Opsin Ubiquitin Merge

COS

CIS

ONL

COS

CIS

ONLM-o

psin

S-o

psin

P18

M-o

psin

S-o

psin

P30

Opsin Ubiquitin Merge

COS

CIS

ONL

COS

CIS

ONL

Fig. S1. S-opsin and ubiquitin in Lrat+/– cones. P18 and P30 Lrat+/– retinal sections were

double-labeled with antibodies against M-opsin (or S-opsin) (green) and ubiquitin (red).

Nuclei were stained with DAPI (blue). Scale bar = 10 µm.

SI Appendix

WT Lrat–/–

P14

COSCIS

ONL

OPL

IgG2a

DAPI

IgG2a

DAPI

COSCIS

ONL

WT Lrat–/–

A Ventral

P18

B Far-dorsal

COSCIS

ONL

OPL

IgG2a

DAPI

IgG2a

DAPI

COSCIS

ONL

OPL

P18

P14

IgG2a

DAPI

IgG2a

DAPI

IgG2a

DAPI

IgG2a

DAPI

Fig. S2. Immunolabeling with the isotype control mouse IgG2a for the CHOP antibody

(L63F7) showed no non-specific signals on the WT and Lrat–/– retinas. The ventral (A) and

far-dorsal (B) retinal sections of WT and Lrat–/– mice (P14 and P18) were labeled with

mouse IgG2a (red) and DAPI (blue). Red signals in the OPL were due to the labeling of

retinal vessels by the Cy3-conjugated goat anti-mouse secondary antibody. Scale bar = 10

µm.

Fig. S3. Bovine rhodopsin showed uniform membrane expression in COS-7 cells. COS-7

cells were transfected with 1 µg bovine rhodopsin encoding plasmid (in pRK5 vector), and

immunostained for rhodopsin with 1D4. Scale bar = 10 µm.

Anti-HA

MBO AB5405 JH6108

1D4

Fig. S4. Immunolabeling of vector (pRK5)-transfected COS-7 cells with antibodies against

various opsins and HA-tag showed no non-specific signals. Cells were labeled with MBO

(rabbit antibody for mouse S-opsin), AB5405 (rabbit antibody for mouse M-opsin and

human red/green opsins), JH6108 (chicken antibody for human blue opsin), 1D4 (mouse

monoclonal antibody for rhodopsin), and anti-HA (mouse monoclonal antibody for HA-

tagged ubiquitin). Primary antibodies were detected with Alexa 488 conjugated goat anti-

rabbit IgG, Cy3-conjugated goat-mouse IgG, and Alexa 488 conjugated goat anti-chicken

IgG secondary antibodies. Nuclei were stained with DAPI (blue). Scale bar = 5 µm.

BlueC

HO

P-G

FP

S-opsinM-opsin Red Green VectorO

psin

or

mC

herr

yM

erg

eRho

Fig. S5. Mouse S-opsin and human blue opsin induced CHOP activation in COS-7 cells. Cells were co-transfected with plasmids encoding CHOP-GFP and various opsins (or control vector pRK5 and pmCherry-N1). Cells were labeled with opsin antibodies (red). Rho, bovine rhodopsin; Red, green, blue opsins are human cone opsins. Scale bar = 150 µm.

CHOP

COSCIS

ONL

OPL

Fig. S6. No CHOP activation in the P14 Rho–/–Lrat–/– retina. The retinal sections of P14 Rho–/–Lrat–/– mice were labeled with a CHOP antibody or isotype control (mouse IgG2a). Nuclei were stained with DAPI. Scale bar = 10 µm.

Dorsal VentralCentral

COSCIS

ONL

OPL

Isotype

control

P14

Rho–/–Lrat–/–

P21

Fig. S7. Comparison of rod degeneration in S-opsin+Rho–/–Lrat–/– and Rho–/–Lrat–/– mice at the indicated ages. Retinas were embedded in plastic, and 1 µm cross sections were stained by hematoxylin and eosin. Scale bar = 20 µm.

RIS

ONL

OPL

INL

RIS

ONL

OPL

INL

S-opsin+Rho–/–Lrat–/–

WT

Lrat–/–

MBO AB5407

COSCIS

ONL

OPL

COSCIS

ONL

OPL

Fig. S8. Comparison of anti-S-opsin antibodies, MBO and AB5407 (Millipore), in detecting S-opsin on P18 WT and Lrat–/– mouse retinal sections. MBO recognized S-opsin from both WT and Lrat–/– cones whereas AB-5407 only recognized S-opsin in WT cones. Scale bar = 10 µm.

A S-opsin Human blue

M-opsin Human red

Human green

Fig. S9. The aggregation profiles of human red, green, blue, M-opsin, and S-opsin at pH 7 predicted by Zyggregator. A line at Zagg = 1 is drawn to identify the sensitive regions. The aggregation-prone regions in S-opsin and human blue are boxed in red.

Mouse 70 ILVNVSLGGFLFCIFSVFTVFIASCHGYFLFGRHVCALEAFLGSVAGLVTGWSLAF 125 9Human 72 ILVNVSFGGFLLCIFSVFPVFVASCNGYFVFGRHVCALEGFLGTVAGLVTGWSLAF 127 9Macaca 73 ILVNVSFGGFLLCIFSVFPVFVNSCKGYFVFGRHVCAFEAFLGTVAGLVTGWSLAF 128 10Bovine 73 ILVNVSLGGFIYCIFSVFIVFITSCYGYFVFGRHVCALEAFLGCTAGLVTGWSLAF 128 8Elephant 74 ILVNISLGGFLSCIFSVFIVFISSCKGYFIFGRYVCALEAFVGSVAGLVTGWSLAF 129 8Boar 75 ILVNVSLGGFIYCIFSVFSVFIASCHGYFVFGRRVCAMEAFLGSAAGLVTGWSLAF 130 8Hamster 29 ILVNISLGGFLFCSFSVFTVFIASCHGYFLFGRHVCALEAFLGSVAGLVTGWSLAF 84 9Squirrel 70 ILVNVSLGGFIYCMFSVFVVFVNSCHGYFVFGRHVCALEAFLGSAAGLVTGWSLAF 125 8D. Wallaby 70 ILVNISLAGFIYCIFSVFTVFISSSQGYFIFGRHVCAMEGFLGSVAGLVTGWSLAF 125 8Bat 73 ILVNVSLGGFLFCIFSVFTVFIASCQGYFVFGRHVCALEAFLGSTAGLVTGWSLAF 128 9Parrot 70 ILVNISFCGFLACIFCIFTVFVSSSQGYFVFGKHVCAFEGFMGATAGLVTGWSLAF 125 10X. laevis 70 ILVNITVGGFLMCIFSIFPVFVSSSQGYFFFGRIACSIDAFVGTLTGLVTGWSLAF 125 9Lizards 70 ILVNISFAGFLFCVFSVFTVFLASSQGYFFFGRHICALEAFLGSVAGLVTGWSLAF 125 11Salamander 70 ILVNVSLAGFTFCIFSVFTVFVSSSQGYFIFGKTICELEAFLGSVSGLVTGWSLAF 125 9Bull Frog 70 ILVNISVGGLLICIFSNFVVFINSWQGYFFFGKAFCAIEAFVGTLAGLVTGWSLAF 125 9

****::. *: * *. * **: * ***.**: * ::.*:* :**********

F

Fig. S10. The phenylalanine-rich region of S-opsin (70-125) is conserved in the SW1 family of vertebrate opsins. “*”, identical; “:”, conserved substitutions; “.”, semi-conserved substitution. The number of phenylalanine residues (underlined) in each region is indicated at the right. The secondary structures of SW1 opsins from different species are modeled from the rhodopsin crystal structure (39).

H-II E-I H-III

Human LWS 91 ILVNLAVADLAETVIASTISIVNQVSGYFVLGHPMCVLEGYTVSLCGITGLWSLAI 146 1 Macaca LWS 91 ILVNLAVADLAETVIASTISVVNQVSGYFVLGHPMCVLEGFTVSLCGITGLWSLAI 146 2Chimpanzee LWS 204 ILVNLAVADLAETVIASTISVVNQVSGYFVLGHPMCVLEGYTVSLCGITGLWSLAI 259 1Bovine LWS 91 ILVNLAIADLAETIIASTISVVNQMYGYFVLGHPLCVVEGYTVSLCGITGLWSLAI 146 1Elephant LWS 91 ILVNLAVADLAETVIASTISVVNQIYGYFILGHPLCVVEGYTVSLCGITGLWSLAI 146 1Cat LWS 91 ILVNLAVADLAETIIASTISVVNQIYGYFVLGHPMCVLEGYTVSLCGITGLWSLAI 146 1Horse LWS 91 ILVNLAVADLAETIIASTISVVNQIYGYFVLGHPMCVVEGYTVSLCGITGLWSLAI 146 1Rabbit LWS 90 ILVNLAIADLGETIIASTISVINQIYGYFILGHPMCVLEGYTVSLCGITGLWSLAI 145 1Bat L/M 47 ILVNLAVADLAETVIASTISIVNQIYGYFVLGHPLCIVEGYTVSLCGITGLWSLAI 102 1Chicken LWS 88 ILVNLAVADLGETVIASTISVINQISGYFILGHPMCVVEGYTVSACGITALWSLAI 143 1Canaria LWS 64 ILVKLSVAELGETVIASTISVVNQIFGYFILGHPMCVIEGYTVSACGITALWSLAI 119 2Dolphin LWS 91 MLVNLAVADLAETVIASTISVVNQMYGYFVLGHPLCIVEGFTVSLCGITGLWSLAI 146 2Whale LWS 91 MLVNLAVADLAETVIASTISVVNQMYGYFVLGHPLCIVEGFTVSLCGITGLWSLAI 146 2Pigeon LWS 85 ILVNLAVADLGETVIASTISVVNQISGYFVLGHPMCVLEGYTVSACGITALWSLAI 140 1Lizard LWS 94 ILVNLAIADLGETVIASTISVINQISGYFILGHPMCVLEGYTVSTCGISALWSLAV 149 1Salamander LWS 90 ILVNLAIADIAETLIASTISVINQIFGYFILGHPLCVIEGYTVSVCGITGLWSLTI 145 2X. Trop LWS 90 ILVNMAIADLGETVIASTISVCNQIFGYFVLGHPMCILEGYTVSVCGIAALWSLTV 145 2Carp LWS 88 ILVNLAIADIGETLLASTISVINQVFGYFILGHPMCVFEGFTVSVCGIAGLWSLTV 143 4 Zebrafish LWS 87 ILVNLAIADLGETLFASTISVINQVFGYFILGHPMCIFEGYTVSVCGIAGLWSLTV 142 4Human MWS 91 ILVNLAVADLAETVIASTISVVNQVYGYFVLGHPMCVLEGYTVSLCGITGLWSLAI 146 1Squirrel MWS 91 ILVNLAIADLAETVIASTISVVNQLYGYFVLGHPLCVVEGYTVSVCGITGLWSLAI 146 1Macaca MWS 91 ILVNLAVADLAETVIASTISVVNQVYGYFVLGHPMCVLEGYTVSLCGITGLWSLAI 146 1Rabbit MWS 91 ILVNLAVADLAETVIASTISVVNQFYGYFVLGHPLCVVEGYTVSLCGITGLWSLAI 146 2Boar MWS 91 ILVNLAIADLAETIIASTISVVNQMYGYFVLGHPLCVVEGYTVSLCGITGLWSLAI 146 1

:**::::*::.**::*****: **. ***:****:*:.**:*** ***:.****::

F

Fig. S11. Alignment of the HII-EI-HIII region (91-146 in human red) of the long-wavelength sensitive (LWS) and medium-wavelength sensitive (MWS) opsin family. The number of phenylalanine residues (underlined) in each region is indicated at the right. “*”, identical; “:”, conserved substitutions; “.”, semi-conserved substitution.

H-II E-I H-III

P18 Lrat+/–

COS

CIS

ONL

COS

CIS

ONL

Rabbit IgG

Rabbit IgG

Mouse IgG1

Mouse IgG1

Do

rsa

lC

en

tra

lV

en

tra

l

COS

CIS

ONL

P18 Lrat–/–

COS

CIS

ONL

COS

CIS

ONL

Rabbit IgG

Rabbit IgG

Mouse IgG1

Mouse IgG1

Do

rsa

lC

en

tra

lV

en

tra

l

COS

CIS

ONL

Rabbit IgG Mouse IgG1 Rabbit IgG Mouse IgG1

P30 Lrat+/–

COS

CIS

ONL

COS

CIS

ONL

Rabbit IgG

Rabbit IgG

Mouse IgG1

Mouse IgG1

Do

rsa

lC

en

tra

lV

en

tra

l

COS

CIS

ONL

Rabbit IgG Mouse IgG1

P30 Lrat–/–

COS

CIS

ONL

COS

CIS

ONL

Rabbit IgG

Rabbit IgG

Mouse IgG1

Mouse IgG1

Do

rsa

lC

en

tra

lV

en

tra

l

COS

CIS

ONL

Rabbit IgG Mouse IgG1

Fig. S12. Immunolabeling with the isotype control mouse IgG1 (for the uniquitin antibody Mab1510) and control rabbit IgG (for the M and S opsin antibodies) showed no non-specific signals on the Lrat+/– and Lrat–/– retinas. Lrat+/– and Lrat–/– (P18 and P30) retinal sections were labeled with control rabbit IgG (green), isotype control mouse IgG1 (red), and DAPI (blue). Scale bar = 10 µm.

S-opsin+Rho–/–Lrat–/–S-opsin+Rho–/–

P14

IgG2a

DAPI

COSCIS

ONL

OPL

Fig. S13. Immunolabeling with the isotype control mouse IgG2a for the CHOP antibody (L63F7) did not show non-specific signals on the S-opsin+Rho–/–Lrat–/– and Rho–/–Lrat–/–

retinas. P14 retinal sections were labeled with mouse IgG2a (red) and DAPI (blue). Red signals in the OPL were due to the labeling of retinal vessels by the Cy3-conjugated goat anti-mouse secondary antibody. Scale bar = 10 µm.

IgG2a

DAPI

Additional Materials and Methods

Plasmids. Mouse M-opsin, mouse S-opsin, human red opsin, human green opsin, human blue

opsin, and bovine rhodopsin in pRK5 were obtained from J. Nathans. 1D4-tagged human red,

green, blue opsins were from D. Oprian. CHOP-GFP (1) and pRK5-HA-Ubiquitin (2) were

purchased from Addgene.

Immunohistochemistry. The immunohistochemical procedure was as previously described (3,

4). Cryostat sections (10-15 μm) were incubated with various primary antibodies, and were

visualized with Alexa 488- or Cy3-conjugated secondary antibodies. Sections were examined by

Olympus FV1000 confocal microscope. The following primary antibodies were used, anti-M-

opsin (AB5405, Millipore) for detecting mouse M-opsin and human red/green opsins, anti-

mouse-S-opsin (MBO) (from J. Chen) (5), anti-mouse-S-opsin (AB5407, Millipore), anti-

human-blue-opsin (JH-6108 from J. Nathans), anti-ubiquitin (Mab1510, mouse IgG1, Millipore),

anti-CHOP (L63F7, mouse IgG2a, Cell Signaling Technology), anti-HA-tag (32-6700,

Invitrogen), anti-cone-arrestin (CARR from W. Baehr), and 1D4 (From R. Molday). AB5407

was only used in Fig. S8. Isotype control antibodies, mouse IgG1 (Cell Signaling Technology)

and mouse IgG2a (Sigma), were used at the same concentrations as Mab1510 and L63F7,

respectively. The rabbit IgG (Jackson ImmunoResearch Laboratory) for control of MBO,

AB5405, and AB5407 was used at the same concentration of MBO, which is higher than both

AB5405 and AB5407. For experiments in Figs. 1, 3, 4, 7B, S1, S2, S6, S12, and S13, an antigen

retrieval step (10 mM sodium citrate, pH 6.0, 95 °C, 5 min) was added before immunolabeling.

Western blot. Retinas from both eyes of the mouse were sonicated in 150 µl of RIPA buffer

(150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM

Tris, pH 8.0) plus protease inhibitors (Sigma). Protein concentration was measured by Bio-Rad

protein assay kit. 15 µg of protein was loaded for each sample for SDS-PAGE. The primary

antibodies were detected by incubation with goat anti-rabbit or anti-mouse secondary antibodies

conjugated with Horseradish Peroxidase. The primary antibodies were the same as used in

immunohistochemistry. In addition, anti-β-actin (AC-15, Sigma-Aldrich) was used. For

quantification, the blot was analyzed by Bio-Rad Molecular Imager ChemiDoc XRS+ System

with the Quantity One software.

Real-time RT-PCR. Total RNA from mouse retina was isolated with Tri Reagent (Applied

Biosystems), and contaminant genomic DNA was removed by RNase-free DNase I (Fermentas).

Total RNA (5 µg) was reverse transcribed using High Capacity cDNA Reverse Transcription Kit

(Applied Biosystems) in a total volume of 20 µL. 0.5% of this reaction was used as a template

for real-time PCR reaction using Power SYBR Green PCR Master Mix (Applied Biosystems)

following the manufacturer’s instruction. The primers and conditions used to amplify M-opsin

and S-opsin were described previously (6). The following primers were used for mouse Gapdh:

5’- GTG AAG GTC GGT GTG AAC GG -3’; reverse, 5’- GCC GTT GAA TTT GCC GTG AG

-3’. Primers were separated by at least one intron to distinguish possible products from genomic

DNA.

Cell Culture. COS-7 cells were grown in DMEM supplemented with 10% FBS, 100 U/mL

penicillin and 0.1 mg/mL streptomycin. Cells were seeded on cover slips placed in 24-well plate.

24 hours after seeding, cells were transfected with 1 µg of various opsin-encoding plasmids

(non-tagged human cone opsin plasmids in Fig. 5, 1D4-tagged human cone opsin plasmids in

Figs. 6 & S5), 0.25 µg pRK5-HA-Ubiquitin or 0.3 µg CHOP-GFP, and 2 µL Turbofect reagent

(Fermentas) following manufacture’s instruction. For vector control, 1 µg pRK5 and 0.25 µg

pmCherry-N1 were used. 55 hours after transfection, cells were fixed in 4% paraformaldehyde in

PBS for 30 min at 37 °C and were permeated with 0.6% Triton X-100 in PBS for 30 min at room

temperature. Cells were immunostained as described under “Immunohistochemistry”. CHOP-

GFP positive cells and opsin expressing cells (or mCherry positive cells) were manually counted

from 6-12 regions to obtain the percentage of CHOP-GFP positive cells in opsin-expressing cells

in Fig. 6.

Cone-cell-number analysis. Before enucleation, the dorsal side of a mouse eye was marked

with a red marker pen. Mouse eyes were fixed in 4% paraformaldehyde in PBS at room

temperature for 2 hours, and cryoprotected overnight with 30% (w/v) sucrose in PBS before

being embedded in OCT (Tissue-Tek). Cryo-sections were cut sagitally (dorsal to ventral)

through the optic nerve head and stained for cone arrestin. Cone numbers from far-dorsal, central

and ventral retina were counted from 4 sections. Data were expressed as the mean number of

cones/field of view ± SEM.

Measurement of the ONL lengths of S-opsin+Rho–/–Lrat–/– and control S-opsin+Rho–/– or S-

opsin+Rho–/–Lra+/– retinas. The dorsal side of mouse eyes of S-opsin+Rho–/–Lrat–/– (P9, P14, and

P21), S-opsin+Rho–/–Lra+/– (P9), and S-opsin+Rho–/– (P14 and P21) were marked with a red

marker pen before ennucleation. Eyes were fixed for 2h in 4% paraformaldehyde in 1xPBS, and

cryoprotected. Eyes were embedded in OCT (Tissue Tek), frozen, and sectioned sagitally

through the optic nerve head. Retinal sections were stained with DAPI and imaged with

Olympus FV1000 confocal microscope. The ONL lengths of the dorsal, central, and ventral

regions of retina were measured. The average ONL (n≥14 for each region) were used to

calculate the ONL ratio between S-opsin+Rho–/–Lrat–/– and control S-opsin+Rho–/– or S-

opsin+Rho–/–Lra+/– retinas in each region.

Histology. Mouse eyes were immersion-fixed overnight in a fixative containing 2.5%

glutaraldehyde/1% formaldehyde and resin embedded as previously described (7). Samples were

sectioned at 1 µm and stained with hematoxylin and eosin (H&E) for light microscopy.

Statistics. Data were expressed as mean ± SEM. Statistics was calculated by unpaired two-

sample Student’s t-test. Significant difference was defined as a P-value < 0.05.

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3. Fu Y, Kefalov V, Luo DG, Xue T, & Yau KW (2008) Quantal noise from human red cone pigment. Nat Neurosci 11(5):565-571.

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