Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains
- Laboratoire de Biogenèse Membranaire (LBM), Unité Mixte de Recherche UMR 5200, CNRS, Université de Bordeaux, France
- GX ABT, Université de Liège, Belgium
- Institute of Chemistry and Biology of Membranes and Nanoobjects (UMR5248 CBMN), CNRS, Université de Bordeaux, Institut Polytechnique Bordeaux, France
- Université de Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, France
- Interdisciplinary Institute for Neuroscience, CNRS, University of Bordeaux, France
- LIPM, Université de Toulouse, INRA, CNRS, France
- Agroécologie, AgroSup Dijon, INRA, Université Bourgogne Franche-Comté, F-21000 Dijon, ERL 6003 CNRS, France
Figures
Figure 1 with 6 supplements
REMORIN localization into highly ordered PM nanodomains is mediated by sterols and PI4P.
(A) Explanatory schematic of the secant or surface views of N. benthamiana leaf abaxial epidermal cell plasma membrane (PM) used throughout the article. (B) Confocal imaging surface views of Nicotina…
Figure 1—figure supplement 1
Sequence alignment of 51 group 1 REMORIN C-terminal Anchor sequences.
https://doi.org/10.7554/eLife.26404.003
Figure 1—figure supplement 2
Nicotiana benthamiana Group 1b REMORINs are expressed in leaf epidermal cells, encode for PM nanodomain localized proteins and are functional homologs of StREM1.3 toward PVX propagation.
(A) REMORIN genes of N. benthamiana expressed in Reads Per Kilobase of transcripts per million mapped reads (RPKM). RNAseq data were retrieved using SRA toolkit (see experimental section). (B) …
Figure 1—figure supplement 3
YFP-StREM1.3 is targeted to the PM-domains by a mechanism independent of the COP-I/COP-II secretory pathway.
(A) Secant view of confocal dual-colour imaging of N. benthamiana expressing YFP-StREM1.3 or proton pump PMA4-GFP (used as positive control) with or without dominant-negative Sar1H52N / SKL-CFP 24 …
Figure 1—figure supplement 4
Spatial clustering index calculated as the max-to-min ratio of fluorescence intensity in the PM.
(A, B). Surface view confocal images of N. benthamiana epidermal cells expressing YFP-StREM1.3 or PMA4-GFP 48 hr after agroinfiltration. (C) Fluorescence Intensity plots through the indicated lines. …
Figure 1—figure supplement 5
Modification of the sterol pool of N.benthamiana leaves by the drug Fenproprimorph (fen).
(A) Quantification by GC-MS of the Δ5 phytosterols and cycloartenol from control and fen-treated N. benthamiana leaves (n = 3, error bars indicate SEM). Cycloartenol accumulation is similar to what …
Figure 1—figure supplement 6
Myristoylation and Palmitoylation (MAP)-mTurquoise2-SAC1p localizes at PM of N.benthamiana leaf epidermal cells and specifically depletes PM PI4P but not PI(4,5)P2 or PS.
(A) Secant view confocal images of N. benthamiana leaf epidermal cells expressing either Dead or Active MAP-mTURQUOISE2-SAC1p from yeast constructs and P19 to increase expression (Baulcombe and …
Figure 2 with 4 supplements
| REMORIN C-terminal anchor peptide is an unconventional PM-binding domain embedded in the bilayer that folds upon specific lipid interaction.
(A) Primary sequence of StREM1.3 showing the two putative regions 1 and 2 (R1 and R2) composing the REM-CA. Hatched domain represents the putative coiled-coil helix. (B) Order parameter of the …
Figure 2—figure supplement 1
Solution NMR and 31P and 2H solid-state NMR analysis.
Thin-layer chromatography analysis of Phosphoinositides mix (PIPs). (A) REM-CA folds in alpha helix in hydrophobic environment. 2D 1H-15N correlation spectra of REM-CA recorded at 800MHz using the …
Figure 2—figure supplement 2
In silico analysis of REM-CA from StREM1.3 suggests the existence of two distinct structural regions.
(A) Primary sequence of StREM1.3. (B) Sequence and predicted structure of the StREM1.3 REM-CA peptide predicted by different methods, the consensus secondary structure prediction is indicated below; …
Figure 2—figure supplement 3
Biophysical studies evidence the interaction of REM-CA with lipids.
(A) Plots of the maximal surface pressure variation (ΔΠ) vs. the initial surface pressure (Πi) and of the corresponding maximal insertion pressure (MIP) obtained from the adsorption experiments with …
Figure 2—figure supplement 4
Molecular dynamics (MD) simulation reveals interactions between REM-CA residues and lipids in the ternary lipid mixture.
(A) Snapshots of MD simulations of REM-CA in the presence of a bilayer composed of 1-palmitoyl-2-linoleyl-sn-glycerol-3-phosphocholine (PLPC), sitosterol and PI4P (also see Video 1). (B) Atomistic …
Figure 3
Positively charged residues of REMORIN C-terminal anchor are essential for PM targeting.
(A) Sequence Logo obtained from 51 Group 1 REM-CA sequences presented in Figure 1—figure supplement 1, and StREM1.3 REM-CA sequence. (B) Summary of the 20 REM-CA mutants of StREM1.3 generated in …
Figure 4 with 1 supplement
Positively charged residues of REMORIN C-terminal Anchor are essential for PM nanodomain localization and REMORIN function in cell-to-cell permeability.
(A) Surface view confocal images of the localization of REM-CA single and double mutants. Scale bar, 10 µm. (B) Tukey boxplot showing the Spatial Clustering Index of the REM-CA single and double …
Figure 4—figure supplement 1
Effect of StREM1.3 REM-CA mutant over-expression on plasmodesmata permeability and PVX cell-to-cell movement.
(A) Plasmodesmata permeability test performed by visualizing cell-to-cell movement of GFP from a single-cell to its neighbors. Epifluorescence microscopy images represent a single-cell where GFP has …
Figure 5
REMORIN C-terminal anchor defines protein mobility in the PM.
(A) Super-resolved trajectories (trajectories > 20 points;) of EOS-StREM1.3 WT and REM-CA mutants (K183S, K192A, K183S/K192A and K192A/K193A) visualized by high-resolution microscopy spt-PALM VAEM. …
Figure 6
REMORIN C-terminal anchor defines protein segregation in nanodomains.
(A) Live PALM analysis of molecules localization by tessellation-based automatic segmentation of super-resolution images. (B) Diameter distributions of the cluster of EOS fusion proteins (line shows …
Videos
Video 1
Molecular dynamics (MD) simulation reveals interactions between REM-CA residues and lipids in the ternary lipid mixture.
MD coarse-grained simulations propose a model of the insertion of REM-CA in the lipid bilayer (PLPC/PI4P/sitosterol), where peptide-lipid interaction would be mediated by the interaction of REM-CA …
Video 2
Live-cell single-particle tracking-photoactivable localization microscopy in variable angle epifluorescence microscopy mode.
RAW DATA spt-PALM VAEM was performed on N. benthamiana leaf epidermal cells expressing EOS-StREM1.3.
