Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission

Abstract

One of the fundamental features of biomembranes is the ability to fuse or to separate. These processes called respectively membrane fusion and fission are central in the homeostasis of events such as those related to intracellular membrane traffic. Proteins that contain amphipathic helices (AHs) were suggested to mediate membrane fission via shallow insertion of these helices into the lipid bilayer. Here we analyze the AH-containing proteins that have been identified as essential for membrane fission and categorize them in few subfamilies, including small GTPases, Atg proteins, and proteins containing either the ENTH/ANTH- or the BAR-domain. AH-containing fission-inducing proteins may require cofactors such as additional proteins (e.g., lipid-modifying enzymes), or lipids (e.g., phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], phosphatidic acid [PA], or cardiolipin). Both PA and cardiolipin possess a cone shape and a negative charge (-2) that favor the recruitment of the AHs of fission-inducing proteins. Instead, PtdIns(4,5)P₂ is characterized by an high negative charge able to recruit basic residues of the AHs of fission-inducing proteins. Here we propose that the AHs of fission-inducing proteins contain sequence motifs that bind lipid cofactors; accordingly (K/R/H)(K/R/H)xx(K/R/H) is a PtdIns(4,5)P₂-binding motif, (K/R)x₆(F/Y) is a cardiolipin-binding motif, whereas KxK is a PA-binding motif. Following our analysis, we show that the AHs of many fission-inducing proteins possess five properties: (a) at least three basic residues on the hydrophilic side, (b) ability to oligomerize, (c) optimal (shallow) depth of insertion into the membrane, (d) positive cooperativity in membrane curvature generation, and (e) specific interaction with one of the lipids mentioned above. These lipid cofactors favor correct conformation, oligomeric state and optimal insertion depth. The most abundant lipid in a given organelle possessing high negative charge (more negative than -1) is usually the lipid cofactor in the fission event. Interestingly, naturally occurring mutations have been reported in AH-containing fission-inducing proteins and related to diseases such as centronuclear myopathy (amphiphysin 2), Charcot-Marie-Tooth disease (GDAP1), Parkinson's disease (α-synuclein). These findings add to the interest of the membrane fission process whose complete understanding will be instrumental for the elucidation of the pathogenesis of diseases involving mutations in the protein AHs.

Keywords: amphipathic helix; fission-inducing protein; lipid cofactor; lipid-binding site; membrane fission; membrane scission; neck-hemifission model; shallow insertion.

FIGURE 1

Nomenclature of leaflets (monolayers) during different kinds of membrane fission reaction. Only membranes decorated with fission-inducing proteins (yellow circles) are considered. When membrane fission takes place, separation of proximal leaflets is followed by separation of distal leaflets. During normal topology fission (membrane-enclosed compartment buds toward the cytoplasm), distal leaflet is cytoplasmic, whereas proximal leaflet is exoplasmic. During reverse topology fission (membrane-enclosed compartment buds away from the cytoplasm), distal leaflet is exoplasmic, and proximal leaflet is cytoplasmic. During both normal and reverse topology fission reactions, disjoint or joint union of membrane-enclosed compartments can form. If disjoint union forms, outer leaflet is distal, and inner leaflet is proximal. When joint union forms, outer leaflet is proximal, whereas inner leaflet is distal. Fission-inducing proteins are usually present on the cytoplasmic side of the membrane.

FIGURE 2

Mechanisms of membrane fission. Membrane fission mechanisms can be classified as active, that require consumption of cellular energy by nucleoside triphosphate (ATP or GTP) hydrolysis (upper panel; blue line) or passive that do not require the direct use of energy (lower panel; green line). Each of these above-mentioned categories can be further divided into two types: normal topology, when the vesicle buds toward the cytoplasm (left panel; purple dashed line) and reverse topology, when the vesicle buds away from the cytoplasm (right panel; dark green dashed line). Consequently, membrane fission mechanisms are divided into four classes: Class I, active mechanism with normal topology; Class II, passive mechanism with normal topology; Class III, active mechanism with reverse topology; Class IV, passive mechanism with reverse topology. The details are in the text.

FIGURE 3

Lipid cofactors are required for fission-inducing proteins. Many AH-containing and AH-free fission-inducing proteins need specific lipids, named lipid cofactors, to promote and complete membrane fission. Thus, the ability of the fission-inducing proteins to support this process is strictly dependent on membrane lipid composition. Of note, proteins are not able to induce membrane fission in the absence of these lipids (left panel; purple dashed line). Conversely, in the presence of such lipid cofactors, fission occurs through the following steps: membrane neck, hemifission and formation of two separate membranes (right panel; green dashed line). Lipid cofactors include: PtdIns(4,5)P₂, PA, cardiolipin, MMPE, PtdIns3P, PtdIns(3,5)P₂ and cholesterol. See text for details.

FIGURE 4

Configurations and helical wheel representations of some of the (K/R)x₆(F/Y) motif-containing AHs belonging to the fission-inducing proteins that use cardiolipin as cofactor. Helical wheel projections were generated using Heliquest software (http://heliquest.ipmc.cnrs.fr; Gautier et al., 2008). Residues belonging to the (K/R)x₆(F/Y) motifs are highlighted. Color coding for residues: yellow for hydrophobic, purple for Ser (S) and Thr (T), blue for Lys (K) and Arg (R), red for acidic, pink for Asn (N) and Gln (Q), gray for small residues (Ala, A and Gly, G), green for Pro (P), and light blue for His (H). The arrow in helical wheels corresponds to the hydrophobic moment. Species names and UniProt accession numbers: Acholeplasma laidlawii MGS, Q93P60; Escherichia coli MinD, P0AEZ3; A. laidlawii DGS, Q8KQL6; Penicillium chrysogenum Pex11p, B6GZG8; Agrobacterium tumefaciens AtPmtA, A0A2L2L7Q9.

FIGURE 5

Configurations and helical wheel representations of the (K/R/H)(K/R/H)xx(K/R/H) motif-containing AHs belonging to the fission-inducing proteins that use PtdIns(4,5)P₂ as cofactor. Helical wheel projections were generated using Heliquest software (http://heliquest.ipmc.cnrs.fr; Gautier et al., 2008). Residues belonging to the (K/R/H)(K/R/H)xx(K/R/H) motifs are highlighted. Color coding for residues: yellow for hydrophobic, purple for Ser (S) and Thr (T), blue for Lys (K) and Arg (R), red for acidic, pink for Asn (N) and Gln (Q), gray for small residues (Ala, A and Gly, G), green for Pro (P), and light blue for His (H). The arrow in helical wheels corresponds to the hydrophobic moment. Species names and UniProt accession numbers: Homo sapiens epsin 1, Q9Y6I3; Rattus norvegicus amphiphysin 2, O08839; R. norvegicus PICK1, Q9EP80; R. norvegicus endophilin A1, O35179; Drosophila melanogaster amphiphysin, Q7KLE5.

FIGURE 6

Configurations and helical wheel representations of some of the KxK motif-containing AHs belonging to the PA-binding proteins. Helical wheel projections were generated using Heliquest software (http://heliquest.ipmc.cnrs.fr; Gautier et al., 2008). Residues belonging to the KxK motifs are highlighted. Color coding for residues: yellow for hydrophobic, purple for Ser (S) and Thr (T), blue for Lys (K) and Arg (R), red for acidic, pink for Asn (N) and Gln (Q), gray for small residues (Ala, A and Gly, G), green for Pro (P), and light blue for His (H). The arrow in helical wheels corresponds to the hydrophobic moment. Species names and UniProt accession numbers: Homo sapiens ATG3, Q9NT62; Saccharomyces cerevisiae Opi1, P21957; Zea mays DHN1, P12950; S. cerevisiae Spo20, Q04359; Schizosaccharomyces pombe Tam41, O74339.