Shaping Membranes with Disordered Proteins T ∗ Mohammad A.A

Archives of Biochemistry and Biophysics 677 (2019) 108163

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Archives of Biochemistry and Biophysics

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Shaping membranes with disordered proteins T ∗ Mohammad A.A. Fakhree, Christian Blum, Mireille M.A.E. Claessens

Nanobiophysics Group, University of Twente, 7522, NB, Enschede, the Netherlands

ARTICLE INFO ABSTRACT

Keywords: Membrane proteins control and shape membrane trafficking processes. The role of protein structure in shaping Disordered proteins cellular membranes is well established. However, a significant fraction of membrane proteins is disordered or Membranes contains long disordered regions. It becomes more and more clear that these disordered regions contribute to the Membrane curvature function of membrane proteins. While the fold of a structured protein is essential for its function, being dis- Membrane tethering ordered seems to be a crucial feature of membrane bound intrinsically disordered proteins and protein regions. Membrane domains 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 membrane can also be thought of as a matrix and two-dimensional anisotropic solvent for membrane proteins. Membrane proteins are In dilute solutions interactions between molecules are unlikely, yet present in or on the membrane to fulfil their function as enzymes, such interactions are essential to life. To make life possible, specific transporters, electron carriers, messenger/ligands, lipid clamps/mem- molecules have to be accumulated and contained in well-defined but brane anchors, and are involved in membrane remodeling processes responsive and dynamic compartments. In this respect, it is thought [6,9,10]. that the partitioning of bio(macro)molecules into compartments with different solvent properties, such as amphipathic micelles, complex 2. Disordered proteins and membranes coacervates and membrane enclosed organelles, plays an important role in the emergence of life [1–5]. The advantages associated with com- For the human genome, 5500 to 7500 genes have been predicted to partmentalization and the subsequent appearance of physiochemically translate into proteins which localize or interact with membranes. This different sub-compartments resulted in the evolution of cellular mem- means that around one third of the protein encoding human genome branes. translates into membrane proteins [11,12]. Considering the relative Cellular membranes are composed of lipid bilayers and their main amount of membrane proteins and their role in all intra- and inter- role is to create a physical separation between milieus that differ in cellular communication, the maintenance of chemical gradients, and chemical composition. This started from simply separating cellular membrane remodeling processes, it comes as no surprise that mem- content from the surrounding environment, and further developed into brane proteins are major players in human physiology, the pathology of compartmentalization as observed in eukaryotes. diseases, and are targets for pharmaceutical interventions [13]. Compartmentalization provides the possibility to execute functions that The function of a protein and its interaction with the membrane is require a different chemical environment without interfering with each determined by the protein's amino acid sequence. Until recently, a other. Although membranes provide a physical barrier, they are also protein's function was thought to be dependent on the formation of a responsible for the communication with the outside world. Transfer of single, well defined structure. This tight relation between structure and information and mass over the membrane is essential for cell signaling function can be observed for many membrane proteins or protein re- processes and cell function [6]. Local and dynamic membrane de- gions [14]. For these proteins it is often observed that the formation of formations enable processes such as the formation of trafficking vesicles their secondary, tertiary, and quaternary structures is facilitated by and pseudopodia [7,8]. Communication with the outside environment interacting with the membrane [15]. In recent decades, it has however and response to external stimuli is typically made possible by the pre- become clear that a large fraction of the eukaryotic proteome does not sence of proteins in or on the membrane [9,10]. In this respect, the adopt a single well defined structure but stays disordered in solution. Of

∗ Corresponding author. E-mail address: [email protected] (M.M.A.E. Claessens). https://doi.org/10.1016/j.abb.2019.108163 Received 19 July 2019; Received in revised form 23 October 2019; Accepted 27 October 2019 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/). M.A.A. Fakhree, et al. Archives of Biochemistry and Biophysics 677 (2019) 108163 the human protein-coding genes, 44% were reported to contain dis- the retention of the disordered protein in the lumen of endoplasmic ordered regions of more than 30 amino acids (AAs) in length [16]. reticulum, the C-terminally located KKXX is responsible for cytoplasmic These AA sequences that do not adopt a single well defined fold are or transmembrane localization [37]. called intrinsically disordered regions (IDRs) or intrinsically disordered proteins (IDPs) if the whole protein is disordered. IDPs and IDRs re- 3.2. Molecular recognition features (MoRFs) cently gained much attention because of their often unknown physiological function and their involvement in protein aggregation diseases MoRFs are longer (typically around 10–70 AAs) sequences than linear [17–19]. Membrane proteins, as a major fraction of the human pro- motifs. Although disordered in solution, MoRFs can adopt alpha-helix (α- teome, also contain IDPs and proteins with IDRs. It has been calculated MoRF), beta-sheet (β-MoRF), and irregular but rigid (ι-MoRF) structures, or that more than 40% of transmembrane proteins have IDRs of significant a mixture of mentioned structures upon binding a specific target [16]. Si- length [20–23]. milar to structured proteins, the secondary structure of MoRFs depend on In this review, after a short introduction into functional motifs in the AA sequence. The N-terminal IDR of the protein p53 is an example of disordered proteins, we discuss mechanisms that enable membrane an α-MoRF when it interacts with the Mdm2 protein. The C-terminal IDR anchored disordered proteins to modulate membrane properties which region of p53 is also a MoRF and can turn to an α-MoRF or ι-MoRF, upon finally defines membrane trafficking processes. interaction with S100B or Cdk2-cyclin A, respectively [38–40].

3. Disordered but functional motifs 3.3. Intrinsically disordered domains (IDDs)

By folding, a structured protein reaches a global minimum energy IDDs are the regions of the IDPs/IDRs that always remain un- state which makes the protein structure stable and functional. During structured. The IDD comprises the often bulky, yet flexible part of the the folding process, the energy minimization results in the burying of disordered protein. For IDDs, being disordered is (part of) their function hydrophobic AAs inside the protein core rather than exposing these AAs [16,41]. An example of IDDs are the intra/extra-cellular loops of to the aqueous environment [14]. Unlike structured proteins, most polytopic transmembrane proteins. In addition to their role as flexible IDPs/IDRs are enriched in AAs with charged or polar side chains for linker, it has been suggested that the orientation of the positively which exposure to the aqueous environment is not energetically pena- charged amino acids in the loop regions plays a role in stabilizing the lized, so they do not need to be buried inside the protein structure overall protein structure [42]. [24,25]. Due to the absence of structural constraints, the number of Note that a single disordered protein can contain all and multiple of accessible conformations of disordered proteins in solution is larger the above mentioned motifs. The functional motifs can even (partly) than for structured proteins [26] In comparison to structured proteins, overlap, which makes the function of the disordered protein dependent IDPs/IDRs do not have a global minimum energy state [26]. They are on the presence of interaction partners and/or the physicochemical solvated and depending on the interaction with other molecules, they characteristics of the environment [16]. Below we will discuss how can interchangeably adopt structure to reach local energy minima these disordered motifs modulate membrane properties and affect [27,28]. This implies that the use of IDPs/IDRs is flexible and that some membrane trafficking processes. disordered proteins can adopt more than one functional structure and may even have multiple functions. Similar to structured proteins, the 4. Disordered proteins modulate membrane properties presence of specific motifs in the amino acid sequence allows IDPs/IDRs to interact with other molecules and thus makes them functional. The Biological lipid bilayers are crowded with proteins. The protein to function(s) of an unstructured protein mainly results from the presence lipid mass ratio of the plasma membrane is ~1:1, while for the inner of one or more of the three structural motifs mentioned below [16]. 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 3.1. Linear motifs mean center-to-center distance between proteins is around 10 nm [44]. The interplay between lipids, the lipid bilayer, and proteins in de- Linear motifs are short sequences of 3–10 AAs. These sequences termining membrane properties is complex [45,46]. On the one hand, provide a single or multiple low affinity recognition site(s) for their some features of biological membranes – such as sub-cellular localiza- interaction partner [29]. The small size of the linear motifs makes them tion, (local) lipid composition, and curvature – are determined by the easily accessible to substrates [30]. A good example of linear motifs in presence of specific membrane proteins. On the other hand, the struc- IDPs/IDRs, are the recognition sites for post translational modification ture, sub-cellular localization, and surface density of membrane pro- (PTM) [25,31]. For example, immunoreceptor tyrosine-based activation teins is determined by the lipid bilayer [44,47]. This complex interplay motifs (ITAMs) are two short sequence of 4 amino acids (YxxL/I) se- between the lipid bilayer and proteins is controlled by feed forward parated by 6–8 amino acids on the intracellular disordered tails of and/or feedback mechanisms and results in the organization of proteins certain membrane proteins of the immune system (e.g. ζ and CD3ε by membranes and vice versa, the organization of membranes by pro- signaling subunits of T cell receptor, and γ signaling subunit of FcεRI teins [10,44]. The large number of different membrane proteins and receptor), which are targets for phosphorylation [32]. The phosphor- local or organelle specific lipid compositions add to this complexity, but ylation of ITAMs regulates the interaction between immune system also provide specificity to interactions and membrane processes. Inthe receptors and their protein target. Ligand binding or homo/hetero di- rest of this review paper, we will focus on how disordered membrane merization sites on proteins can also be linear motifs. An example of a proteins are used to regulate or control membrane processes. We will linear motif that serves as an interaction site is the LC3 interaction discuss how membrane bound disordered proteins: a) modulate mem- motif (LIR). The LIR motif has the general sequence of [W/F/Y]-X1X2- brane curvature, b) tether membranes together and c) affect (local) [I/L/V] and is found in protein complexes that facilitate the interaction lipid composition and domain formation. With this, we evaluate how with autophagosomes [33]. FAM134B has a LIR sequence and is in- disordered regions determine localization, shape, and formation of volved in changing membrane properties of the endoplasmic reticulum membrane bound organelles and vesicles inside the cell. 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 4.1. IDPs/IDRs and membrane curvature motifs also play a role in the targeting of proteins to specific cellular compartments. Two examples of such sequences are C-terminally lo- IDPs and IDRs play a role in the membrane remodeling processes cated KDEL and KKXX sequences. While the KDEL sequence results in that are required for vesicle trafficking. In this trafficking, the vesicle

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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 hindrance 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 tothe Web version of this article.)

content is transported from one cell compartment to another by alpha-helix in the presence of highly curved membranes. At low con- membrane fission and fusion processes. The mechanism by which IDPs centrations, the disordered-to-ordered transition results in the binding and IDRs remodel membrane depends on the presence of MoRFs, cel- of the ArfGAP1 to the target membrane. While at high concentrations, lular localization, surface density, and post translational protein mod- similar to αS, the asymmetric insertion of ALPS sequence into only one ifications. Below we discuss how 1) MoRFs, 2) the density of IDPs/IDRs of the membrane leaflets increases membrane curvature [7,57,58]. on the membrane surface, and 3) the combined effect of 1 and 2 con- The ability to adopt alpha-helical structure upon binding mem- tributes to membrane remodeling processes. 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- 4.1.1. MoRFing disordered proteins and membrane curvature spectively. The interaction of these proteins with membranes and Membranes can induce coupled binding-folding reactions in a generation of curvature by MoRFing into an alpha-helical structure in number of disordered proteins. The resulting newly formed structure an intermediate step towards aggregation has been suggested to be can be transmembrane or inserted into only one of the membrane toxic [59,60]. leaflets. If this insertion is asymmetric, it will result in a unilateral increase in the surface area. This changes the ratio between the outer and 4.1.2. Density of disordered proteins on the membrane surface inner surface area of the membrane. In order to adopt a topography that It has been shown that disordered proteins do not only induce corresponds to this new ratio, the membrane remodels by increasing its curvature by the MoRF mechanism described in 4-1-1. The asymmetric curvature (Fig. 1A). Clearly, how much the curvature increases depends coupling of disordered proteins to one of the leaflets of a phospholipid on two factors: a) the size of the inserted MoRF, b) surface density of the membrane can induce spontaneous membrane curvature even in the disordered protein. An example of a disordered-to-ordered structural absence of a mechanism that generates an area difference between the change that induces curvature happens in the N-terminal region of leaflets of the bilayer. The mechanism by which surface adsorbed or alpha-synuclein (αS) in the presence of anionic lipid bilayers. Binding anchored disordered proteins generate curvature is generic and can also of αS to these lipid bilayers results in the formation of amphipathic be exploited by coupling DNA or synthetic polymers and even struc- alpha-helix [48–50]. When this alpha-helix inserts into the outer leaflet tured proteins to model membranes [61–63]. Although the mechanism of the lipid bilayer it increases the surface area of this leaflet with ~15 is not specific, it has been suggested to contribute to membrane re- nm2/αS and induces curvature [51]. The transition to the alpha-helical modeling in biological systems. To account for the observed membrane state and the resulting generation of curvature has been demonstrated curvature generation by membrane bound disordered proteins, two in in vitro experiments with model lipid vesicles [52–54]. Following closely related mechanisms have been put forward. 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- 4.1.2.1. High IDP/IDR surface density – steric pressure. The first structured cytosolic form of the protein [55]. In a tested SH-SY5Y cell mechanism by which membrane anchored IDPs/IDRs can induce model, the surface density of αS on cellular vesicles was found to be curvature describes that the steric hindrance among the adsorbed high enough to induce curvature by the MoRF insertion mechanism disordered proteins on the membrane at very high surface density [56]. results in a lateral pressure. For asymmetric IDP/IDR adsorption or Another example of membrane curvature sensing/modulation by anchoring, increasing the curvature of the membrane will result in the the MoRF motif of a disordered protein is ADP-ribosylation factor increase in the area of the (now) outer leaflet of the bilayer which GTPase-activating protein 1, ArfGAP1 [57]. In the absence of lipid reduces the pressure (Fig. 1B). Examples of proteins with IDRs for membranes, ArfGAP1 is mainly disordered. The ArfGAP1 lipid packing which this mechanism of action has been suggested are epsin1, p180, sensor region (ALPS), which consists of ~40 amino acids, MoRF into an disordered regions of BAR proteins, and αS [64–66].

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4.1.2.2. Lower IDP/IDR surface density – lateral pressure. The second surface density of the proteins will result in the saturation of the mechanism described for curvature induction by disordered proteins is membrane with proteins. At the saturating concentrations, steric pres- closely related to the first, but ascribes the generation of curvature to sure starts to play a role in membrane remodeling. As one can see, the the thermal motion of these macromolecules on the membrane magnitude of the contribution of the different mechanism to generating (Fig. 1C). The membrane is a fluid and the anchored disordered curvature depends on: a) size of the structured region, b) size of the proteins are free to diffuse in the membrane leaflets. This thermal disordered region, c) and surface density of the protein. This last motion on the membrane surface will result in collisions with other parameter depends on the concentration in the bulk and affinity to the membrane anchored macromolecules. The collisions generate a lateral membrane. pressure on the membrane. Similar to the mechanism described for steric hindrance, increase in the membrane curvature will help to 4.2. Tethering membranes together using disordered proteins reduce this pressure. The main difference between the two mechanisms is that much lower protein surface densities are required for the second The flexibility and charged nature of membrane anchored dis- mechanism to work. The second mechanism has also been modeled ordered proteins gives them the ability to extend in to the environment assuming that the disordered regions of the membrane inserted proteins surrounding the membrane and fish for target molecules in solution or can be described as hard disks that diffuse in a 2D liquid and taking into on neighboring vesicles. The capturing of the target by disordered account the mechanical properties of the lipid bilayer [62]. For both membrane bound protein results in conformational changes in this structured and disordered proteins, good agreement has been found protein, which brings the target molecule closer to the opposing between experiments and model calculations [62,64,67]. membrane. Interestingly, this structural change in the disordered pro- For both high and low surface density mechanisms, since the mag- tein upon target binding can involve both disorder-to-order and order- nitude of the lateral pressure depends on the distance of the center of to-disorder transitions. Below we describe some examples in which mass of the anchored protein to the bilayer, disordered proteins are membrane anchored disordered proteins exploit these transitions. much more efficient in generating curvature than structured proteins with the same number of AAs. Disordered proteins are less compact 4.2.1. Disorder-to-order transition which makes them more efficient in generating lateral pressure. The For relatively short disordered MoRF containing proteins, approxi- presence of a large fraction of charged and polar amino acids in dis- mately smaller than 200 AAs, the largest dimension of the IDR in the ordered proteins contributes to their bulkiness. A possible additional structured state is smaller than the radius of gyration of the protein in advantage of using disordered chains is that their radius of gyration can the disordered state. Consequently, the disordered form is more effi- be dynamically addressed. The volume that the disordered chain oc- cient than structured form in fishing for target molecules. In the dis- cupies can be modulated by interacting with other molecules. Any ordered form the protein samples a larger volume compared to the changes in the effective (spherical) volume occupied by the disordered protein in the structured form. Coupled target binding and adopting regions of the protein, will result in a change in the generated pressure, structure, results in the compaction of the disordered region. The dis- and can consequently be used to help change membrane curvature. For ordered proteins can thus be used to catch the target molecule and example, αS has been suggested to bind synaptic vesicles via its N- bring it closer to the membrane. terminal MoRF region [49] while the disordered C-terminal region in- The Soluble N-ethylmaleimide-sensitive factor attachment receptor teracts with calcium ions [68,69] and the vesicle bound SNARE-protein (SNARE) complex is a good example of a disordered protein system that synaptobrevin [56,70–72]. These C-terminal interactions may result in tethers membrane bound compartments together using this mechanism. compaction of the bulky disordered region. Such a compaction would There are two sets of SNARE proteins: one on the target membrane result in a decrease in the lateral pressure exerted by the disordered known as t-SNAREs. On the active zone membrane of synapses of region of αS and thus make synaptic vesicles more fusion prone. neurons, these t-SNAREs include syntaxin-1 and SNAP-25. The other set, located on vesicles, is known as v-SNAREs and include synapto- 4.1.3. Cooperation of ordered and disordered regions brevin (VAMP2). Membrane bound monomeric SNAP-25 and syntaxin- It has been shown that membrane curvature modulation can be a 1 on the target membrane are disordered proteins [75–78], but upon result of the cooperative action of structured and disordered regions of interaction with synaptobrevin on the vesicle membrane, they turn into membrane proteins. In this sense, curvature is induced by combining parallel alpha-helices to form the SNARE complex core [79]. Synapto- the effect of the asymmetric binding of a (partially) structured curva- brevin is a transmembrane protein with a disordered region extending ture scaffold protein or the insertion of a (partially structured) protein into the cytosol. The interaction with SNAP-25 and syntaxin-1 also into one of the bilayer leaflets and creating lateral pressure bythe causes the disordered region of synaptobrevin to adopt an alpha-helical unstructured, solvent exposed part (Fig. 2A). Examples of cooperation structure [79–81]. The formation of the SNARE complex thus involves a between structured and disordered regions of a protein in membrane disorder-to-order transition that brings the target and vesicle mem- remodeling include, but are not limited to, the curved BAR domain branes in very close proximity. This initiates the fusion of the vesicle membrane scaffold proteins and αS which partly inserts into mem- with the target membrane. In Fig. 3ii this process is schematically de- branes via the formation of an amphipathic alpha-helix [65,73,74]. picted. In our recent work, we have studied the ability of αS to remodel The SNARE machinery not only facilitates the fusion of vesicles with membranes using the combined action of structured and disordered a target membrane, it also provides specificity to the fusion process. regions [74]. From this work we derive that at low concentrations of Different sets of t-SNARE and v-SNARE proteins are found on thedif- proteins on the membrane, the interaction between the proteins is ferent membrane bound organelles in cells. Only specific combinations negligible. Consequently, the lateral pressure generated by membrane of t-SNAREs and v-SNAREs result in the efficient formation of an alpha- bound proteins is very small and the membrane remodeling is domi- helical coiled-coil. For example, the syntaxin-16/Vti1a/syntaxin-6/ nated by the structured region of the protein (Fig. 2B). Fig. 2B shows VAMP4 SNARE complex is involved in retrograde transport from early that for an IDR with a radius of gyration (Rg) < ~4 nm, insertion of an endosomes to trans-Golgi network, while the syntaxin-16/Vti1a/syn- amphipathic alpha-helix (MoRF) is the dominant mechanism of cur- taxin-10/VAMP3 complex provides specificity in retrograde transport vature induction for all protein concentrations shown. At higher protein from the late endosomes to the trans-Golgi network [82,83]. The en- concentrations, interactions between the membrane bound proteins dosome bound VAMP3 and VAMP4 encode the origin of these vesicle, becomes significant which increases the contribution of the lateral while syntaxin 6 and syntaxin 10 are specifically found on the Golgi pressure to the generation of curvature and makes curvature less de- membrane [83]. There are some differences between syntaxin6/syn- pendent on the size of the MoRF (Fig. 2B). Further increasing the taxin10 and VAMP3/VAMP4 in single or short sequences of amino acids

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Fig. 2. A Schematic of how IDPs induce curvature by MoRFing into amphipathic alpha-helices and by generating lateral pressure with their large unstructured domains. Assuming equilibrium 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 lateral 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 membrane. Panel B depicts calculations showing

which combinations of MoRF area, Rg, and protein concentration result in the formation of vesicles with a radius of 45 nm taking into account the above mentioned mechanisms. The parameters in this figure span a physiologically relevant range. We calculated Rg of the IDR assuming a self-avoiding random walk, and the Rg values 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 Rg of 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 curvaturethan amphipathic alpha-helix insertion. in alpha-helices. However, the major pair-wise difference between the target molecule closer to the membrane surface (Fig. 3i). An ex- these proteins is in their disordered regions. Syntaxin 6 and syntaxin 10 ample of a disordered protein that tethers two membrane bound com- mainly differ from each other in the disordered region which linksthe partments using this mechanism is EEA1. According to D2P2 database, two alpha-helices in their SNARE complexed structures. In the case of early endosome antigen-1 (EEA1) is disordered [84] and binds to PI(3)P VAMP4 and VAMP3, the latter is missing a large portion of disordered on early endosome membranes via its C-terminal FYVE domain [85]. amino acids in comparison to VAMP4. These examples indicate that the Upon binding to PI(3)P, two monomeric EEA1 proteins adopt a stiff disordered regions are important to infer specificity to the tethering coiled-coil structure which extends up to 200 nm into the cytosol. This interactions. 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- 4.2.2. Order-to-disorder transition coil structure of the EEA1 which results in the collapse of the extended For disordered MoRF containing proteins longer than ~1000 AAs, EEA1 homodimer [87]. The magnitude of this entropic collapse force the longest structured length of the protein can be significantly larger was measured to be around 14kBT for a single homodimer of EEA1 than the radius of gyration of the protein in disordered form. [87]. The force exerted by this order-to-disorder transition brings two Consequently, the ordered form enables fishing by sampling a distance early endosomes closer together and facilitates the fusion of early en- further away from the membrane in comparison to the unstructured dosomes via the SNARE machinery. This fusion subsequently results in form, while the transition to the disordered state can be used to bring

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 proteins 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.

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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 D2P2 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. 4 and 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] the formation of larger early endosomes [87]. These processes are 4.3. Lipid domains and IDPs shown schematically in Fig. 3. Biological membranes consist of more than one lipid type and the 4.2.3. Tethering membranes using both order-disorder and disorder-order composition of membranes differs between different organelles. This transitions diversity in lipid composition is generated and maintained by mem- For disordered proteins with an approximately equal length of dis- brane proteins which in return help to organize membrane proteins ordered and ordered structures, the structural flexibility (due to the selectively. Lipid composition of the membrane determines the speci- presence of disordered regions or interchange between the ordered and ficity of the interaction between proteins (that are involved in curvature disordered form) enables fishing and tethering of target molecules to and tethering mechanisms) and membranes. In the next two subsec- the membrane. Golgin proteins are set of disordered tethering proteins tions, we discuss how disordered proteins are involved in generating that use this mechanism and localize at Golgi apparatus (GA) mem- specific (local) lipid compositions and how they affect the formation of brane. Prior to or upon localization at the GA membrane, golgins form lipid domains. dimers and adopts a coiled-coil structure [88,89]. Golgins have a significant amount of disordered regions, both before and after thefor- 4.3.1. Disordered proteins and lipid composition mation of the coiled-coil dimers (See Table 1). Most of the golgins bind The lipid composition differs among cellular organelles and be- to GA via their C-terminal region and tether vesicles to the GA mem- tween their outer and inner leaflets. Membrane proteins are involved in brane via their N-terminal region. Depending on the golgin species, introducing and maintaining these differences in two ways. The first either one or both the C-terminal and N-terminal region are disordered. mechanism is to cause newly synthesized lipids to be incorporated As can be seen from Table 1, a number of membrane bound target asymmetrically in the lipid bilayer. A good example of proteins that molecules/proteins has been identified for each golgin. From the play a role in maintaining an asymmetry in the lipid composition of the summary of mechanisms by which Golgins tether membrane surfaces bilayer leaflets are flippases [124,125]. Flippases are enzymes that help presented in Table 1 and Fig. 4, it can be seen that being or becoming the inter-leaflet translocation of certain lipids. Their action conse- disordered is an essential feature of most of the proposed mechanisms quently results in the asymmetric distribution of the lipids between the [90,91]. However, the exact mechanism(s) by which golgin proteins inner and outer leaflets of the membranes. Flippases contain IDRs bring the captured vesicle closer to the GA membrane is understudied which link the structured regions to each other. It has been shown that [91]. Towards unraveling mechanisms, Cheung and coworkers showed targeting the disordered region of a flippase (i.e. PglK), inhibits its flip- that the formation of a disordered “bubble” in the middle of the flop activity [126]. This shows that the disordered regions of these homodimer coiled-coil of GCC185 is responsible for the bending of the proteins are essential for their activity possibly by providing freedom to coiled-coil structure [92] (see Fig. 4). For GMAP-210 homodimer the relative motion of the structured parts of the enzyme. coiled-coil structure, structural flexibility along with interaction with a The second mechanism is to modify the head groups of the lipids on GTPase protein (i.e. Rab2) has been reported to be involved in trapping the membrane. For example, phosphatidylinositol phosphate kinases the coiled-coil structure in a bended state which brings the vesicle (PIPKs) are kinase proteins that phosphorylate the phosphatidylinositol closer to GA membrane [93] (see Fig. 4). (PIP) lipids on the membranes. PIPKs have a “specificity” loop which In addition to the tethering function of golgin proteins, their loca- consists of a disordered region and determines the specificity of the lization on the GA membrane, length, and interacting targets have been PIPK towards the substrate [127]. Depending on the class and type of reported to determine the shape and specific morphology of the GA the PIPK, the product of the phosphorylation can be either PI(3,4)P2, PI [91,94]. Golgins seem to fully exploit the functional features of the (3,5)P2, or PI(4,5)P2. Each of these phosphorylated PIs act as a cue for combination of rigid membrane bound structures and the presence of specific protein-membrane interaction. For example, PI(3,5)P2 is a disordered flexible regions. specific cue to early endosomes [128], while PI(4,5)P2 is a cue on the Another example of an IDP that may use mixed mechanisms to te- inner leaflet of the plasma membrane. ther vesicle together is αS. αS has been reported to be able to bridge Protein membrane interactions are not only governed by the lipid two neighboring vesicles [123]. The N-terminal AAs of αS can adopt an composition of the membrane, but the density of the cues on the alpha-helical structure upon binding membranes. Of this alpha-helix membrane also plays an important role. Feedback/feed forward me- the first 20 N-terminal AAs bind strongly to the membrane, the restof chanisms result in changes of the cue density on the membrane. An the helix has been reported to have a lower membrane affinity [123]. example for feedback control that involves proteins with a disordered This lower affinity makes it possible to detach part of the helix fromthe region is the interplay between Rab5 and phosphatidylinositol 3- vesicle. This part becomes disordered, and tethers to a neighboring phosphate, PI(3)P, on early endosomes. Rab5 is a small GTPase that vesicle using the transition to an α-MoRF [123]. binds to PI(3)P on early endosomes via a phosphate-binding disordered

6 M.A.A. Fakhree, et al. Archives of Biochemistry and Biophysics 677 (2019) 108163

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 representations of the reported tethering mechanisms for golgins. Once a tether has been established, 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.

region [129]. There are two more functional disordered loops in Rab5 5. Conclusions and outlook called switch I and switch II, which mediate the interaction of Rab 5 with phosphoinositide 3-kinase (PI3K). PI3K increases the phosphor- In comparison to structured proteins disordered proteins of similar ylation of the phosphatidylinositol headgroup on the surface of the AA length occupy large volumes, are flexible, and can undergo coupled membrane which increases the local density of PI(3)P. This conse- binding-folding reactions. These feature are exploited by cells for quently increases the recruitment of more Rab5 on the early endo- functional purposes. Here we discussed how IDPs and IDRs are used to somes. The resulting high surface density of PI(3)P and Rab5 is one of modulate membrane properties and thus affect membrane trafficking. the specific features of early endosomes. The interplay between proteins and membranes is a two way process; proteins not only affect membrane properties, the membrane lipids also 4.3.2. Lipid domains and disordered proteins affect the binding and organization of proteins. In spite of this, the Biological membranes contain lipids that differ considerably in tail present view is rather membrane centered. The membrane is a matrix in length and headgroup properties. In in vitro model membranes this which IDPs are embedded or to which IDRs are anchored that help the difference in properties can result in phase separation. In equilibrium membrane perform its functions. However in some cases a more IDP/ multi-component lipid bilayers are thus expected to macroscopically IDR centric view on membrane trafficking processes is justified. phase separate. However, the coarsening process that follows the nu- More and more IDPs are reported to be able to undergo liquid- cleation of a new phase is typically very slow. This slow coarsening liquid phase separation/transitions both in vitro and in vivo [135–137]. combined with membrane asymmetry, active processes, and local an- This phase separation is responsible for the formation of so-called choring to the supporting cytoskeleton probably prevents global phase membraneless organelles. These membraneless organelles play a role in separation of membranes in biology [130–132]. However, nano-do- various cellular tasks and have e.g. been shown to drive the formation mains of specific lipids have been reported to exist in biological of nucleolar subcompartments [138]. Recently, a phase separating membranes [131,133]. protein, synapsin, has been shown to play a role in the organization of Besides the mechanism mentioned above, the presence of proteins membranes [139]. Synapsin plays a role in the uptake and release of in the membrane has also been put forward as a reason why phase vesicles from the synaptic vesicle pool. The disordered region of sy- separation does not occur at larger length scales in biological mem- napsin undergoes liquid-liquid phase separation and forms droplets. At branes. Experiments on giant unilamellar vesicles with a lipid compo- the same time synapsins interact with synaptic vesicles via their sitions at which a liquid-ordered and liquid-disordered phase coexist membrane binding region. It has been suggested that these droplets act indicate that membrane anchored disordered proteins may also con- as a matrix for the organization of the synaptic vesicle pool [140]. tribute to preventing phase separation. When PEGylated phospholipids Phosphorylation of synapsin by calcium/calmodulin dependent protein are added to these vesicles, lipid phase separation is suppressed [134]. kinase II (CaMKII) dissolves the droplet and makes the synaptic vesicles The mechanism responsible for this suppression is argued to involve available for release [139,140]. Similar phase separation phenomena crowding and the generation of lateral pressure by the membrane an- may play a role in the physiology of other membrane binding dis- chored polymer [134]. However, it is not clear if in a cellular context ordered proteins. the lateral pressure generated by disordered proteins significantly In conclusion, although it is clear that disordered proteins help contributes to the prevention of membrane phase separation. Further shaping membranes, insights into the mechanisms exploited by specific research to quantify the different contributions and asses the effec- proteins are often lacking. Additionally the relative contributions of tiveness and regulatory roles of disordered proteins in this process, is disordered regions compared to the structured regions in biology is still required. underinvestigated. Studies into these aspects will not only improve our

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