dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors

Abstract

In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). We screened the mutants affecting rhabdomeric expression of rhodopsin 1 (Rh1) in the Drosophila photoreceptors and found that dPob/EMC3, EMC1, and EMC8/9, Drosophila homologs of subunits of ER membrane protein complex (EMC), are essential for stabilization of immature Rh1 in an earlier step than that at which another Rh1-specific chaperone (NinaA) acts. dPob/EMC3 localizes to the ER and associates with EMC1 and calnexin. Moreover, EMC is required for the stable expression of other multi-pass transmembrane proteins such as minor rhodopsins Rh3 and Rh4, transient receptor potential, and Na(+)K(+)-ATPase, but not for a secreted protein or type I single-pass transmembrane proteins. Furthermore, we found that dPob/EMC3 deficiency induces rhabdomere degeneration in a light-independent manner. These results collectively indicate that EMC is a key factor in the biogenesis of multi-pass transmembrane proteins, including Rh1, and its loss causes retinal degeneration.

Keywords: D. melanogaster; EMC; cell biology; chaperon; rhodopsin; transmembrane protein.

Figure 1.. Identification of CG6750 as an essential gene for rhodopsin 1 (Rh1) biosynthesis.

(A) Observation of fluorescent protein localizations in CG6750^e02662 mosaic retinas by the water immersion technique. RFP (red) indicates wild-type photoreceptors (R1–R8). Arrestin2::GFP (green) shows endogenous Rh1 localization in R1–R6 peripheral photoreceptors. (B) Schematic drawing of CG6750 and insertion/deletion mutants. The dPob^-null mutant allele, dPob^∆4, was created by the recombination of two FRTs on dPob^f07762 and dPob^{CB−0279−3} using an FRT/FLP-based deletion method. (C, D) Immunostaining of dPob^e02662 (C) and dPob^∆4 (D) retinas expressing RFP as a wild-type cell marker (magenta) by anti-Rh1 antibody (green). Asterisks show mutant cells. Scale bar: 5 μm (A, C, D). DOI: http://dx.doi.org/10.7554/eLife.06306.003

Figure 2.. Construction of antisera against dPob.

(A) Immunoblotting of wild-type (+/+) and dPob^e02662 homozygous (−/−) extracts from whole larvae using antiserum against dPob N- and C-terminal polypeptides. (B) Immunostaining of a dPob^e02662 mosaic retina expressing RFP (red) as a wild-type cell marker (not shown) by rat anti-dPob-C1 antiserum (blue) and phalloidin (green). Asterisks show dPob^e02662 homozygous photoreceptors. (C, D) Immunostaining of wild-type retinas by anti-dPob (green) and anti-NinaA (C) or anti-HDEL (D) antisera. Scale bar: 5 μm (B–D). DOI: http://dx.doi.org/10.7554/eLife.06306.004

Figure 3.. dPob stabilizes rhodopsin 1 (Rh1) apoprotein.

(A) Immunostaining of a dPob^∆4 mosaic retina from a fly reared in vitamin A (VA)-deficient medium by anti-Rh1 antibody. Asterisks show dPob^∆4 homozygous photoreceptors. (B–D) Immunostaining of a wild-type (B), ninaA^p263(C), or dPob^∆4 (D) ommatidium of flies reared in normal vitamin A-containing medium. (E) Immunostaining of a dPob^e02662 mosaic retina in ninaA^p263 homozygous mutant background from a fly reared in normal medium. Asterisks show dPob^∆4 homozygous photoreceptors. Scale bar: 5 μm (A–E). DOI: http://dx.doi.org/10.7554/eLife.06306.005

Figure 4.. Loss of rhodopsin 1 (Rh1) apoprotein in EMC1 and EMC8/9 deficiency.

Immunostaining of a EMC1^655G mosaic retina (A, B) or a EMC8/9^008J mosaic retina (C, D) reared in normal (A, C) and vitamin A-deficient media (B, D). Asterisks show EMC1^655G or EMC8/9^008J homozygous photoreceptors. RFP (red) indicates wild-type photoreceptors (R1–R8). (A, C) Na⁺K⁺-ATPase, green; Rh1, blue; RFP, red. (B, D) Rh1, green; RFP, magenta. Scale bar: 5 μm (A–D). DOI: http://dx.doi.org/10.7554/eLife.06306.006

Figure 5.. Co-immunoprecipitation of EMC1::GFP with dPob and calnexin (Cnx).

Immunoblotting of precipitates with anti-GFP antibody from the head extract was prepared from Rh1-Gal4/UAS-EMC1::GFP or sec61::GFP flies reared in a vitamin A (VA)-deficient medium (left) or heat shock (hs)-Gal4/UAS-EMC1::GFP or sec61::GFP flies reared in a vitamin A-containing normal medium (right). The mature form of rhodopsin 1 (Rh1) is accumulated in the rhabdomeres in normal medium but not in vitamin A-deficient medium. Instead of the mature form, an N-glycosylated immature form of Rh1 with a larger molecular weight accumulated in the endoplasmic reticulum of flies reared in the vitamin A-deficient medium. In both input extracts prepared from Rh1-Gal4/UAS-EMC1::GFP or sec61::GFP flies there is a band with the same position as EMC1GFP; this band will be the protein cross-reacting to anti-GFP antibody. DOI: http://dx.doi.org/10.7554/eLife.06306.007

Figure 6.. Essential role of dPob in the biosynthesis of multi-pass transmembrane proteins.

Immunostaining of a dPob^∆4 mosaic retina (A–H) or a dPob^e02662 mosaic retina (I). Asterisks show dPob homozygous photoreceptors. (A) Na⁺K⁺-ATPase, green; Rh1, magenta. (B) Crb, green; TRP1, magenta. (C, D) Rh3 (C) and Rh4 (D), green; RFP (wild-type cell marker), magenta. Although the boundary between dPob^∆4 and wild-type cells is unclear, all green signals are attached to RFP-expressing cell bodies, indicating that mutant R7 cells do not express Rh3 (C) or Rh4 (D). (E) DE-Cad staining. (F) Nrv, the beta subunit of Na⁺K⁺-ATPase, green; dMPPE, magenta. (G) Nrt staining. (H) Syx1A staining. (I) Eys staining. (J) Nrg, blue; F-actin, red; GFP-nls (wild-type cell marker), green. (K) FasIII staining. (L) Na⁺K⁺-ATPase, green; Rh1, magenta. Scale bar: 2 μm (A, B), 10 μm (C, D), 2 μm (E–I). DOI: http://dx.doi.org/10.7554/eLife.06306.008

Figure 7.. Essential role of EMC1 and EMC8/9 in the biosynthesis of multi-pass transmembrane proteins.

Immunostaining of a EMC1^655G mosaic retina (A, B, C) or a EMC8/9^008J mosaic retina (D, E, F). (A, D) Left: Rh3, middle: Rh4, right: TRP in green, RFP in magenda. (B, E) Eys in green, Crb in blue, and RFP, wild-type cell marker in red. (C, F) Left: dMPPE, middle: Nrt, right: Syx1A in green, RFP in magenda. Scale bar: 10 μm (left and middle in A, D), 5 μm (right in A, D), 5 μm (B, C, E, F). DOI: http://dx.doi.org/10.7554/eLife.06306.009

Figure 8.. Endoplasmic reticulum membrane amplification and unfolded protein response (UPR) induced in dPob^∆4 photoreceptor.

(A–C) Electron microscopy of late pupal photoreceptors: wild-type (A), dPob^e02662 (B), and dPob^∆4 photoreceptors (C). Arrow indicate adherens junctions. Insets show Golgi bodies. (D, E) Immunostaining of a dPob^e02662 mosaic retina. dPob is shown in green and KDEL (D) or HDEL (E) are shown in magenta. Asterisks show dPob^∆4 homozygous photoreceptors. Scale bar: 1 μm (A–C), 5 μm (D, E). DOI: http://dx.doi.org/10.7554/eLife.06306.010

Figure 9.. Unfolded protein response (UPR) induced in dPob^∆4 photoreceptor.

(A) Projection image from the Z-series section with a 1 μm interval of dPob^∆4 mosaic retina expressing RFP (magenta) as a wild-type cell marker and Xbp1:GFP as a UPR sensor. The Xbp1:GFP signal (green) is enhanced by immunostaining using anti-GFP antibody. Asterisks show dPob^∆4 homozygous photoreceptors. (B) Immunostaining of a dPob^∆4 mosaic retina expressing RFP (magenta) as a wild-type cell marker. Phosphorylated eukaryotic translation Initiation Factor 2α is shown in green. Asterisks show dPob^∆4 homozygous photoreceptors. DOI: http://dx.doi.org/10.7554/eLife.06306.011

Figure 10.. Development and degeneration of dPob^∆4 photoreceptor rhabdomeres.

Electron microscopy of pupal and adult dPob^∆4 mosaic retinas. Asterisks show dPob^∆4 homozygous photoreceptors. Scale bar: 1 μm. (A, B) dPob^∆4 mosaic ommatidia from 58% pupal development (A) and 73% pupal development (B) under constant light (L) condition. (C–F) dPob^∆4 mosaic ommatidia from flies reared in complete darkness (D) (C, E) or under 12 hr light/12 hr dark conditions (D, F). Ommatidia from 3-day-old (C, D) and 17-day-old (E, F) flies. (D, inset) dPob^∆4 R5 photoreceptor rhabdomere at higher magnification. DOI: http://dx.doi.org/10.7554/eLife.06306.012