Mechanisms determining the morphology of the peripheral ER

Abstract

The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by the curvature-stabilizing proteins reticulons and DP1/Yop1p, but how the sheets are formed is unclear. Here, we identify several sheet-enriched membrane proteins in the mammalian ER, including proteins that translocate and modify newly synthesized polypeptides, as well as coiled-coil membrane proteins that are highly upregulated in cells with proliferated ER sheets, all of which are localized by membrane-bound polysomes. These results indicate that sheets and tubules correspond to rough and smooth ER, respectively. One of the coiled-coil proteins, Climp63, serves as a "luminal ER spacer" and forms sheets when overexpressed. More universally, however, sheet formation appears to involve the reticulons and DP1/Yop1p, which localize to sheet edges and whose abundance determines the ratio of sheets to tubules. These proteins may generate sheets by stabilizing the high curvature of edges.

Figure 1. Localization of proteins to different ER domains

(A) The localization of endogenous luminal ER protein calreticulin is compared with that of the stably overexpressed membrane protein GFP-Sec61β using confocal microscopy in BSC1 cells. Calreticulin was detected with specific antibodies by indirect immunofluorescence (left panel) and Sec61β by GFP fluorescence (middle panel). The right panel shows a merged image. Junction between peripheral ER sheets and tubules are highlighted in the magnified view of the boxed area (inset). Scale bar, 10 μm. (B) As in (A), but comparing the localization of the ER membrane protein calnexin with that of GFP-Sec61β. (C) The localization of endogenous Sec61β is compared to that of the endogenous ER luminal proteins BiP and GRP94 (anti-KDEL), using indirect immunofluorescence with specific antibodies and confocal microscopy. (D) As in (A), but comparing the localization of the translocon membrane protein TRAPα with that of GFP-Sec61β. Also note that TRAPα is noticeably depleted from the nuclear envelope. (E) The localization of stably expressed GFP-Dad1 in a BHK cell line lacking endogenous Dad1 is compared with that of endogenous ER luminal proteins detected by calreticulin antibodies. See also Figure S1.

Figure 2. Membrane proteins enriched in ER sheets

(A) The endogenous localization of the membrane protein Climp63 is compared with that of the luminal ER protein calreticulin in COS7 cells, using indirect immunofluorescence with specific antibodies. The right most panel shows a merged image. Junction between peripheral ER sheets and tubules are highlighted in the magnified view of the boxed area (inset). Scale bar, 10 μm. (B) As in (A), but comparing the localization of kinectin (KTN) and calreticulin. (C) As in (A), but comparing the localization of p180 and calreticulin. (D) Climp63, kinectin, and p180 were depleted in COS7 cells by RNAi, and Climp63, TRAPα, and calreticulin were visualized using indirect immunofluorescence with specific antibodies. Scale bar, 10 μm. (E) As in (D), but with cells transfected with control siRNA oligonucleotides. See also Figure S2 and S5.

Figure 3. Polysome-dependent membrane protein enrichment in ER sheets

(A) The localization of the translocon component TRAPα is compared with that of stably expressed GFP-Sec61β after 15 min of treatment with puromycin (PURO). The right most panel shows a merged image. Junction between peripheral ER sheets and tubules are highlighted in the magnified view of the boxed area (inset). Scale bar, 10 μm. (B) As in (A), but after 15 min of treatment with cycloheximide (CHX). (C) As in (A), but comparing the localization of Climp63 with calreticulin after puromycin treatment. (D) As in (C), but after cycloheximide treatment. (E) Quantification of sheet enrichment of different ER proteins in untreated cells (blue bars) and in cells treated with puromycin (PURO; green) or cycloheximide (CHX; red). The ratio of the average fluorescence intensity in sheets versus tubules was determined for calnexin (CNX), BAP31, calreticulin (CRT), TRAPα, and kinectin, and divided by the sheet to tubule ratio for stably expressed GFP-Sec61β, a protein that shows no preference for either ER domain. A similar analysis was done for GFP-Dad1 and Climp63, but with calreticulin as reference. Shown are the means and standard errors of data obtained from 7 to 30 cells for each condition. See also Figure S3 and S4.

Figure 4. Climp63 affects the luminal width of peripheral ER sheets

(A) Rough ER sheets in a COS7 cell visualized by thin-section electron microscopy. Scale bar, 0.5 μm. (B) As in (A), but after treatment with puromycin (PURO) for 15 min. (C) As in (A), but after RNAi-depletion of Climp63, p180, and kinectin (C/P/K siRNA). (D) As in (A), but after RNAi-depletion of Climp63. (E) Quantification of the luminal width of peripheral ER sheets and the nuclear envelope (NE) in differently treated COS7 cells. For comparison, Drosophila S2R+ cells were also analyzed. Shown are the mean and standard error of n cells analyzed.

Figure 5. Climp63 and reticulon overexpression change the abundance of sheets and tubules

(A) FLAG-Climp63 overexpressed at relatively high levels in a COS7 cell was visualized by indirect immunofluorescence using FLAG antibodies. A 3D image was generated from a complete series of z-sections (step size 0.25 um) taken with a confocal microscope. (B) As in (A), but in a cell expressing FLAG-Climp63 at the highest observed levels. (C) Quantification of the effect of Climp63 overexpression on ER sheet abundance. Shown are the percentages of cells with normal reticular ER (blue bars), of cells with both large sheets and reticular ER (red), and of cells with large ER sheets lacking reticular ER (green) at different expression levels of FLAG-Climp63. The cells were divided into five groups according to their expression levels, determined by overall average fluorescence intensity. (D) Thin-section electron micrograph of a COS7 cell overexpressing GFP-Climp63. The inset shows an enlargement of the boxed region. Scale bar, 0.5 μm. (E) HA-Rtn4b (red) was expressed in COS7 cells at relatively low levels and localized with HA-antibodies by indirect immunofluorescence and confocal microscopy. Endogenous Climp63 (green) was localized in the same cells with specific antibodies. (F) As in (G), but with the highest observed expression level of HA-Rtn4b. Note that Climp63 appears in bright punctae and in the nuclear envelope. (G) Quantification of the peripheral ER sheet area relative to the total ER area for different expression levels of HA-Rtn4b. The areas of ER sheets and tubules were determined from the fluorescence of Climp63 and Rtn4b, respectively, after subtraction of background. The cells were divided into five groups according to their expression levels of HA-Rtn4b, determined by overall average fluorescence intensity, and the mean and stardard error were calculated. (H) Quantification of the effect of Rtn4b overexpression on ER sheet morphology, as determined by Climp63 staining. Shown are the percentages of cells with normal ER sheets (blue bars), of cells with disc-like ER sheets (red), and of cells with punctae (green) at different expression levels of Rtn4b. The cells were divided into five groups according to their expression levels. (I) Quantification of the effect of Rtn4b overexpression on ER tubule morphology, as determined by HA-Rtn4b staining. Shown are the percentages of cells with normal reticular ER (blue bars), of cells with an abnormally dense ER network (red), and of cells with unbranched, long tubules (green) at different expression levels of Rtn4b. The cells were divided into five groups according to their expression levels. (J) Myc-Rtn4a and FLAG-Climp63 were both highly expressed in COS7 cells. The right most panel shows a merged image. Note that the ER morphology is almost normal. See also Figure S6 and S7.

Figure 6. The reticulons localize to the edges of ER sheets

(A) The localization of endogenous Rtn4a and 4b is compared with that of Climp63 using indirect immunofluorescence with specific antibodies in COS7 cells. The lower row panels show enlargements of the boxed region. Arrows point to reticulons lining the sheets. The right most panel shows merged images. (B) As in (A), but with cells overexpressing FLAG-Climp63. (C) GFP-Rtn1p (green) and ssRFP-HDEL (red) were co-expressed in wild type S. cerevisiae cells and the cortical ER was visualized by fluorescence microscopy. Scale bar, 5 μm. (D) As in (C), except that the cells had proliferated ER sheets caused by deletion of SEY1 and YOP1 (sey1Δyop1Δ). (E) As in (C), except the cells had proliferated ER sheets caused by deletion of OPI1 (opi1Δ). The cells also contained an empty vector as a control for panels (F) and (G). (F) As in (E), except that untagged Rtn1p was expressed under the endogenous promoter from a CEN plasmid. (G) As in (E), except that untagged Rtn1p was expressed under the endogenous promoter from a 2μ plasmid. (H) Quantification of the experiments in (C) and (E–G). The relative area of ER sheets was determined from the area of ssRFP-HDEL fluorescence that did not co-localize with GFP-Rtn1p fluorescence and divided by the total area of ssRFP-HDEL fluorescence. 14 to 38 cells were analyzed and the mean and standard error were calculated.

Figure 7. Modeling of the effect of curvature-stabilizing and sheet-promoting proteins on ER morphology

(A) The reticulons and DP1/Yop1p (yellow arcs) are assumed to localize exclusively to tubules and sheet edges, generating and stabilizing these high curvature membranes. Stabilization of sheet edges enables the upper and lower membranes of the sheet to adopt planar shapes. (B) Top view of membrane shapes computed by the theoretical model for increasing Γ values. The computation was performed for a total membrane area corresponding to 1μm radius of the initial disc-like shape, a 15nm cross-section radius of the tubules and edges, and a 40 nm optimal distance between the arc-like proteins at the edge (see Supplemental Information). Change of Γ from 1 to 2.1(blue to red) corresponds to increasing the number of curvature-stabilizing proteins N_c from 140 to 290. (C) Γ values and membrane shapes were calculated for different numbers of curvature-stabilizing and sheet-promoting proteins, N_c and N_s. The colors correspond to the membrane shapes shown in Figure 7B. The colored lines on the bottom plane of the diagram represent the relationship between N_c and N_s for a given shape of the system. (D) Γ values and membrane shapes were computed for different N_s values at N_c = 290. The shapes refer to N_s = 0, 500, and 1000.