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Membrane shape or geometrical curvature emerged recently as a potent regulator of membrane recruitment during protein trafficking and sorting. Cellular membranes display distinct curvature geometries e.g. spherical (trafficking- and synaptic vesicles) or cylindrical (tubes in the ER and Golgi), however quantitative studies of protein recruitment by membrane curvature typically focus on a single geometry. Thus the biological implications of different curvature geometries remain largely unexplored.We recently used our single liposome assay to show that the N-Ras lipid anchor (tN-Ras) is recruited by spherical membrane curvature. Here we report the development of a novel membrane tube assay, allowing us to quantitatively compare the recruitment of tN-Ras by spherical and cylindrical membrane curvature. Furthermore we expand the study to include representatives of the four most common families of membrane-binding domains (MBDs); the lipid anchor of N-Ras (tN-Ras), the C2AB-domain of Synaptotagmin1 (Syt), AnnexinB12 (Anx) and Amphiphysin NBAR (Amph). Our data revealed an increased recruitment of all four MBDs by spherical as compared to cylindrical curvature. Molecular field theory calculations attributed this trend to the greater perturbation of lipid packing parameters by spherically curved membranes. Importantly, the four MBDs displayed markedly distinct abilities to discriminate the two curvature geometries e.g. tN-Ras had a moderate 2-fold discrimination while Amph a remarkable absolute preference for spherical membranes. This demonstrated that discrimination of curvature geometry can be modulated and likely adapted to specific biological functions. Our results reveal membrane curvature geometry as a novel regulator of protein recruitment during trafficking and sorting for a plethora of membrane-binding proteins, and thus suggest a novel functional role to the diversity of conserved organelle morphologies.

Precise control of the oligomeric state of proteins is of central importance for biological function and for the properties of biopharmaceutical drugs. Here, the self-assembly of 2,2'-bipyridine conjugated monomeric insulin analogues, induced through coordination to divalent metal ions, was studied. This protein drug system was designed to form non-native homo-oligomers through selective coordination of two divalent metal ions, Fe(II) and Zn(II), respectively. The insulin type chosen for this study is a variant designed for a reduced tendency toward native dimer formation at physiological concentrations. A small-angle X-ray scattering analysis of the bipyridine-modified insulin system confirmed an organization into a novel well-ordered structure based on insulin trimers, as induced by the addition of Fe(II). In contrast, unmodified monomeric insulin formed larger and more randomly structured assemblies upon addition of Fe(II). The addition of Zn(II), on the other hand, led to the formation of small quantities of insulin hexamers for both the bipyridine-modified and the unmodified monomeric insulin. Interestingly, the location of the bipyridine-modification significantly affects the tendency to hexamer formation as compared to the unmodified insulin. Our study shows how combining a structural study and chemical design can be used to obtain molecular understanding and control of the self-assembly of a protein drug. This knowledge may eventually be employed to develop an optimized in vivo drug release profile.

Ras proteins are small GTPases that are post-translationally modified by the attachment of lipid moieties (1). This modification is essential for the correct trafficking and sorting of Ras proteins through the vesicular pathway from the Golgi to the plasma membrane (2).Traditionally the sorting of Ras is primarily discussed in the context of membrane domains in flat membranes, neglecting the influence of membrane shape. Recently we have demonstrated, utilizing our single liposome curvature (SLiC) assay that the minimal anchoring motif of N-Ras (tN-Ras) up-concentrates in areas of high membrane curvature (not published), suggesting that curvature might act as a cue for the spatial localization of Ras proteins.In the SLiC assay, curvature-sensing molecules are added from aqueous solution to vesicles of different curvatures, but the vesicles are not in diffusive contact (3, 4). In vivo Ras proteins are anchored to membranes and laterally sorts between curved and planar membranes, which are in diffusive contact. To study the curvature-sensing ability of tN-Ras in a setup mimicking the in vivo scenario we developed a membrane tube based assay in which the tubes are in diffusive contact with a lipid bilayer.Membrane tubes are formed by heating a confined lipid bilayer (5). The tubes eventually adsorb to the flat membrane, which enable imaging by confocal fluorescence microscopy. After addition of tN-Ras we observed a preferential sorting into curved tubes rather than the flat bilayer. This observation further implies a pivoting role of membrane shape as a regulator of Ras-protein localization.1. Prior & Hancock, Semin Cell Dev Biol 23:145(2012).2. Choy et al., Cell 98:69(1999).3. Kunding et al., Biophysical Journal 95:1176(2008).4. Hatzakis et al., Nat Chem Biol 5:835(2009).5. Weirich & Fygenson, Poster Abstract 2731, Biophys. Soc. 2011.