Monomeric synucleins generate membrane curvature - PubMed (original) (raw)

Monomeric synucleins generate membrane curvature

Christopher H Westphal et al. J Biol Chem. 2013.

Abstract

Synucleins are a family of presynaptic membrane binding proteins. α-Synuclein, the principal member of this family, is mutated in familial Parkinson disease. To gain insight into the molecular functions of synucleins, we performed an unbiased proteomic screen and identified synaptic protein changes in αβγ-synuclein knock-out brains. We observed increases in the levels of select membrane curvature sensing/generating proteins. One of the most prominent changes was for the N-BAR protein endophilin A1. Here we demonstrate that the levels of synucleins and endophilin A1 are reciprocally regulated and that they are functionally related. We show that all synucleins can robustly generate membrane curvature similar to endophilins. However, only monomeric but not tetrameric α-synuclein can bend membranes. Further, A30P α-synuclein, a Parkinson disease mutant that disrupts protein folding, is also deficient in this activity. This suggests that synucleins generate membrane curvature through the asymmetric insertion of their N-terminal amphipathic helix. Based on our findings, we propose to include synucleins in the class of amphipathic helix-containing proteins that sense and generate membrane curvature. These results advance our understanding of the physiological function of synucleins.

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Figures

FIGURE 1.

Quantitative proteomics reveals the repertoire of proteins exhibiting increased levels in αβγ-synuclein KO synapses. A, DIGE gel of the synaptic vesicle (LP2) fraction. The image is a scan of the αβγ-synuclein KO sample, and the spots corresponding to the location of α- and β-synuclein are highlighted in red. A total of 6 DIGE experiments were performed and quantitatively compared all synaptic fractions. On average, we analyzed ∼50 proteins/DIGE experiment. B, volcano plot of iTRAQ experiment on synaptic cytosol fraction (LS2). The frequency distribution of peptides showing differences between wild type and αβγ-synuclein KO samples, plotted as log10(WT/KO), are overlaid with the protein score for each protein. The largest changes were observed for α- and β-synuclein and are noted. Note that most protein levels are unchanged and thus center around log10(WT/KO) = 0. A total of 6 iTRAQ experiments were conducted, and we analyzed ∼500 proteins/iTRAQ experiment. C, table showing proteins that were increased in αβγ-synuclein KO synapses. Only proteins that were increased beyond cut-offs whose change was replicated in at least two biological and technical replicates are included. The average fold increase ± S.E., the number of peptides used to identify a given protein, the method by which the protein change was determined, if the change was validated, the lipid binding properties, and known functions of the proteins are listed. We validated the increase in protein levels for endophilin A1, endophilin B2, annexin A5, and synapsin IIb by quantitative immunoblotting, which is indicated by a + sign (See Fig. 2 and Ref. 12).

FIGURE 2.

Proteomic analysis reveals that synucleins and endophilin A1 levels are inversely related. A, validation of endophilin levels by quantitative immunoblotting. Wild type (green bar) and αβγ-synuclein KO (black bar; n = 4/genotype) brain homogenates (20 μg protein) were separated on SDS-PAGE gels and immunoblotted for the denoted proteins. The levels of individual proteins were determined by quantitative Western blotting with IRDye conjugated secondary antibodies on a LI-COR infrared imaging system, using actin and tubulin as internal loading controls. Abbreviations: Endo, endophilin; *, p < 0.05, **, p < 0.01. B, representative Western blot of synaptic protein expression of endophilin A1, annexin A5, and FCHO1 in age-matched αβγ-synuclein KO (αβγ −/−), wild type (αβγ +/+), transgenic human (αβγ −/−; htg) and mouse (αβγ −/−; mtg) α-synuclein overexpression mouse models (n = 3/genotype). Actin is used as a loading control. C, quantification of endophilin A1 levels in αβγ-synuclein KO, wild type, and α-synuclein overexpression mouse models. *, p < 0.05, **, p < 0.01. D, comparison of synaptic protein expression shows an inverse relationship between α-synuclein and endophilin A1 levels. r2 = 0.848. See

supplemental Fig. S2

for a similar analysis using total brain homogenates. E, levels of FCHO1 are unchanged as a function of α-synuclein levels. r2 = 0.119.

FIGURE 3.

All synucleins generate membrane curvature in vitro. Images of liposomes only and liposomes mixed with BSA, α-, β-, γ-synuclein and endophilin A1. Tubules were generated by α-, β-, γ-synucleins and endophilin A1 (1.4 μ

) from rhodamine-labeled liposomes composed of either: A, 50% DOPE/40% DOPS/10% cholesterol (1 mg/ml) or B, brain polar lipid (BPL; 1 mg/ml). Scale bar = 1 μm and applies to all images in a given row. C, quantification of tubulation activity, as measured by the ratio of tubule area to total lipid area for BSA, synucleins, and endophilin A1 using BPL-derived liposomes. BSA and endophilin A1 were used as negative and positive controls, respectively. Liposome-only and synuclein ratios are calculated from 900 images each, BSA and endophilin A1 from 540 images each; *, p < 0.05; **, p < 0.01 for comparisons to the liposomes only condition. D, Alexa-fluor 488-labeled α-synuclein localizes on the membrane tubules generated. Scale bar = 1 μm.

FIGURE 4.

Electron microscopic analysis of synuclein-generated tubules. Representative electron micrographs of membrane tubules generated by: A, liposomes; B, α-synuclein; C, β-synuclein; D, γ-synuclein; and E, endophilin A1 using the 50%DOPE/40%DOPS/10% cholesterol liposomes. Scale bar = 100 nm. Similar results were obtained in three independent experiments.

FIGURE 5.

Concentration dependence of α-synuclein generated membrane tubulation. A, dose dependence of α-synuclein-mediated membrane tubulation. The tubulation activity of α-synuclein at the denoted protein concentrations was quantified as described in Fig. 3. Membrane tubulation shows a linear relationship to α-synuclein concentration until ∼7 μ

and saturates around 15 μ

. Ratios are calculated from at least 540 images for each concentration. The green-shaded area represents the concentration of α-synuclein at the synapse. B, representative Western blot of α-synuclein standards, along with undiluted (1×) and diluted (4×) synaptosomes and total brain homogenate, blotted with a mouse α-synuclein specific antibody. These blots were used to compare and estimate the concentration of α-synuclein at synapses and in brain homogenate. C, standard curve for synaptosome samples generated from quantifying blots shown in B. (r2 = 0.991). Blue lines indicate the amount of α-synuclein in undiluted (1×) and diluted (4×) synaptosomes. See

supplemental Fig. S4

for a similar analysis using synaptobrevin 2 standards.

FIGURE 6.

Purification and characterization of the native α-synuclein tetramer. A, crosslinking of recombinantly purified monomeric and blood purified tetrameric α-synuclein. The purified proteins were crosslinked with glutaraldehyde according to Ref. and separated on SDS-PAGE. The gel shows a band of ∼50 kDa corresponding to the tetramer (arrow), while the monomer remains at ∼14kD. B, circular dichroism (CD) of tetrameric and monomeric α-synuclein. Spectra show the tetramer is folded in solution, and its folding is unchanged by the addition of BPL liposomes, while the recombinantly purified, monomeric α-synuclein is unfolded in solution. C, quantification of α-synuclein tetramer tubulation. Graph shows that the tetramer is unable to generate membrane curvature. Quantification of tubulation activity was done as described in Fig. 3 from 540 images for each condition. **, p < 0.01.

FIGURE 7.

PD mutants of α-synuclein have varied ability to generate membrane curvature. A, representative images of membrane tubules generated by human wild type α-synuclein and the three PD mutants. B, quantification of membrane tubulation activity of the three human PD mutants as compared with human wild type α-synuclein. While A53T and E46K tubulation activity is similar to wild type, the A30P mutant has no membrane tubulation activity. Ratios were quantified from 540 images for each condition. *, p < 0.05.

FIGURE 8.

Model of α-synuclein membrane curvature generation and its possible synaptic functions. A, at rest, α-synuclein is on the synaptic vesicle in an α-helical conformation. B, upon stimulation, it generates curvature during exocytosis, after which it comes off the flattened membrane and becomes unfolded. C, at the start of endocytosis, α-synuclein binds to the incipiently curved membrane to generate further curvature at the endocytic stalk. D, after one round of neurotransmitter release, it is bound to the synaptic vesicle again. E, unused α-synuclein is stored as a folded tetramer. F, the A30P PD mutant of α-synuclein does not participate in any of these membrane functions.

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Monomeric synucleins generate membrane curvature - PubMed (original) (raw)