lead to a
severe molecular phenotype forV81D rhodopsin.
We characterized V81D, A164E, and A164V using theyeast-based
methods for investigating rhodopsin molecularphenotype and
function, with experiments conducted in thesamemanner as previously
described (Figure 7, B–E). Plasmamembrane localization of all three
mutants were poor, with28–40% of yeast cells displaying patchy
expression of rho-dopsin. Light-activated signal transduction in
yeast was com-pletely abolished for each, similar to the other
mutants westudied known to be unstable or misfold. UPR
activationwas elevated for all three mutants, with A164E higher
thanany other rhodopsin mutant we studied. Together, our re-sults
suggest that all three of these mutants have a severephenotype in
yeast, based on decreased function, subcellularlocalization, and
UPR activation, where we may expect adifference in severity between
the A164 mutants based onUPR activation.
Yeast assays comparable to mammalian cell data forrhodopsin
mutations V81D, A164E, and A164V
We compared our yeast-based assays of V81D, A164E, andA164V to
expression in mammalian cells. A164V colocalizedwith the plasma
membrane marker, suggesting a less severephenotype in mammalian
cells, which contrasted with theyeast data (Figure 8A). However,
A164E and V81D werecompletely retained inside mammalian cells,
colocalizingwith the ER marker, indicating they were severely
misfolded.Following immunoaffinity purification from mammalian
cells,the three RP-associated mutations also showed a range intheir
response to light (Figure 8B). Heterologous expressionof V81D
produced no functional protein, demonstrating avery severe
phenotype. A164E, while not as severe a pheno-type as V81D,
expressed poorly and produced limited func-tional protein. A164V
expression did produce a high amountof functional protein,
consistent with another in vitro study ofthis mutation (Stojanovic
et al. 2003). The sum of this cellu-lar and functional data
suggested the same functional trendwe first predicted using
yeast-based methods, although therewas more evidence of functional
A164V protein in mamma-lian cells.
Patient clinical data supports functional trend predictedby
yeast and mammalian cells
Next, we looked at patient clinical data. The
comparativeseverity in vitro was found to follow the same trend as
avail-able patient phenotype information (Table S3). The mostsevere
phenotype was exhibited by the patient with theV81D mutation (Case
1). This patient first had symptoms�10 years of age, with
difficulty adapting to a dim litenvironment (nyctalopia). This
slowly progressed, and at39 years she has moderate visual acuity
loss (20/50), mildlyabnormal color vision, constriction of the
visual field to thecentral 5�. At age 26 years, electroretinography
already
Figure 6 UPR activation in yeast relative to wild-type
rhodopsin. GFP expression is a reporter of UPR upregulation, while
RFP is expressed constitutivelyto help correct for changes in
global protein expression. WT: Wild-type human rhodopsin; Vector:
yeast transformed with a plasmid not containing therhodopsin gene;
Strain: the yJW1200 strain not transformed with plasmid and grown
in rich media; arRP: autosomal recessive retinitis pigmentosa.
Datapoints represent results of nine individual colonies, each in a
600 ml culture, normalized to wild-type. Boxes extend from the 25th
to 75th percentile, theline across the box represents the median
value. Bars represent the min and max recorded values. * P , 0.05
vs. WT.
Rhodopsin-Coupled Yeast Signaling 607
-
documented severe reduction of rod and cone function.
Thephenotype of the patient with the A164Vmutation (Case 2)
ismilder than that of the patient with the A164E mutation.Although
Case 2 had symptoms of nyctalopia since child-hood, the progression
of his disease was extremely slow. Atage 64 years his
electroretinogram was recordable and onlymildly abnormal. His
central visual acuity at 67 years was20/40 and, despite a
paracentral scotoma (area of decreasedvision), he maintained a
peripheral field. In contrast, the pa-tient with the A164E mutation
(Case 3) has a good centralvisual acuity and normal color vision at
age 53 years. How-ever, her paracentral scotoma were more severe
and pro-gressed to form an annular scotoma at the age of 53
years.Unlike Case 1 (V81D), she also preserved some good
periph-eral field of vision at the age of 45 years, and her ERG
wasonly moderately abnormal.
The V81D patient is the youngest of the threewith a
highlyreduced retinal function, while the A164V patient is the
el-dest of the three with the best retinal function (Figure
9).A164E again appeared intermediate to both. These results
show that the overall trend of clinical severity was
accuratelypredicted by combining both sets of yeast-derived and
mam-malian cell-derived data. The yeast methods provided
addi-tional information on UPRupregulation, which also supportedthe
difference in severity between A164E and A164V mu-tations, while
being less labor intensive than mammalianmicroscopy and expression
methods.
Discussion
Yeast provide new methods to investigate rhodopsinstructure and
function
In this study, we have engineered the yeast S. cerevisiae
tocharacterize both known and novel pathogenic mutations ofthe
visual pigment rhodopsin. In comparing our new assaysto traditional
mammalian cell-based approaches, we demon-strate that the molecular
phenotypes of this light-activatedhuman GPCR are similar in yeast,
and that these phenotypesreflect patient clinical data. There are a
number of advantagesand differences when compared to traditional
techniques,as these yeast-based rhodopsin assays are performed in
acellular context, which provides a new perspective on
signaltransduction pathways, subcellular localization, and
UPRupregulation.
A direct measurement of downstream pathway activationin response
to light was achieved by functionally couplingrhodopsin to the
yeast mating pathway. This required pro-ductive in vivo activation
of a G protein, mimicking the initialstep that occurs in human
photoreceptors, even if down-stream signaling differs. By using
yeast, many rhodopsin mu-tations could be studied in the same
experiment, withmultiple replicates, without the laborious
purification stepsthat are traditionally required for in vitro
transducin activa-tion assays (Reeves et al. 1996). Not only did
yeast-expressedrhodopsin maintain its ability to respond to light,
but themagnitude of signal pathway activation was comparable tomany
previous studies of mutations expressed in mammaliansystems. We
were also able to observe rhodopsin mutationsthat resulted in the
activation of dark state rhodopsin, whichhas previously required
sensitive spectroscopic assays usingimmunoaffinity purified
rhodopsin (Liu et al. 2013), or geneknock-in animal models (Zhang
et al. 2013). This includedproviding new data on L125R signaling in
the dark, a mutantpoorly expressed in mammalian cells, which has
made pre-vious characterization of function challenging
(Stojanovicet al. 2003). Mutations at site L125 have been shown to
re-duce the thermal stability of rhodopsin (Andrés et al.
2001),which could lead to dark noise through thermal isomeriza-tion
of the bound chromophore (Luo et al. 2011). This mech-anism was
supported by our finding that L125R dark noisewas retinal-dependent
in yeast, which could be a result ofthermal isomerization. Thus,
yeast may serve as a platformfor studying difficult-to-express
rhodopsin proteins, allowingthe rapid quantification of downstream
G protein activationunder various conditions.
Figure 7 Characterization of novel rhodopsin mutants using
yeast. (A)Crystal structure of rhodopsin, highlighting residues V81
and A164 in red.The other rhodopsin mutants characterized in this
study are highlighted incyan, and the approximate location of the
membrane is indicated by thedotted line (1U19.pdb). Quantified
phenotype and functional assays ofV81D, A164E, and A164V in yeast
(B) Representative subcellular locali-zation, Bar, 5 mm, (C)
quantified consistency of rhodopsin localization onthe yeast plasma
membrane, (D) light-activated signal transduction, and(E) UPR
activation. WT: Wild-type human rhodopsin. The number of
bi-ological replicates and error bars are identical to previous
figures. * P ,0.05 vs. WT in all panels unless otherwise
indicated.
608 B. M. Scott et al.
-
Mutations known to disrupt rhodopsin folding or stabilitytended
to have incomplete localization on the yeast plasmamembrane,
similar to mammalian-cell-expressed rhodopsin.Although there was
significant heterogeneity between theinvestigated mutants, this
observation suggests that thebiochemical properties of rhodopsin
were conserved inyeast, despite known differences in glycosylation
patterns(Mollaaghababa et al. 1996). We quantified membrane
local-ization using a novel automated image analysis
procedure,which may be useful for studies of other GPCRs and
associ-ated pathogenic mutations when expressed in yeast. How-ever,
not all human GPCRs are expressed productively inyeast (O’Malley et
al. 2009), so these methods should firstbe validated with the
wild-type receptor.
The majority of disease-linked rhodopsin mutations thathave been
identified cause the protein to misfold, which can ac-tivate theUPR
in the ER, and eventually lead to photoreceptor
cell death (Athanasiou et al. 2018). Modulating the UPRhas been
investigated as a potential treatment for RP (Tamet al. 2010;
Chiang et al. 2012a; Parfitt et al. 2014), butthe effect on UPR
upregulation had only been determinedfor three rhodopsinmutations
(Lin et al. 2007; Kunte et al. 2012;Marsili et al. 2015).
Determining UPR upregulation by mu-tant rhodopsin has previously
required microscopy, immuno-blot, and qPCR methods (Kunte et al.
2012; Marsili et al.2015). We took advantage of an engineered yeast
strain thatpossesses a reporter linked to the IRE1UPR pathway,
which isthe only one of three UPR pathways that is conserved
be-tween mammals and yeast (Kimata and Kohno 2011). Thisyeast-based
reporter of UPR activity enables the use of flowcytometry, a more
simple and high-throughput method, andoffers the advantage of
measuring UPR upregulation directlyin live cells. We found that
upregulation of this pathway wasassociated with certain rhodopsin
mutants known to misfoldor with reduced stability, which may be
predictive of themolecular mechanism contributing to retinal
degeneration.
However, yeast do not contain the PERK and ATF6 UPRpathways
found in mammalian cells (Kimata and Kohno2011). Thus, although the
yeast-based methods provide in-sight into UPR upregulation, these
studies would need to becombinedwithmammalian cells to better
understand how allUPR pathways may be affected by rhodopsin and
other GPCRmutations. This may also explain why nine of the 15
mutantsthat are believed to misfold did not cause an increase
UPRactivation in yeast, suggesting significant heterogeneity
be-tween rhodopsin mutations. Determining the contribution
amissense mutation makes to UPR upregulation is highly rel-evant to
pharmacogenomics, as an inability of rhodopsin torespond to light
may not necessarily indicate that the patho-genic mutation can be
rescued by UPR modulation.
Overall, many known rhodopsin phenotypes were recapit-ulated in
yeast, a requirement for any assay of human genefunction seeking to
determine the clinical relevance of patientderived missense
mutations (Amendola et al. 2016). Thereare also important
differences to consider when comparingour yeast-based assays to
mammalian cell-based assays. Im-portantly, when a lack of signaling
is observed in yeast, it isdifficult to separate rhodopsin mutants
that misfold frommutants that fold properly but do not productively
activatethe downstream pathway. Although our quantified micros-copy
data supported the notion that rhodopsin mutants withinconsistent
plasma membrane expression in yeast are alsomutants known to be
unstable or misfolded, unique aspectsof yeast cellular machinery or
post-translational processingmay influence these mutants. Results
from the R135G muta-tion highlight these differences, where this
mutant activatedthe UPR in yeast but does not aggregate in the ER
of mam-malian cells (Chuang et al. 2004).
The signaling phenotypes of the G90D andA292Emutantsin yeast
also differed from the reported constitutive activityin vitro when
using purified protein (Gross et al. 2003).That A292E signaled
higher and G90D lower than expectedsuggests that constitutive
activity in yeast is dependent on
Figure 8 Characterization of novel rhodopsin mutants using
mammaliancell expression. (A) Comparative subcellular localization
of rhodopsin mu-tants in SK-N-SH cells. PM: Fluorescence of a
plasma membrane marker;ER: fluorescence of a ER-specific marker.
Bar, 30 mm. (B) Difference spec-tra of mammalian cell-expressed
rhodopsin mutants in response to light.The peak at 500 nm indicates
a light-dependent response.
Rhodopsin-Coupled Yeast Signaling 609
http://www.yeastgenome.org/locus/S000001121/overview
-
Figure 9 Clinical assessment of patients with rhodopsin
mutations V81D, A164E, and A164V. (A) Goldmann visual fields of the
right eye at two timepoints. Normal fields would reach the gray
dotted line. The solid blue line outlines the actual field. The
hatched areas are scotoma, i.e., areas of loss insensitivity.
Darker areas refer to denser scotoma. (B) Structural retinal
phenotype of the right eye from cases carrying the A164E and V81D
mutations.Optical coherence tomography (OCT) above showing the
different retinal layers. Brackets show area of preserved outer
retina; A164E . V81D. Unlikefor A164E, the OCT of V81D shows
disturbed lamination of the retina with degenerative cysts,
reflecting more advanced disease. The retinalphotograph below
centered on the posterior pole. Photograph on the right is taken
with a wider field camera. ON: Optic nerve. The dotted whiteline
indicated the foveal area at the center of the macula. Double white
arrow indicates vessel attenuation, while single arrow shows
typical pigmentarydeposits (few in these cases). The width of the
central visual field corresponds to the area of preserved outer
retina on the OCT.
610 B. M. Scott et al.
-
cellular conditions that would not be revealed in an in
vitroassay using purified protein, such as a renewing supply ofboth
rhodopsin and G protein. It is interesting to note, how-ever, that
A292E has the highest constitutive activity reportedof a
CSNB-associated mutation in vitro (Gross et al. 2003),which fits
the trend observed in yeast. That light-dependentsignaling in yeast
was affected by the V104I, F220L, andV345M mutations was
unexpected, which provides interest-ing new data for these
previously uncharacterized mutantsthat should be followed up in
mammalian cell-based assays.Thus, these yeast-based methods provide
complimentary butindependent data to traditional mammalian-cell and
in vitrobiochemical techniques, offering a unique perspective on
rho-dopsin structure and function in a cellular context.
Characterizing novel rhodopsin mutations with yeast
Determining mutant function rapidly and accurately has be-come
increasingly important with the rise of whole genomesequencing, and
the ever-expanding rise in gene mutationswith an unknown impact on
human health. Rhodopsin mu-tations linked to inherited retinal
disease have been used asexamples of howmany genemutations
discovered in patientsare rarely characterized, and that the
molecular basis forpathology is poorly understood (Davies 2014;
Chiang andGorin 2016). Animal models and traditional in vitro
assaysprovide detailed information, but they have not kept pacewith
the hundreds (.350) of rhodopsin mutations identifiedto date. This
is true of many other genetic diseases, but newmethods of
functional characterization are helping to addressthis (Starita et
al. 2017), including using yeast-based assays(Sun et al. 2016; Yang
et al. 2017).
We investigated the use of yeast to characterize novel
andunderstudied pathogenic mutations, and compared to clinicaldata
for patients with varying severity of RP. Our yeast-basedapproaches
predicted severe phenotypes for the V81D andA164 mutations, which
included determinations of their light-activation, subcellular
localization, and UPR upregulation. Bycombining yeast and mammalian
cell-based assays, the rela-tive severity of these mutations was
revealed, as comparedwith clinical phenotypes measuring decline in
visual functionin patients. The V81D rhodopsin mutation showed the
mostsevere phenotype in our combined assays and the most ex-tensive
visual deterioration clinically, in contrast to A164V,which had the
mildest phenotype both clinically and experi-mentally, and A164E,
which was found to be intermediate.
The difference in severity for the two missense mutationsfound
at site 164 highlights the heterogenic nature of RP, andthe
importance of characterizing individual disease pheno-types. These
results indicate that yeast-based approachescould be useful not
only for investigating the molecular basisof retinal disease, but
also for better prediction of mutationpathogenicity, to help
improve the accuracy of prognoses forpatients associated with
specific mutations in rhodopsin. In-tegrating the results of both
UPR and light-activation assays(Figure S10) may also help determine
the molecular mech-anism of disease, to differentiate between
mutations that
cause severe misfolding, vs. disruptions in retinal binding
orstability that prevent activation but do not cause cell
stress.
Recent successes in gene therapy for inherited retinaldisease
means that mutation classification is of utmost im-portance, to
determine if such therapy is required (U.S. Foodand Drug
Administration 2017). This issue is particularlyimportant to
address for degenerative diseases, such asinherited retinal
disease, where early intervention is crucial.A method to determine
the functional consequences of mu-tations throughout rhodopsin,
rapidly and accurately, wouldtherefore be highly beneficial.
The methods presented here could also extend to func-tionally
characterizing mutations of many other GPCRs. In-deed, although
over 30 human GPCRs have been functionallylinked to the yeast
mating pathway, no previous yeast-basedstudy has focused on direct
functional characterization ofhuman GPCR mutations. As discussed in
a recent review ofGPCR pharmacogenomics, characterizing GPCR
mutationscould lead to a better understanding of disease and
drugresponses in patients (Hauser et al. 2018). Missense muta-tions
that modulate interactions with downstream signalingand regulatory
proteins are known to play a role in this, soassays that accurately
reflect GPCR function and interactionsin a cellular context, such
as the yeast assays presented here,will be key to understanding the
impact of GPCR mutationson human health.
Acknowledgments
This study was supported by NSERC Discovery Grants toB.S.W.C.
(RGPIN-2015-06279), and S.P (RGPIN-2015-05114),and a Canada
Foundation for Innovation Grant to S.P.(#34473).
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