Wang N et al. (2021), Mechanism of membrane-curvature generation by E...

XB-ART-57756

Nat Commun 2021 Jan 25;121:568. doi: 10.1038/s41467-020-20625-y.

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Mechanism of membrane-curvature generation by ER-tubule shaping proteins.

Wang N , Clark LD , Gao Y , Kozlov MM , Shemesh T , Rapoport TA .

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The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane. When overexpressed or reconstituted at high concentrations with phospholipids, REEPs cause extreme curvature through their TMs, generating lipoprotein particles instead of vesicles. The APH facilitates curvature generation, as its mutation prevents ER network formation of reconstituted proteoliposomes, and synthetic L- or D-amino acid peptides abolish ER network formation in Xenopus egg extracts. In Schizosaccharomyces japonicus, the APH is required for reticulon's exclusive ER-tubule localization and restricted mobility. Thus, the TMs and APH cooperate to generate high membrane curvature. We propose that the formation of splayed REEP/reticulon dimers is responsible for ER tubule formation.

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Species referenced: Xenopus laevis
Genes referenced: mtor ncr3 reep1 rtn1
GO keywords: endoplasmic reticulum

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	Fig. 1. REEPs form dimers.a Proposed membrane topology of the REEPs and Rtns. The four hydrophobic transmembrane segments (TMs) are numbered and shown in blue; the predicted amphipathic helix (APH) is shown in purple. b SEC of S. japonicus Yop1 and Xenopus laevis REEP5 purified in the detergent DDM. The experiment was performed four times. The top panel shows the absorbance at 280 nm. Fractions of the eluate were analyzed by SDS-PAGE, and stained with Coomassie blue (lower panels). c SEC-MALS analysis of purified REEP5 and Yop1-3C-SBP, analyzed in DDM. Absorbance at 280 nm (UV) was normalized relative to the maximum reading of REEP5 (left axis). The calculated protein mass is shown across the peaks (right axis). The table on top shows the calculated and predicted monomer masses. d SBP-tagged full-length Yop1 or Yop1 lacking the APH (Yop1ΔAPH) was co-expressed with His-tagged full-length Yop1 or Yop1ΔAPH. DDM-solubilized membrane fractions were incubated with streptavidin beads and the bound material analyzed by SDS-PAGE and Coomassie-blue staining. As a control, SBP- and His-tagged Yop1 were individually expressed and purified. The experiment was performed three times.
	Fig. 2. REEP/Yop1 monomers interact in membranes through TM2.a Photoreactive Bpa probes were incorporated into Yop1 at the indicated positions of different TMs. The purified protein was irradiated with UV light either in DDM or after reconstitution in liposomes containing S. cerevisiae lipids at a 1:200 molar ratio of protein to lipid. The samples were analyzed by SDS-PAGE and Coomassie-blue staining. The asterisk indicates the position of the cross-linked Yop1F65Bpa dimer. The right panel shows quantification of the intensity of dimer cross-links relative to total protein. b As in a, but with mutants in the APH reconstituted into proteoliposomes. c Quantification of experiments as carried out in b. Data are presented as mean ± the standard deviation (SD) from n = 10 (wt) and n = 3 (I145R, I145K, I145E, ΔAPH) independent experiments. d Dimer formation was tested with purified REEPs from Schizosaccharomyces japonicus (S. jap), Xenopus laevis (X. lae), Thermothelomyces thermophila (T. the) and Thielavia terrestris (T. ter). In each case, a Bpa probe was incorporated at the position of the conserved Phe residue in TM2. The samples were analyzed as in a. The asterisks show the positions of cross-linked dimers.
	Fig. 3. Reconstitution of purified REEPs generates high-curvature structures.a Yop1 or Yop1ΔAPH were purified from DDM-solubilized membranes and reconstituted with S. cerevisiae lipids at a molar ratio of 1:200 protein:lipid. The samples were analyzed by negative-stain EM. Short tubules (1), vesicles with diameters <12 nm (2), vesicles with diameters 12–30 nm (3), and vesicles with diameter >30 nm (4) are highlighted. b Quantification of experiments as shown in a (n = 2664 for wt and 2632 for ΔAPH from three independent experiments). c Yop1 or Xenopus REEP5 were reconstituted with S. cerevisiae lipids at a molar ratio of 1:200 protein:lipid. The samples were subjected to flotation in a Nycodenz gradient, and fractions were analyzed by SDS-PAGE and Coomassie-blue staining. The band at the bottom is lipid. d The indicated fractions of the Nycodenz flotation of Yop1 in c were analyzed by negative-stain EM. e Negative-stain EM analysis of Xenopus REEP5 after reconstitution, but before flotation. f Fractions of the Nycodenz flotation of Xenopus REEP5 in c were analyzed by negative-stain EM. Dashed circles in e and f highlight LPPs budding off from large proteoliposomes. All experiments were performed three times. Bars, 100 nm.
	Fig. 4. Overexpression of REEP/Yop1 in E. coli generates lipoprotein particles (LPPs).a SBP-tagged REEP5, REEP5ΔAPH, or Sey1 were expressed in E. coli. Lysates (L) were fractionated by centrifugation into a non-sedimentable fraction (NS) and membrane fraction (M). Equivalent aliquots of these fractions were analyzed by SDS-PAGE, followed by blotting with Dylight 800-labeled streptavidin. b Quantification of the data in a. The experiment was performed two times. c Xenopus REEP5 was expressed in E. coli and purified from the NS fraction. It was then subjected to size-exclusion chromatography (SEC) and its elution followed by the absorbance at 280 nm. The inset shows a representative negative-stain image of the peak fraction. The visualized structures are referred to as lipoprotein particles (LPPs). Bar, 100 nm. d Fractions of the SEC run in c were analyzed by SDS-PAGE and Coomassie-blue staining. The experiment was performed four times. e Lipids were extracted from the purified LPPs, and different amounts of the extract were analyzed by thin-layer chromatography (TLC) and Primuline-staining. Pure PC, PS, PE, PI, and PG were run in parallel as markers. The asterisk indicates an unidentified lipid. The experiment was performed two times. f Purified LPPs were co-reconstituted with the GTPase Sey1 into liposomes containing POPC, DOPE, DOPS, and fluorescent rhodamine-PE (Rh-PE). The samples were incubated with or without GTP and visualized with a fluorescence microscope. The experiment was performed two times. Bar, 10 µm.
	Fig. 5. The APH of REEPs is required for the reconstitution of a tubular network.a Xenopus REEP5 or REEP5 lacking the APH (REEP5ΔAPH) were purified from DDM-solubilized membranes and co-reconstituted with the GTPase Sey1 into liposomes containing POPC, DOPE, DOPS, and fluorescent Rh-PE. The samples were incubated with or without GTP, as indicated. The experiment was performed four times. b As in a, but with purified wild-type Yop1, a Yop1 mutant lacking the APH (Yop1ΔAPH), Yop1 variants with point mutations in the APH (Yop1(I145S) and Yop1(I145K)), or a Yop1 variant, in which the APH was replaced by that of sjRtn1 (Yop1ΔAPH-APHRtn1). Bars (on the right), 10 µm. The experiment was performed two times.
	Fig. 6. Synthetic APH peptides bind to liposomes and generate high curvature.a Peptides corresponding to the APH of wild-type Yop1, to APH harboring a point mutation (Yop1 I145K), or to the APH of REEP5, with either L- or D-amino acids (REEP5 L-APH and REEP5 D-APH), were incubated with liposomes containing S. cerevisiae lipids at a 1:40 molar ratio of peptide to lipid. The samples were subjected to flotation in a Nycodenz gradient and analyzed by silver staining. The experiment was performed two times. The sequences of the peptides are shown in Supplementary Table 3. b The indicated peptides were analyzed by CD spectroscopy in the buffer, DDM, or after incubation with liposomes. The experiment was performed two times. c Yop1 was reconstituted into proteoliposomes and incubated with the indicated peptides. The samples were analyzed by negative-stain EM. Bar, 100 nm. d Quantification of the structures seen in c. Short tubules (1), vesicles with diameters <12 nm (2), vesicles with diameters 12–30 nm (3), and vesicles with diameter >30 nm (4) were counted (n = 1662 for a buffer, 1490 for Yop1 APH, and 1800 for Yop1 I145K from two independent experiments). e Alexa 546-labeled Yop1 (Yop1A546) was reconstituted into liposomes containing S. cerevisiae lipids at a molar ratio of 1:200 protein to lipid. The samples were incubated with buffer or Yop1 peptide (1:20 molar ratio of peptide to lipid) and subjected to flotation in a Nycodenz gradient. Fractions were analyzed with a fluorescence scanner. The lower band corresponds to lipid. The experiment was performed three times.
	Fig. 7. APH peptides abolish membrane networks in a reconstituted system and Xenopus egg extracts.a Alexa 488-labeled Yop1 (Yop1A488) was co-reconstituted with the GTPase Sey1 into liposomes containing S. cerevisiae lipids and fluorescent Rh-PE at the protein to lipid ratios given on the left. Synthetic peptides corresponding to the APH of wild-type Yop1 or to a mutant in the APH (Yop1 I145K) were added at the molar ratios (peptide to lipid) given on the top. All samples were incubated with Mg2+ and GTP, added 10 min before the peptides. The samples were analyzed for fluorescence of lipid and protein. Arrows point to residual short tubules. The experiment was performed two times. b An interphase tubular network was generated with Xenopus egg extract and stained with the fluorescent dye DiD. The network was visualized with a fluorescence microscope with excitation at 642 nm. Bar, 10 µm. c As in b, but in the presence of 100 µM of a synthetic peptide corresponding to wild-type Yop1 (Yop1 APH). d As in b, but with a peptide carrying a point mutation in the APH (Yop1 I145K). e As in b, but with a peptide corresponding to the APH of wild-type Xenopus REEP5 (REEP5 L-APH). f As in e, but with a peptide containing D-amino acids (REEP5 D-APH). These experiments were performed four times. g Network generated with Xenopus egg extract in metaphase. h As in g, but in the presence of REEP5 L-APH. i As in g, but in the presence of REEP5 D-APH. These experiments were performed three times.
	Fig. 8. The APH is required for Rtn1’s exclusive ER-tubule localization and restricted mobility in S. japonicus cells.a Fusions of mNeonGreen with wild-type Rtn1 or APH mutants (ΔAPH, and I388K, I392K) were expressed in S. japonicus cells under rtn1’s native promoter. The cells also expressed a mCherry-fusion of Pom152 as a marker for nuclear pores and the nuclear envelope (NE). The cells were visualized with a confocal fluorescence microscope. Shown are representative images focused on the center of the cells obtained from three independent experiments. Arrowheads point to NE-localized mutant Rtn1. b Quantification of total fluorescence intensity. Background fluorescence acquired from untagged cells was subtracted. The total fluorescence of the mutants was normalized to that of wild-type cells. c Quantification of NE-localized fluorescence intensity. The Rtn1 fluorescence co-localizing with Pom152 was determined, the cytoplasmic background was subtracted, and the difference divided by the total intensity. Data are presented as mean ± SD of n = 49 (wt), 43 (ΔAPH), and 46 (I388K, I392K) cells for each strain from two independent experiments. d Cells expressing Rtn1-mNeonGreen or Rtn1ΔAPH-mNeonGreen were bleached at their periphery and the recovery of fluorescence followed over time (FRAP). The box shows the bleached area. The lower panels show magnified views. The arrows point to the bleached region of the peripheral ER. Bars, 10 µm. e Quantification of FRAP experiments. Fluorescence intensity before and immediately after bleaching was set to 100% and 0%, respectively. Error bars are standard error of the mean (SEM) of each time point from nine Rtn1-mNeonGreen-expressing cells and ten Rtn1ΔAPH-mNeonGreen-expressing cells. f Recovery half-times (± SEM) were calculated from FRAP experiments, such as shown in d. The analysis for Rtn1, Rtn1ΔAPH, Yop1, and Yop1 (I145K) is based on 9, 10, 14, 11 cells from three independent experiments, respectively. g Model for membrane curvature generation by REEP dimers. The TMs of REEPs can form either straight dimers (monomers represented by green and blue rods) that span the lipid bilayer (gray) or splayed dimers that sit predominantly in the outer leaflet of the bilayer. The APH (red/orange circle) promotes the formation of splayed dimers by generating local curvature through hydrophobic insertion. APH mutants can still generate membrane curvature when their local concentration is sufficiently high.

References [+] :

Aoki, Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus. 2010, Pubmed