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. 2020 Feb;27(2):646-661.
doi: 10.1038/s41418-019-0378-6. Epub 2019 Jul 1.

ER complex proteins are required for rhodopsin biosynthesis and photoreceptor survival in Drosophila and mice

Liangyao Xiong #  1   2 , Lin Zhang #  3 , Yeming Yang  3 , Na Li  4 , Wenjia Lai  5 , Fengchao Wang  2   6 , Xianjun Zhu  7   8   9   10 , Tao Wang  11   12
Affiliations

ER complex proteins are required for rhodopsin biosynthesis and photoreceptor survival in Drosophila and mice

Liangyao Xiong et al. Cell Death Differ. 2020 Feb.

Abstract

Defective rhodopsin homeostasis is one of the major causes of retinal degeneration, including the disease Retinitis pigmentosa. To identify cellular factors required for the biosynthesis of rhodopsin, we performed a genome-wide genetic screen in Drosophila for mutants with reduced levels of rhodopsin. We isolated loss-of-function alleles in endoplasmic reticulum membrane protein complex 3 (emc3), emc5, and emc6, each of which exhibited defective phototransduction and photoreceptor cell degeneration. EMC3, EMC5, and EMC6 were essential for rhodopsin synthesis independent of the ER associated degradation (ERAD) pathway, which eliminates misfolded proteins. We generated null mutations for all EMC subunits, and further demonstrated that different EMC subunits play roles in different cellular functions. Conditional knockout of the Emc3 gene in mice led to mislocalization of rhodopsin protein and death of cone and rod photoreceptor cells. These data indicate conserved roles for EMC subunits in maintaining rhodopsin homeostasis and photoreceptor function, and suggest that retinal degeneration may also be caused by defects in early biosynthesis of rhodopsin.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Rhodopsin biosynthesis defects in the emc3, emc5, and emc6 mutants. a Isolation of the emc3G7, emc3G10, emc5G2, and emc6N10 mutations via a forward genetic screen. Rh1-GFP fluorescence in the deep pseudopupil of 1-day-old wild-type (ey-flp rh1-GFP;FRT40A/GMR-hid CL FRT40A), emc3G7 (ey-flp rh1-GFP;emc3G7 FRT40A/GMR-hid CL FRT40A), emc3G10 (ey-flp rh1-GFP;emc3G10 FRT40A/GMR-hid CL FRT40A), emc5G2 (ey-flp rh1-GFP;emc5G2 FRT40A/GMR-hid CL FRT40A), and emc6N10 (ey-flp rh1-GFP; FRT82B emc6N10/FRT82B GMR-hid CL) flies are shown. Scale bar is 100 μm. b The emc3, emc5, and emc6 loci and mutations associated with the emc3G7, emc3G10, emc5G2, and emc6N10 alleles. c Rhodopsin levels were reduced in emc3G7, emc3G10, emc5G2, and emc6N10 flies, and were restored by ninaE-emc3, ninaE-emc5, and ninaE-emc6 transgenes, respectively. Protein extracts from 1/4 head of each genotype (1 d after eclosion) were loaded and probed with antibodies against Rh1, ß-actin, TRP, and INAD
Fig. 2
Fig. 2
Loss of PDA in emc3, emc5, and emc6 mutants. a ERG recordings in wild-type flies (ey-flp rh1-GFP;FRT40A/GMR-hid CL FRT40A) showed that PDA was induced by blue light and terminated by orange light (arrows). b PDA was eliminated in ninaEP332 flies and in c emc3G7, e emc3G10, g emc5G2, and i emc6N10 flies. PDA was restored in (d) emc3G7;ninaE-emc3, f emc3G10;ninaE-emc3, h emc5G2;ninaE-emc5, and j ninaE-emc6;emc6N10 flies. Flies ~1day after eclosion were dark-adapted for 2 min and subsequently exposed to 5-s pulses of orange (O) or blue (B) light. At least 10 flies of each genotype were tested
Fig. 3
Fig. 3
EMC3, EMC5, and EMC6 were not required for maturation of the Rh1 protein. a Rh1 localized to the rhabdomere region in emc3, emc5, and emc6 mutant photoreceptor cells. Tangential resin-embedded retina sections of compound eyes from ~1-day-old wt, emc3G7, emc3G7;ninaE-emc3, emc5G2, emc5G2;ninaE-emc5, emc6N10, and ninaE-emc6;emc6N10 flies were labeled using antibodies against Rh1. Rhabdomeres are indicated by arrows. Scale bar is 2 μm. Illustration of photoreceptor cells within a single ommatidium is on the top right corner, and rhabdomeres of R1-R7 photoreceptor cells are indicated by numbers. b Deglycosylation of Rh1 is normal in emc3, emc5, and emc6 mutants. Head extracts prepared from 1-day-old wild-type, ninaA, dmppee02905, emc3G7, emc5G2, and emc6N10 flies were probed with antibodies against Rh1, and mature and immature bands are indicated. Extracts from ninaA and emc6N10 flies (10 heads), emc3G7 and emc5G2 flies (5 heads), and wild-type and dmppee02905 flies (0.25 of a head) were loaded. c–e Similar Rh1 levels were seen in (c) emc3G7 and emc3G7;hrd11 flies (ey-flp rh1-GFP;emc3G7 FRT40A/GMR-hid CL FRT40A;FRT82B hrd11/FRT82B GMR-hid CL), d emc5G2 and emc5G2;hrd11 flies (ey-flp rh1-GFP;emc5G2 FRT40A/GMR-hid CL FRT40A;FRT82B hrd11/FRT82B GMR-hid CL), and e emc6N10 and emc6N10 hrd11 (ey-flp rh1-GFP; FRT82B emc6N10 hrd11/FRT82B GMR-hid CL) flies. Protein extracts from 0.25 of a head from ~1-day-old flies were loaded
Fig. 4
Fig. 4
Interaction among EMC subunits. a S2 cells were transiently transfected with emc3-RFP, emc5-mCherry, or emc6-GFP, or co-transfected with emc3-RFP/emc6-GFP, emc5-mCherry/emc6-GFP, emc6-GFP/RFP-rab7. EMC6 puncta are indicated by arrows. Scale bar is 5 μm. b Flowchart of Tandem Mass Tags Labeling coupled with LC-MS/MS for the comparative analysis of protein levels in retina of emc2 (GMR-Gal4/UAS-emc2ARNAi), emc4 (GMR-Gal4/UAS-emc4RNAi), emc5 (GMR-Gal4/UAS-emc5RNAi), and emc7 (FRT42D emc71) mutant flies, as well as wild-type (GMR-gal4) flies. c Venn diagrams showing the overlap of proteins that were downregulated > 50% among the four analyzed genotypes: emc2, emc4, emc5, and emc7
Fig. 5
Fig. 5
Retinal degeneration in emc3, emc5, and emc6 mutant photoreceptors. TEM images of representative ommatidia from (a) wild-type, (b) emc3G7, (c) emc5G2, and (d) emc6N10 flies. Flies were raised in a 12h-light/12h-dark (L/D) cycle for indicated time points. The degenerating photoreceptor cells are indicated by arrows. Scale bars are 2 μm
Fig. 6
Fig. 6
Loss of Emc3 in mammalian cone cells led to cone cell death. a, b Retina cryosections from (a) WT and (b) cone-KO littermate mice at P30. Sections were immunostained for M-Opsin (green) and the cone marker, peanut agglutinin (PNA, red). Cone cell number did not decrease at P30. c, d Retina cryosections from (c) WT and (d) cone-KO mice at P50. Sections were immunostained for M-Opsin (green) and PNA (red). There were almost no cone cells in cone-KO retinas. Nuclei were counterstained with DAPI. ONL outer nuclear layer, OS outer segment, IS inner segment. Scale bar is 25 μm
Fig. 7
Fig. 7
Retinal degeneration and RHO mislocalization in Emc3 inducible KO mice. a Paraffin sections of mice retinas 11 days after induction (P31). Sections were stained with H&E, and quantification of outer nuclear layer is shown on the left. Scale bar is 50 μm. H&E staining of paraffin sections and quantification of outer nuclear layer (ONL) nuclei revealed that the mutant ONL was reduced by ~50% compared to controls 11 days after induction. On top is an illustration of the whole mouse retina. b Immunofluorescence labeling of retina cryosections for control (WT) and mutant (KO) littermates at P31. Sections were labeled using antibodies against RHO (green) and DAPI (blue). NaK ATPase antibodies were used to label the inner segment. RHO was mislocalized to the inner segment and cell bodies in KO retinas (arrows). Scale bar is 25 μm
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