Shaping membranes with disordered proteins

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Shaping membranes with disordered proteins

Mohammad A.A. Fakhree, Christian Blum, Mireille M.A.E. Claessens

A R T I C L E I N F O Keywords: Disordered proteins Membranes Membrane curvature Membrane tethering Membrane domains A B S T R A C T

Membrane proteins control and shape membrane trafficking processes. The role of protein structure in shaping cellular membranes is well established. However, a significant fraction of membrane proteins is disordered or contains long disordered regions. It becomes more and more clear that these disordered regions contribute to the function of membrane proteins. While the fold of a structured protein is essential for its function, being dis-ordered seems to be a crucial feature of membrane bound intrinsically disdis-ordered proteins and protein regions. Here we outline the motifs that encode function in disordered proteins and discuss how these functional motifs enable disordered proteins to modulate membrane properties. These changes in membrane properties facilitate and regulate membrane trafficking processes which are highly abundant in eukaryotes.

1. Biological membranes

In dilute solutions interactions between molecules are unlikely, yet such interactions are essential to life. To make life possible, specific molecules have to be accumulated and contained in well-defined but responsive and dynamic compartments. In this respect, it is thought that the partitioning of bio(macro)molecules into compartments with different solvent properties, such as amphipathic micelles, complex coacervates and membrane enclosed organelles, plays an important role in the emergence of life [1–5]. The advantages associated with com-partmentalization and the subsequent appearance of physiochemically different sub-compartments resulted in the evolution of cellular mem-branes.

Cellular membranes are composed of lipid bilayers and their main role is to create a physical separation between milieus that differ in chemical composition. This started from simply separating cellular content from the surrounding environment, and further developed into compartmentalization as observed in eukaryotes. Compartmentalization provides the possibility to execute functions that require a different chemical environment without interfering with each other. Although membranes provide a physical barrier, they are also responsible for the communication with the outside world. Transfer of information and mass over the membrane is essential for cell signaling processes and cell function [6]. Local and dynamic membrane de-formations enable processes such as the formation of trafficking vesicles and pseudopodia [7,8]. Communication with the outside environment and response to external stimuli is typically made possible by the pre-sence of proteins in or on the membrane [9,10]. In this respect, the

membrane can also be thought of as a matrix and two-dimensional anisotropic solvent for membrane proteins. Membrane proteins are present in or on the membrane to fulfil their function as enzymes, transporters, electron carriers, messenger/ligands, lipid clamps/mem-brane anchors, and are involved in memclamps/mem-brane remodeling processes [6,9,10].

2. Disordered proteins and membranes

For the human genome, 5500 to 7500 genes have been predicted to translate into proteins which localize or interact with membranes. This means that around one third of the protein encoding human genome translates into membrane proteins [11,12]. Considering the relative amount of membrane proteins and their role in all intra- and inter-cellular communication, the maintenance of chemical gradients, and membrane remodeling processes, it comes as no surprise that mem-brane proteins are major players in human physiology, the pathology of diseases, and are targets for pharmaceutical interventions [13].

The function of a protein and its interaction with the membrane is determined by the protein's amino acid sequence. Until recently, a protein's function was thought to be dependent on the formation of a single, well defined structure. This tight relation between structure and function can be observed for many membrane proteins or protein re-gions [14]. For these proteins it is often observed that the formation of their secondary, tertiary, and quaternary structures is facilitated by interacting with the membrane [15]. In recent decades, it has however become clear that a large fraction of the eukaryotic proteome does not adopt a single well defined structure but stays disordered in solution. Of

https://doi.org/10.1016/j.abb.2019.108163

Received 19 July 2019; Received in revised form 23 October 2019; Accepted 27 October 2019 ∗_{Corresponding author.}

E-mail address:m.m.a.e.claessens@utwente.nl(M.M.A.E. Claessens).

Available online 29 October 2019

0003-9861/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

the human protein-coding genes, 44% were reported to contain dis-ordered regions of more than 30 amino acids (AAs) in length [16]. These AA sequences that do not adopt a single well defined fold are called intrinsically disordered regions (IDRs) or intrinsically disordered proteins (IDPs) if the whole protein is disordered. IDPs and IDRs re-cently gained much attention because of their often unknown physio-logical function and their involvement in protein aggregation diseases [17–19]. Membrane proteins, as a major fraction of the human pro-teome, also contain IDPs and proteins with IDRs. It has been calculated that more than 40% of transmembrane proteins have IDRs of significant length [20–23].

In this review, after a short introduction into functional motifs in disordered proteins, we discuss mechanisms that enable membrane anchored disordered proteins to modulate membrane properties which finally defines membrane trafficking processes.

3. Disordered but functional motifs

By folding, a structured protein reaches a global minimum energy state which makes the protein structure stable and functional. During the folding process, the energy minimization results in the burying of hydrophobic AAs inside the protein core rather than exposing these AAs to the aqueous environment [14]. Unlike structured proteins, most IDPs/IDRs are enriched in AAs with charged or polar side chains for which exposure to the aqueous environment is not energetically pena-lized, so they do not need to be buried inside the protein structure [24,25]. Due to the absence of structural constraints, the number of accessible conformations of disordered proteins in solution is larger than for structured proteins [26] In comparison to structured proteins, IDPs/IDRs do not have a global minimum energy state [26]. They are solvated and depending on the interaction with other molecules, they can interchangeably adopt structure to reach local energy minima [27,28]. This implies that the use of IDPs/IDRs is flexible and that some disordered proteins can adopt more than one functional structure and may even have multiple functions. Similar to structured proteins, the presence of specific motifs in the amino acid sequence allows IDPs/IDRs to interact with other molecules and thus makes them functional. The function(s) of an unstructured protein mainly results from the presence of one or more of the three structural motifs mentioned below [16].

3.1. Linear motifs

Linear motifs are short sequences of 3–10 AAs. These sequences provide a single or multiple low affinity recognition site(s) for their interaction partner [29]. The small size of the linear motifs makes them easily accessible to substrates [30]. A good example of linear motifs in IDPs/IDRs, are the recognition sites for post translational modification (PTM) [25,31]. For example, immunoreceptor tyrosine-based activation motifs (ITAMs) are two short sequence of 4 amino acids (YxxL/I) se-parated by 6–8 amino acids on the intracellular disordered tails of certain membrane proteins of the immune system (e.g. ζ and CD3ε signaling subunits of T cell receptor, and γ signaling subunit of FcεRI receptor), which are targets for phosphorylation [32]. The phosphor-ylation of ITAMs regulates the interaction between immune system receptors and their protein target. Ligand binding or homo/hetero di-merization sites on proteins can also be linear motifs. An example of a linear motif that serves as an interaction site is the LC3 interaction motif (LIR). The LIR motif has the general sequence of [W/F/Y]-X1X2-[I/L/V] and is found in protein complexes that facilitate the interaction with autophagosomes [33]. FAM134B has a LIR sequence and is in-volved in changing membrane properties of the endoplasmic reticulum in the process of ER-phagy [34,35]. The linear motif GxxxG has been observed to play a role in the dimerization of IDPs/IDRs [36]. Linear motifs also play a role in the targeting of proteins to specific cellular compartments. Two examples of such sequences are C-terminally lo-cated KDEL and KKXX sequences. While the KDEL sequence results in

the retention of the disordered protein in the lumen of endoplasmic reticulum, the C-terminally located KKXX is responsible for cytoplasmic or transmembrane localization [37].

3.2. Molecular recognition features (MoRFs)

MoRFs are longer (typically around 10–70 AAs) sequences than linear motifs. Although disordered in solution, MoRFs can adopt alpha-helix (α-MoRF), beta-sheet (β-(α-MoRF), and irregular but rigid (ι-MoRF) structures, or a mixture of mentioned structures upon binding a specific target [16]. Si-milar to structured proteins, the secondary structure of MoRFs depend on the AA sequence. The N-terminal IDR of the protein p53 is an example of an α-MoRF when it interacts with the Mdm2 protein. The C-terminal IDR region of p53 is also a MoRF and can turn to an α-MoRF or ι-MoRF, upon interaction with S100B or Cdk2-cyclin A, respectively [38–40].

3.3. Intrinsically disordered domains (IDDs)

IDDs are the regions of the IDPs/IDRs that always remain un-structured. The IDD comprises the often bulky, yet flexible part of the disordered protein. For IDDs, being disordered is (part of) their function [16,41]. An example of IDDs are the intra/extra-cellular loops of polytopic transmembrane proteins. In addition to their role as flexible linker, it has been suggested that the orientation of the positively charged amino acids in the loop regions plays a role in stabilizing the overall protein structure [42].

Note that a single disordered protein can contain all and multiple of the above mentioned motifs. The functional motifs can even (partly) overlap, which makes the function of the disordered protein dependent on the presence of interaction partners and/or the physicochemical characteristics of the environment [16]. Below we will discuss how these disordered motifs modulate membrane properties and affect membrane trafficking processes.

4. Disordered proteins modulate membrane properties

Biological lipid bilayers are crowded with proteins. The protein to lipid mass ratio of the plasma membrane is ~1:1, while for the inner mitochondrial membrane ratios as high as ~3:1 have been reported [43]. Based on 1:1 protein:lipid ratio, it has been estimated that the mean center-to-center distance between proteins is around 10 nm [44]. The interplay between lipids, the lipid bilayer, and proteins in de-termining membrane properties is complex [45,46]. On the one hand, some features of biological membranes – such as sub-cellular localiza-tion, (local) lipid composilocaliza-tion, and curvature – are determined by the presence of specific membrane proteins. On the other hand, the struc-ture, sub-cellular localization, and surface density of membrane pro-teins is determined by the lipid bilayer [44,47]. This complex interplay between the lipid bilayer and proteins is controlled by feed forward and/or feedback mechanisms and results in the organization of proteins by membranes and vice versa, the organization of membranes by pro-teins [10,44]. The large number of different membrane proteins and local or organelle specific lipid compositions add to this complexity, but also provide specificity to interactions and membrane processes. In the rest of this review paper, we will focus on how disordered membrane proteins are used to regulate or control membrane processes. We will discuss how membrane bound disordered proteins: a) modulate mem-brane curvature, b) tether memmem-branes together and c) affect (local) lipid composition and domain formation. With this, we evaluate how disordered regions determine localization, shape, and formation of membrane bound organelles and vesicles inside the cell.

4.1. IDPs/IDRs and membrane curvature

IDPs and IDRs play a role in the membrane remodeling processes that are required for vesicle trafficking. In this trafficking, the vesicle

content is transported from one cell compartment to another by membrane fission and fusion processes. The mechanism by which IDPs and IDRs remodel membrane depends on the presence of MoRFs, cel-lular localization, surface density, and post translational protein mod-ifications. Below we discuss how 1) MoRFs, 2) the density of IDPs/IDRs on the membrane surface, and 3) the combined effect of 1 and 2 con-tributes to membrane remodeling processes.

4.1.1. MoRFing disordered proteins and membrane curvature

Membranes can induce coupled binding-folding reactions in a number of disordered proteins. The resulting newly formed structure can be transmembrane or inserted into only one of the membrane leaflets. If this insertion is asymmetric, it will result in a unilateral in-crease in the surface area. This changes the ratio between the outer and inner surface area of the membrane. In order to adopt a topography that corresponds to this new ratio, the membrane remodels by increasing its curvature (Fig. 1A). Clearly, how much the curvature increases depends on two factors: a) the size of the inserted MoRF, b) surface density of the disordered protein. An example of a disordered-to-ordered structural change that induces curvature happens in the N-terminal region of alpha-synuclein (αS) in the presence of anionic lipid bilayers. Binding of αS to these lipid bilayers results in the formation of amphipathic alpha-helix [48–50]. When this alpha-helix inserts into the outer leaflet of the lipid bilayer it increases the surface area of this leaflet with ~15 nm2_{/αS and induces curvature [51]. The transition to the alpha-helical} state and the resulting generation of curvature has been demonstrated in in vitro experiments with model lipid vesicles [52–54]. Following these in vitro experiments, it has been shown that, also in cells, the structure of the αS on cellular vesicles differs from the mainly un-structured cytosolic form of the protein [55]. In a tested SH-SY5Y cell model, the surface density of αS on cellular vesicles was found to be high enough to induce curvature by the MoRF insertion mechanism [56].

Another example of membrane curvature sensing/modulation by the MoRF motif of a disordered protein is ADP-ribosylation factor GTPase-activating protein 1, ArfGAP1 [57]. In the absence of lipid membranes, ArfGAP1 is mainly disordered. The ArfGAP1 lipid packing sensor region (ALPS), which consists of ~40 amino acids, MoRF into an

alpha-helix in the presence of highly curved membranes. At low con-centrations, the disordered-to-ordered transition results in the binding of the ArfGAP1 to the target membrane. While at high concentrations, similar to αS, the asymmetric insertion of ALPS sequence into only one of the membrane leaflets increases membrane curvature [7,57,58].

The ability to adopt alpha-helical structure upon binding mem-branes has also been associated with disease. hIAPP and Aβ play a role in the development of type II diabetes and Alzheimer's disease, re-spectively. The interaction of these proteins with membranes and generation of curvature by MoRFing into an alpha-helical structure in an intermediate step towards aggregation has been suggested to be toxic [59,60].

4.1.2. Density of disordered proteins on the membrane surface

It has been shown that disordered proteins do not only induce curvature by the MoRF mechanism described in 4-1-1. The asymmetric coupling of disordered proteins to one of the leaflets of a phospholipid membrane can induce spontaneous membrane curvature even in the absence of a mechanism that generates an area difference between the leaflets of the bilayer. The mechanism by which surface adsorbed or anchored disordered proteins generate curvature is generic and can also be exploited by coupling DNA or synthetic polymers and even struc-tured proteins to model membranes [61–63]. Although the mechanism is not specific, it has been suggested to contribute to membrane re-modeling in biological systems. To account for the observed membrane curvature generation by membrane bound disordered proteins, two closely related mechanisms have been put forward.

4.1.2.1. High IDP/IDR surface density – steric pressure. The first

mechanism by which membrane anchored IDPs/IDRs can induce curvature describes that the steric hindrance among the adsorbed disordered proteins on the membrane at very high surface density results in a lateral pressure. For asymmetric IDP/IDR adsorption or anchoring, increasing the curvature of the membrane will result in the increase in the area of the (now) outer leaflet of the bilayer which reduces the pressure (Fig. 1B). Examples of proteins with IDRs for which this mechanism of action has been suggested are epsin1, p180, disordered regions of BAR proteins, and αS [64–66].

Fig. 1. Schematic representations of mechanisms by which IDPs/IDRs can induce membrane curvature. Panel A shows that unilateral insertion of disordered proteins and MoRFing (left) increases the area of the one of the leaflets (indicated by red ovals). In order to compensate for this increase in area, the curvature of the membrane increases (right). Panel B shows how membrane adsorbed macromolecules at super-saturating concentrations (left) can bend membrane (right) to reduce the pressure caused by steric hin-drance between the macromolecules. Panel C shows collisions between the membrane adsorbed/inserted macromolecules (left) that result in lateral pressure. Membrane bending reduces this lateral pressure (right). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4.1.2.2. Lower IDP/IDR surface density – lateral pressure. The second

mechanism described for curvature induction by disordered proteins is closely related to the first, but ascribes the generation of curvature to the thermal motion of these macromolecules on the membrane (Fig. 1C). The membrane is a fluid and the anchored disordered proteins are free to diffuse in the membrane leaflets. This thermal motion on the membrane surface will result in collisions with other membrane anchored macromolecules. The collisions generate a lateral pressure on the membrane. Similar to the mechanism described for steric hindrance, increase in the membrane curvature will help to reduce this pressure. The main difference between the two mechanisms is that much lower protein surface densities are required for the second mechanism to work. The second mechanism has also been modeled assuming that the disordered regions of the membrane inserted proteins can be described as hard disks that diffuse in a 2D liquid and taking into account the mechanical properties of the lipid bilayer [62]. For both structured and disordered proteins, good agreement has been found between experiments and model calculations [62,64,67].

For both high and low surface density mechanisms, since the mag-nitude of the lateral pressure depends on the distance of the center of mass of the anchored protein to the bilayer, disordered proteins are much more efficient in generating curvature than structured proteins with the same number of AAs. Disordered proteins are less compact which makes them more efficient in generating lateral pressure. The presence of a large fraction of charged and polar amino acids in dis-ordered proteins contributes to their bulkiness. A possible additional advantage of using disordered chains is that their radius of gyration can be dynamically addressed. The volume that the disordered chain oc-cupies can be modulated by interacting with other molecules. Any changes in the effective (spherical) volume occupied by the disordered regions of the protein, will result in a change in the generated pressure, and can consequently be used to help change membrane curvature. For example, αS has been suggested to bind synaptic vesicles via its N-terminal MoRF region [49] while the disordered C-terminal region in-teracts with calcium ions [68,69] and the vesicle bound SNARE-protein synaptobrevin [56,70–72]. These C-terminal interactions may result in compaction of the bulky disordered region. Such a compaction would result in a decrease in the lateral pressure exerted by the disordered region of αS and thus make synaptic vesicles more fusion prone.

4.1.3. Cooperation of ordered and disordered regions

It has been shown that membrane curvature modulation can be a result of the cooperative action of structured and disordered regions of membrane proteins. In this sense, curvature is induced by combining the effect of the asymmetric binding of a (partially) structured curva-ture scaffold protein or the insertion of a (partially struccurva-tured) protein into one of the bilayer leaflets and creating lateral pressure by the unstructured, solvent exposed part (Fig. 2A). Examples of cooperation between structured and disordered regions of a protein in membrane remodeling include, but are not limited to, the curved BAR domain membrane scaffold proteins and αS which partly inserts into mem-branes via the formation of an amphipathic alpha-helix [65,73,74].

In our recent work, we have studied the ability of αS to remodel membranes using the combined action of structured and disordered regions [74]. From this work we derive that at low concentrations of proteins on the membrane, the interaction between the proteins is negligible. Consequently, the lateral pressure generated by membrane bound proteins is very small and the membrane remodeling is domi-nated by the structured region of the protein (Fig. 2B).Fig. 2B shows that for an IDR with a radius of gyration (Rg) < ~4 nm, insertion of an amphipathic alpha-helix (MoRF) is the dominant mechanism of cur-vature induction for all protein concentrations shown. At higher protein concentrations, interactions between the membrane bound proteins becomes significant which increases the contribution of the lateral pressure to the generation of curvature and makes curvature less de-pendent on the size of the MoRF (Fig. 2B). Further increasing the

surface density of the proteins will result in the saturation of the membrane with proteins. At the saturating concentrations, steric pres-sure starts to play a role in membrane remodeling. As one can see, the magnitude of the contribution of the different mechanism to generating curvature depends on: a) size of the structured region, b) size of the disordered region, c) and surface density of the protein. This last parameter depends on the concentration in the bulk and affinity to the membrane.

4.2. Tethering membranes together using disordered proteins

The flexibility and charged nature of membrane anchored dis-ordered proteins gives them the ability to extend in to the environment surrounding the membrane and fish for target molecules in solution or on neighboring vesicles. The capturing of the target by disordered membrane bound protein results in conformational changes in this protein, which brings the target molecule closer to the opposing membrane. Interestingly, this structural change in the disordered pro-tein upon target binding can involve both disto-order and order-to-disorder transitions. Below we describe some examples in which membrane anchored disordered proteins exploit these transitions.

4.2.1. Disorder-to-order transition

For relatively short disordered MoRF containing proteins, approxi-mately smaller than 200 AAs, the largest dimension of the IDR in the structured state is smaller than the radius of gyration of the protein in the disordered state. Consequently, the disordered form is more effi-cient than structured form in fishing for target molecules. In the dis-ordered form the protein samples a larger volume compared to the protein in the structured form. Coupled target binding and adopting structure, results in the compaction of the disordered region. The dis-ordered proteins can thus be used to catch the target molecule and bring it closer to the membrane.

The Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex is a good example of a disordered protein system that tethers membrane bound compartments together using this mechanism. There are two sets of SNARE proteins: one on the target membrane known as t-SNAREs. On the active zone membrane of synapses of neurons, these t-SNAREs include syntaxin-1 and SNAP-25. The other set, located on vesicles, is known as v-SNAREs and include synapto-brevin (VAMP2). Membrane bound monomeric SNAP-25 and syntaxin-1 on the target membrane are disordered proteins [75–78], but upon interaction with synaptobrevin on the vesicle membrane, they turn into parallel alpha-helices to form the SNARE complex core [79]. Synapto-brevin is a transmembrane protein with a disordered region extending into the cytosol. The interaction with SNAP-25 and syntaxin-1 also causes the disordered region of synaptobrevin to adopt an alpha-helical structure [79–81]. The formation of the SNARE complex thus involves a disorder-to-order transition that brings the target and vesicle mem-branes in very close proximity. This initiates the fusion of the vesicle with the target membrane. InFig. 3ii this process is schematically de-picted.

The SNARE machinery not only facilitates the fusion of vesicles with a target membrane, it also provides specificity to the fusion process. Different sets of t-SNARE and v-SNARE proteins are found on the dif-ferent membrane bound organelles in cells. Only specific combinations of t-SNAREs and v-SNAREs result in the efficient formation of an alpha-helical coiled-coil. For example, the syntaxin-16/Vti1a/syntaxin-6/ VAMP4 SNARE complex is involved in retrograde transport from early endosomes to trans-Golgi network, while the syntaxin-16/Vti1a/syn-taxin-10/VAMP3 complex provides specificity in retrograde transport from the late endosomes to the trans-Golgi network [82,83]. The en-dosome bound VAMP3 and VAMP4 encode the origin of these vesicle, while syntaxin 6 and syntaxin 10 are specifically found on the Golgi membrane [83]. There are some differences between syntaxin6/syn-taxin10 and VAMP3/VAMP4 in single or short sequences of amino acids

in alpha-helices. However, the major pair-wise difference between these proteins is in their disordered regions. Syntaxin 6 and syntaxin 10 mainly differ from each other in the disordered region which links the two alpha-helices in their SNARE complexed structures. In the case of VAMP4 and VAMP3, the latter is missing a large portion of disordered amino acids in comparison to VAMP4. These examples indicate that the disordered regions are important to infer specificity to the tethering interactions.

4.2.2. Order-to-disorder transition

For disordered MoRF containing proteins longer than ~1000 AAs, the longest structured length of the protein can be significantly larger than the radius of gyration of the protein in disordered form. Consequently, the ordered form enables fishing by sampling a distance further away from the membrane in comparison to the unstructured form, while the transition to the disordered state can be used to bring

the target molecule closer to the membrane surface (Fig. 3i). An ex-ample of a disordered protein that tethers two membrane bound com-partments using this mechanism is EEA1. According to D2_P2_database, early endosome antigen-1 (EEA1) is disordered [84] and binds to PI(3)P on early endosome membranes via its C-terminal FYVE domain [85]. Upon binding to PI(3)P, two monomeric EEA1 proteins adopt a stiff coiled-coil structure which extends up to 200 nm into the cytosol. This stiff structure ends in an N-terminal Zn2+ _{finger that fishes for} Rab5:GTP bound to the surface of other early endosomes [86]. Binding to Rab5:GTP induces an allosteric conformational change in the coiled-coil structure of the EEA1 which results in the collapse of the extended EEA1 homodimer [87]. The magnitude of this entropic collapse force was measured to be around 14kBT for a single homodimer of EEA1 [87]. The force exerted by this order-to-disorder transition brings two early endosomes closer together and facilitates the fusion of early en-dosomes via the SNARE machinery. This fusion subsequently results in Fig. 2. A Schematic of how IDPs induce curva-ture by MoRFing into amphipathic alpha-helices and by generating lateral pressure with their large unstructured domains. Assuming equili-brium binding, the protein density on the membrane is determined by the protein and membrane concentrations and the equilibrium binding constant. If the surface concentration is high, the combined effects of amphipathic alpha-helix insertion and the generation of lat-eral pressure can bend the membrane. How much curvature is generated depends on the size of the helix (MoRF area), the radius of gyration (Rg) of the IDR, the concentration of bound

protein, and the bending rigidity of the mem-brane. Panel B depicts calculations showing which combinations of MoRF area, Rg, and

pro-tein concentration result in the formation of vesicles with a radius of 45 nm taking into ac-count the above mentioned mechanisms. The parameters in this figure span a physiologically relevant range. We calculated Rgof the IDR assuming a self-avoiding random walk, and the Rgvalues represent IDRs of lengths up to ~600 AA. The depicted length of

the alpha-helix ranges from 0 to 350 AAs or 0–~30 nm2_{. The calculations were performed assuming a total lipid concentration of 500 μM, the bending rigidity of the} bilayer was assumed to be 10 kBT, and all protein was assumed to be bound to the membrane [74]. For this set of parameters the effect of the IDR becomes larger than that of the MoRF for Rg> ~8 nm. Please note that Rgof the IDR is presented as 1D parameter (radius), while the size of the MoRF is depicted in 2D (area).

Considering the required number of amino acids, the effect of lateral pressure generated by the IDR represents a more efficient mechanism of inducing curvature than amphipathic alpha-helix insertion.

Fig. 3. Ordered-to-disordered (i) and disordered-to-ordered (ii) transitions bring vesicles closer to the membrane surface. An example of such an ordered-to-disordered transition is given by the entropic collapse of EEA1 which brings the captured vesicle closer to the proteins on the target membrane (i). The disordered-to-ordered transition of SNARE pro-teins subsequently brings the vesicles even closer to the target membrane (ii). The relative sizes of the vesicle, structured and disordered EEA1 and SNARE proteins are to scale.

the formation of larger early endosomes [87]. These processes are shown schematically inFig. 3.

4.2.3. Tethering membranes using both order-disorder and disorder-order transitions

For disordered proteins with an approximately equal length of dis-ordered and dis-ordered structures, the structural flexibility (due to the presence of disordered regions or interchange between the ordered and disordered form) enables fishing and tethering of target molecules to the membrane. Golgin proteins are set of disordered tethering proteins that use this mechanism and localize at Golgi apparatus (GA) mem-brane. Prior to or upon localization at the GA membrane, golgins form dimers and adopts a coiled-coil structure [88,89]. Golgins have a sig-nificant amount of disordered regions, both before and after the for-mation of the coiled-coil dimers (SeeTable 1). Most of the golgins bind to GA via their C-terminal region and tether vesicles to the GA mem-brane via their N-terminal region. Depending on the golgin species, either one or both the C-terminal and N-terminal region are disordered. As can be seen from Table 1, a number of membrane bound target molecules/proteins has been identified for each golgin. From the summary of mechanisms by which Golgins tether membrane surfaces presented inTable 1andFig. 4, it can be seen that being or becoming disordered is an essential feature of most of the proposed mechanisms [90,91]. However, the exact mechanism(s) by which golgin proteins bring the captured vesicle closer to the GA membrane is understudied [91]. Towards unraveling mechanisms, Cheung and coworkers showed that the formation of a disordered “bubble” in the middle of the homodimer coiled-coil of GCC185 is responsible for the bending of the coiled-coil structure [92] (see Fig. 4). For GMAP-210 homodimer coiled-coil structure, structural flexibility along with interaction with a GTPase protein (i.e. Rab2) has been reported to be involved in trapping the coiled-coil structure in a bended state which brings the vesicle closer to GA membrane [93] (seeFig. 4).

In addition to the tethering function of golgin proteins, their loca-lization on the GA membrane, length, and interacting targets have been reported to determine the shape and specific morphology of the GA [91,94]. Golgins seem to fully exploit the functional features of the combination of rigid membrane bound structures and the presence of disordered flexible regions.

Another example of an IDP that may use mixed mechanisms to te-ther vesicle togete-ther is αS. αS has been reported to be able to bridge two neighboring vesicles [123]. The N-terminal AAs of αS can adopt an alpha-helical structure upon binding membranes. Of this alpha-helix the first 20 N-terminal AAs bind strongly to the membrane, the rest of the helix has been reported to have a lower membrane affinity [123]. This lower affinity makes it possible to detach part of the helix from the vesicle. This part becomes disordered, and tethers to a neighboring vesicle using the transition to an α-MoRF [123].

4.3. Lipid domains and IDPs

Biological membranes consist of more than one lipid type and the composition of membranes differs between different organelles. This diversity in lipid composition is generated and maintained by mem-brane proteins which in return help to organize memmem-brane proteins selectively. Lipid composition of the membrane determines the speci-ficity of the interaction between proteins (that are involved in curvature and tethering mechanisms) and membranes. In the next two subsec-tions, we discuss how disordered proteins are involved in generating specific (local) lipid compositions and how they affect the formation of lipid domains.

4.3.1. Disordered proteins and lipid composition

The lipid composition differs among cellular organelles and be-tween their outer and inner leaflets. Membrane proteins are involved in introducing and maintaining these differences in two ways. The first mechanism is to cause newly synthesized lipids to be incorporated asymmetrically in the lipid bilayer. A good example of proteins that play a role in maintaining an asymmetry in the lipid composition of the bilayer leaflets are flippases [124,125]. Flippases are enzymes that help the inter-leaflet translocation of certain lipids. Their action conse-quently results in the asymmetric distribution of the lipids between the inner and outer leaflets of the membranes. Flippases contain IDRs which link the structured regions to each other. It has been shown that targeting the disordered region of a flippase (i.e. PglK), inhibits its flip-flop activity [126]. This shows that the disordered regions of these proteins are essential for their activity possibly by providing freedom to the relative motion of the structured parts of the enzyme.

The second mechanism is to modify the head groups of the lipids on the membrane. For example, phosphatidylinositol phosphate kinases (PIPKs) are kinase proteins that phosphorylate the phosphatidylinositol (PIP) lipids on the membranes. PIPKs have a “specificity” loop which consists of a disordered region and determines the specificity of the PIPK towards the substrate [127]. Depending on the class and type of the PIPK, the product of the phosphorylation can be either PI(3,4)P2, PI (3,5)P2, or PI(4,5)P2. Each of these phosphorylated PIs act as a cue for specific protein-membrane interaction. For example, PI(3,5)P2 is a specific cue to early endosomes [128], while PI(4,5)P2is a cue on the inner leaflet of the plasma membrane.

Protein membrane interactions are not only governed by the lipid composition of the membrane, but the density of the cues on the membrane also plays an important role. Feedback/feed forward me-chanisms result in changes of the cue density on the membrane. An example for feedback control that involves proteins with a disordered region is the interplay between Rab5 and phosphatidylinositol 3-phosphate, PI(3)P, on early endosomes. Rab5 is a small GTPase that binds to PI(3)P on early endosomes via a phosphate-binding disordered Table 1

The predicted content of disordered AA regions of 8 golgin proteins is ranging between 10% and 44%. This percentage of disordered is calculated based on the overlap of > 75% of predictors from D2_P2_{database [84]. All of the listed proteins form parallel homodimer and their monomers show high propensity for being} disordered. Only golgins for which tethering has been reported to involve collapse/bending mechanisms are listed. These mechanisms are shown schematically in Fig. 4and can be generalized to apply to all of the golgins [91]. As it can be seen from this table, for each golgin several interaction partners have been identified as expected from disordered proteins.

Golgin: Length in AA (Disord.

%) Interaction partners:

GM130 1002 (44%) GRASP65, Rab1, Rab2, Rab33b, Syntaxin-5, Rab6, p115, Giantin, COG, ZFPL1, C1GalT1, Importin α [95–103] GMAP-210 1979 (15%) Arf1, Highly curved membranes, Rab2, IFT20, γ-Tubulin, GalNAc-T2, ZFPL1 [93,104–109]

Golgin-84 731 (39%) Rab1, CASP, GalNAc-T2, ZFPL1 [109–112] TMF1 1093 (42%) Rab6, GalNAc-T2, ST6GalT [109,113]

Golgin-97 767 (29%) Arl1, Rab6, Rab19, Rab30, CASP, FIP1/RCP [100,114–117] Golgin-245 2230 (12%) Arl1, Arl3, Rab2, Rab30, Rab6 [100,114,116,117] GCC88 775 (29%) Arl1, Rab6, Rab19, Rab30 [116]

GCC185 1684 (10%) Rab6A/Arl1, STX16, CLASP2, AP-1, CLASP1a, Arl4A, CLASP2g, Rab1A, Rab1B, Rab2A, Rab2B, Rab6B, Rab9A, Rab9B, Rab15, Rab27B, Rab30, Rab33B, Rab35, Rab36 [116–122]

region [129]. There are two more functional disordered loops in Rab5 called switch I and switch II, which mediate the interaction of Rab 5 with phosphoinositide 3-kinase (PI3K). PI3K increases the phosphor-ylation of the phosphatidylinositol headgroup on the surface of the membrane which increases the local density of PI(3)P. This conse-quently increases the recruitment of more Rab5 on the early endo-somes. The resulting high surface density of PI(3)P and Rab5 is one of the specific features of early endosomes.

4.3.2. Lipid domains and disordered proteins

Biological membranes contain lipids that differ considerably in tail length and headgroup properties. In in vitro model membranes this difference in properties can result in phase separation. In equilibrium multi-component lipid bilayers are thus expected to macroscopically phase separate. However, the coarsening process that follows the nu-cleation of a new phase is typically very slow. This slow coarsening combined with membrane asymmetry, active processes, and local an-choring to the supporting cytoskeleton probably prevents global phase separation of membranes in biology [130–132]. However, nano-do-mains of specific lipids have been reported to exist in biological membranes [131,133].

Besides the mechanism mentioned above, the presence of proteins in the membrane has also been put forward as a reason why phase separation does not occur at larger length scales in biological mem-branes. Experiments on giant unilamellar vesicles with a lipid compo-sitions at which a liquid-ordered and liquid-disordered phase coexist indicate that membrane anchored disordered proteins may also con-tribute to preventing phase separation. When PEGylated phospholipids are added to these vesicles, lipid phase separation is suppressed [134]. The mechanism responsible for this suppression is argued to involve crowding and the generation of lateral pressure by the membrane an-chored polymer [134]. However, it is not clear if in a cellular context the lateral pressure generated by disordered proteins significantly contributes to the prevention of membrane phase separation. Further research to quantify the different contributions and asses the effec-tiveness and regulatory roles of disordered proteins in this process, is still required.

5. Conclusions and outlook

In comparison to structured proteins disordered proteins of similar AA length occupy large volumes, are flexible, and can undergo coupled binding-folding reactions. These feature are exploited by cells for functional purposes. Here we discussed how IDPs and IDRs are used to modulate membrane properties and thus affect membrane trafficking. The interplay between proteins and membranes is a two way process; proteins not only affect membrane properties, the membrane lipids also affect the binding and organization of proteins. In spite of this, the present view is rather membrane centered. The membrane is a matrix in which IDPs are embedded or to which IDRs are anchored that help the membrane perform its functions. However in some cases a more IDP/ IDR centric view on membrane trafficking processes is justified.

More and more IDPs are reported to be able to undergo liquid-liquid phase separation/transitions both in vitro and in vivo [135–137]. This phase separation is responsible for the formation of so-called membraneless organelles. These membraneless organelles play a role in various cellular tasks and have e.g. been shown to drive the formation of nucleolar subcompartments [138]. Recently, a phase separating protein, synapsin, has been shown to play a role in the organization of membranes [139]. Synapsin plays a role in the uptake and release of vesicles from the synaptic vesicle pool. The disordered region of sy-napsin undergoes liquid-liquid phase separation and forms droplets. At the same time synapsins interact with synaptic vesicles via their membrane binding region. It has been suggested that these droplets act as a matrix for the organization of the synaptic vesicle pool [140]. Phosphorylation of synapsin by calcium/calmodulin dependent protein kinase II (CaMKII) dissolves the droplet and makes the synaptic vesicles available for release [139,140]. Similar phase separation phenomena may play a role in the physiology of other membrane binding dis-ordered proteins.

In conclusion, although it is clear that disordered proteins help shaping membranes, insights into the mechanisms exploited by specific proteins are often lacking. Additionally the relative contributions of disordered regions compared to the structured regions in biology is underinvestigated. Studies into these aspects will not only improve our Fig. 4. Suggested tethering mechanisms for golgin proteins. Top panel is an artist im-pression of how structural flexibility makes it possible to sample larger volumes. The lower panel gives a schematic representa-tions of the reported tethering mechanisms for golgins. Once a tether has been estab-lished, changes in conformation and/or flexibility of the linker bring the vesicle close to the target membrane surface. The flexibility of the structure subsequently sets the area that will be sampled in the search for fusion factors.

insight into the biophysics of membrane processes, it will also facilitate the use of (biomimetic) disordered proteins in applications including drug delivery systems [141,142].

Acknowledgements

We would like to thank Ine Segers-Nolten for helpful discussions and are grateful to the “Stichting ParkinsonFonds” for financial support. References

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