Rer1p regulates the ER retention of immature rhodopsin and modulates its intracellular trafficking

Abstract

Rhodopsin is a pigment in photoreceptor cells. Some rhodopsin mutations cause the protein to accumulate in the endoplasmic reticulum (ER), leading to photoreceptor degeneration. Although several mutations have been reported, how mutant rhodopsin is retained in the ER remains unclear. In this study, we identified Rer1p as a modulator of ER retention and rhodopsin trafficking. Loss of Rer1p increased the transport of wild-type rhodopsin to post-Golgi compartments. Overexpression of Rer1p caused immature wild-type rhodopsin to accumulate in the ER. Interestingly, the G51R rhodopsin mutant, which has a mutation in the first transmembrane domain and accumulates in the ER, was released to the plasma membrane or lysosomes in Rer1-knockdown cells. Consistent with these results, Rer1p interacted with both wild-type and mutant rhodopsin. These results suggest that Rer1p regulates the ER retention of immature or misfolded rhodopsin and modulates its intracellular trafficking through the early secretory pathway.

Figure 1. Rhodopsin mutants are retained in the ER and degraded in part by the ubiquitin-proteasome system.

(A) The structure of FLAG-rhodopsin-GFP chimeric proteins. The positions of point mutations relevant to this study are also indicated. (B) Subcellular localization of FLAG-rhodopsin-GFP. HeLa cells stably expressing each rhodopsin fusion protein were immunostained with anti-calreticulin antibody (red in the merged image). The GFP signal appears in green in the merged image. Bar: 10 μm. (C) Glycosylation states of FLAG-rhodopsin-GFP. Cell lysates were prepared from HeLa cells or cells expressing each rhodopsin fusion protein and immunoblotted with anti-GFP (top panel) and anti-GSK3β (bottom panel) antibodies. Each cell lysate was treated with PNGase F (middle) and Endo H (right). An arrowhead indicates the bands, which presumably represent SDS-insoluble multimer of FLAG-rhodopsin-GFP derivatives. (D–E) Degradation of rhodopsin mutants by the ubiquitin-proteasome system. HeLa cells expressing each rhodopsin fusion protein were treated with DMSO (D), 2.5 μM MG132 (M), or 200 nM bafilomycin A1 (B) for 20 h. The cell lysates were immunoblotted with anti-GFP (top panel) and anti-GSK3β (bottom panel) antibodies. The lysate of cells expressing FLAG-WT rhodopsin-GFP treated with PNGase F is also shown. * and ** indicate ER and non-glycosylated forms of rhodopsin, respectively. Note that cropped western blots are shown and that full-length images are presented in the supplementary information.

Figure 2. Rer1p contributes to the ER retention of G51R mutant rhodopsin.

(A) Loss of Rer1p increases the cell surface expression of G51R mutant rhodopsin. HeLa cells expressing each FLAG-rhodopsin-GFP construct were treated with control siRNA or Rer1 #1 siRNA for 72 h. Cells were fixed without permeabilization and stained with anti-FLAG antibody. The GFP signal and anti-FLAG staining indicate the localization of total and external rhodopsin, respectively. Bar: 10 μm. (B) Cell surface protein biotinylation assay. HeLa cells expressing each FLAG-rhodopsin-GFP construct were treated with control siRNA or Rer1 #1 siRNA for 72 h and biotinylated. Biotinylated proteins were then pulled down using streptavidin agarose and immunoblotted with an anti-GFP antibody. A fraction (10%) of the total cell lysate was also immunoblotted with anti-GFP (rhodopsin), anti-α-tubulin, and anti-Rer1p antibodies. Note that cropped western blots are shown and that full-length images are presented in the supplementary information. (C) The phenotype induced by Rer1 siRNA is specific to the knockdown of Rer1 expression. Cells were treated with control siRNA or Rer1 #1 siRNA for 72 h. After 48 h of siRNA transfection, cells were co-transfected with FLAG-rhodopsin-GFP and either mRFP or siRNA-resistant mRFP-canine Rer1. Cells were fixed without permeabilization and stained with anti-FLAG antibody. The graph indicates the ratio of anti-FLAG stained cells to the total number of cells.

Figure 3. A portion of G51R rhodopsin is transported to the lysosomes in the absence of Rer1p.

(A) Translocation of G51R mutant rhodopsin to lysosomes in Rer1 knockdown cells. HeLa cells expressing each FLAG-rhodopsin-GFP construct were transfected with control siRNA or Rer1 #2 siRNA. Cells were immunostained with an anti-LAMP1 antibody. The signals of FLAG-rhodopsin-GFP (green) and LAMP-1 (red) are shown in the merged images. Bar: 10 μm. (B) The graph shows the average number of rhodopsin-positive puncta per cell. Thirty cells were counted in each experiment. Three independent experiments were examined. Error bars represent the SE. * indicates p < 0.05.

Figure 4. Rer1p interacts with rhodopsin and modulates its intracellular trafficking.

(A, B) Overexpression of mRFP-Rer1p results in the ER accumulation of rhodopsin. HeLa cells stably expressing each FLAG-rhodopsin-GFP construct (A and B) were co-transfected with mRFP (A) or mRFP-Rer1p (B). The signals of FLAG-rhodopsin-GFP (green) and mRFP-Rer1p (red) are shown in the merged images. (C) Overexpression of mRFP-Rer1p leads to the accumulation of the ER form of rhodopsin. HeLa cells stably expressing FLAG-rhodopsin-GFP were transiently transfected with mRFP or mRFP-Rer1p. The cell lysates were immunoblotted with anti-GFP and anti-mRFP antibodies. * indicates the ER form of rhodopsin-GFP. (D) Overexpressed mRFP-Rer1p interacts with rhodopsin. HeLa cells stably expressing WT or mutant (P23H, L40R, or G51R) FLAG-rhodopsin-GFP were transiently transfected with mRFP or mRFP-Rer1p. The cell lysates were immunoprecipitated with FLAG M2 agarose beads. The immunoprecipitants and cell lysates were immunoblotted with anti-mRFP (upper panel) and anti-GFP (lower panel) antibodies. The signal intensities of co-immunoprecipitated mRFP-Rer1p and rhodopsin-GFP were quantified, and the amount of co-immunoprecipitated mRFP-Rer1p was normalised to the amount of rhodopsin-GFP. To compare the co-immunoprecipitation efficiencies of wild-type and mutant rhodopsins, the binding activities were calculated by expressing each normalised value relative to the normalised value obtained with WT rhodopsin-GFP. (E) Endogenous Rer1p interacts with rhodopsin. Lysates of HeLa cells expressing each FLAG-rhodopsin-GFP construct were immunoprecipitated with anti-FLAG M2 agarose beads. The immunoprecipitants and cell lysates were immunoblotted with anti-Rer1 (top panel), anti-GFP (middle panel), and anti-Hsp70 (bottom panel) antibodies. The binding activities were analysed as described in panel D. Note that cropped western blots are shown and that full-length images are presented in the supplementary information.