Fig 1.
ALPS motifs confer organelle selectivity to a naïve protein.
(A) Schematic of the GMAP-ACC-GFP, GFP133 and Sar1-GFP constructs. (B) Confocal images of live U2OS cells expressing GMAP-ACC-GFP, GFP133 or Sar1-GFP. (C-D) U2OS cells were transfected with GMAP-ACC-GFP, fixed and stained with the cis-Golgi marker anti-GM130. Cells were then imaged by confocal microscopy (C) or structured illumination (D). (D) Upper panels show a maximum projection of a Z-stack, lower panels are single cross-sections. Scale bars are 10μm unless otherwise stated.
Fig 2.
GFP133 and Sar1-GFP recapitulate membrane curvature sensitivity of native proteins.
(A) Confocal image of a cell co-expressing GFP133 and the ER marker Sec61-mCherry. Arrowheads indicate flat cisternae. (B) Confocal image of a cell co-expressing Sec61-GFP and Sec61-mCherry. Arrowheads indicate flat cisternae. (C) Fluorescence intensities of GFP133 and Sec61-mCherry (right panel) along a line spanning an ER tubule and an ER flat sheet (in yellow in the left panel). (D) Several plots as in (C) were analysed in cells co-expressing GFP133 and Sec61-mCherry or Sec61-GFP and Sec61-mCherry. For each plot, paired Student t-tests were run to assess if the GFP and mCherry intensities are significantly different. This analysis was done on plot portions spanning cytoplasm (base line), cisternae or tubules. The dashed red line indicates a p-value of 0.05. Black lines represent median values. (E) Cross-section (left) and nuclear surface (right) of a cell co-expressing GFP133 and Sec61-mCherry. The cyan line delineates the nuclear surface. (F) Cross-section (left) and nuclear surface (right) of a cell expressing Sar1-GFP. (G) Representative intensity plots of GFP133 (upper panel) or Sar1-GFP (lower panel) along a line spanning a portion of nuclear envelope and a portion of cytoplasm. (H) Fluorescence intensity of Sar1-GFP (right panel) along a line spanning ER tubules and an ER flat sheets (in cyan in the left panel). Scale bars are 10μm unless otherwise stated.
Table 1.
Comparison of ALPS motifs and canonic amphipathic helices.
Fig 3.
Reducing its hydrophobicity does not increase membrane curvature sensitivity of Nup133 ALPS motif.
(A) Amino-acid sequence of the ALPS motif of Nup133. The strongest hydrophobic region is highlighted in yellow, and the secondary region is framed in red. The hydrophobic residues mutated in this study are indicated by arrows. (B) L252 in GFP133 was mutated to less hydrophobic residues, namely Valine, Alanine and Glycine. (C) F263 in GFP133 was mutated to less hydrophobic residues, namely Valine, Alanine and Glycine. Scale bars are 10μm. (D) Normalized GLCM contrasts, as an indication of membrane-bound fractions, were measured in peripheral regions of cells transfected with the indicated constructs. Horizontal bars represent median values.
Fig 4.
Increasing its hydrophobicity does not impair membrane curvature sensitivity of Nup133 ALPS motif.
(A) V259 and I265 of GFP133 were mutated to bulkier hydrophobic residues. (B) Intensity plots of GFP133 / Sec61-mCherry along a line spanning an ER tubule and an ER flat sheet. (C) Cross-section and nuclear surface of a cell co-expressing GFP133 and Sec61-mCherry. All scale bars are 10μm. (D) Normalized GLCM contrasts measured in peripheral regions of cells transfected with the indicated constructs. Horizontal bars represent median values. (E) For each mutant, we calculated the side chain volume ratio of the mutated to wt residues, and the average of the corresponding normalized contrast ratios. Contrast ratios were then plotted as a function of volume ratios. Error bars are standard deviations.
Fig 5.
Altering the charges of ALPS Nup133 does not change its specificity.
(A) Helical projection of Nup133 ALPS motif, showing the position of the two basic residues. The projection was generated by the Heliquest software (http://heliquest.ipmc.cnrs.fr). (B) Confocal images of live cells expressing GFP133 and mutants of lysine K258. (C) Confocal images of live cells expressing GFP133 and mutants where the R257 residue has been mutated to uncharged residues. (D) Neutralizing or adding a negative charge at the interface between the polar and hydrophobic faces of the helix reduces its binding to membranes. Scale bars = 10μm. (E) Normalized GLCM contrasts of mutated constructs, as indicated. Bars are median values.
Fig 6.
Geometry of the ALPS motif, imposed by the backbone, influences Nup133 ALPS motif localization.
(A) Confocal images of live cells transfected with GFP133, 133-GFP, GFP-133 or 133-ACC-GFP. (B) Confocal images of cells transfected with GFP133, 133-ACC-GFP or GMAP-ACC-GFP, fixed and stained with anti-GM130 (red). Note that fixation alters ER morphology. (C) Confocal images of live cells transfected with GMAP-ACC-GFP, GFP-GMAP or GFP-GMAP-GMAP. (D) Confocal images of live cells co-transfected with Sec61-mCherry and GMAP-ACC-GFP or GFP-GMAP-GMAP. Scale bars = 10μm.
Fig 7.
The coiled-coil domain provides membrane curvature sensitivity to Sar1 AH.
(A) Confocal images of live cells transfected with Sar1-GFP or Sar1-ACC-GFP. (B) Confocal images of cells transfected with Sar1-GFP or Sar1-ACC-GFP, fixed and stained with anti-GM130 (red). (C) Confocal image of a cell co-transfected with Sar1-ACC-GFP and Sec61-mCherry. Arrowheads indicate ER cisternae. (D) Relative intensities of Sar1-ACC-GFP and Sec61-mCherry along a line spanning ER tubules and a cisternae (shown in cyan in left panel). (E) Confocal images of the nuclear surface of cells expressing Sar1-GFP or Sar1-ACC-GFP. On the right are shown representative intensity plots along a segment spanning portions of nuclear envelope and cytoplasm. Scale bars = 10μm.
Fig 8.
Schematic cellular localization of Sar1 AH and Nup133 ALPS motif within various backbones.
This diagram illustrates that addition of the ACC domain increases membrane curvature sensitivity of the considered helices. But importantly, this gain remains correlated to their initial degree of membrane curvature sensitivity. This supports that the AH physico-chemical properties are determinant for membrane curvature sensitivity even when they are modulated by the surrounding backbone.