Geometry of the ALPS motif, imposed by the backbone, influences Nup133 ALPS motif localization.

<p>(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.</p

GFP¹³³ and Sar1-GFP recapitulate membrane curvature sensitivity of native proteins.

<p>(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.</p

Comparison of ALPS motifs and canonic amphipathic helices.

<p>Comparison of ALPS motifs and canonic amphipathic helices.</p

Increasing its hydrophobicity does not impair membrane curvature sensitivity of Nup133 ALPS motif.

<p>(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.</p

Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone

<div><p>Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests that the chemistry of ALPS motifs is a key parameter for membrane curvature sensitivity, which can be further modulated by the surrounding protein backbone.</p></div

Altering the charges of ALPS Nup133 does not change its specificity.

<p>(A) Helical projection of Nup133 ALPS motif, showing the position of the two basic residues. The projection was generated by the Heliquest software (<a href="http://heliquest.ipmc.cnrs.fr/" target="_blank">http://heliquest.ipmc.cnrs.fr</a>). (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.</p

ALPS motifs confer organelle selectivity to a naïve protein.

<p>(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 <i>cis</i>-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.</p

Schematic cellular localization of Sar1 AH and Nup133 ALPS motif within various backbones.

<p>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.</p

The coiled-coil domain provides membrane curvature sensitivity to Sar1 AH.

<p>(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.</p