membrane association

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OPEN ACCESS
Perspective
The membrane surface as a platform that organizes
cellular and biochemical processes
Thomas A. Leonard,1,2,* Martin Loose,3,* and Sascha Martens1,4,*
1Max Perutz Labs, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 9, 1030, Vienna, Austria
2Medical University of Vienna, Center for Medical Biochemistry, Dr. Bohr-Gasse 9, 1030, Vienna, Austria
3Institute of Science and Technology Austria, Am Campus 1, 3400 Klosterneuburg, Austria
4University of Vienna, Center for Molecular Biology, Department of Biochemistry and Cell Biology, Dr. Bohr-Gasse 9, 1030, Vienna, Austria
*Correspondence: thomas.leonard@meduniwien.ac.at (T.A.L.), martin.loose@ist.ac.at (M.L.), sascha.martens@univie.ac.at (S.M.)

https://doi.org/10.1016/j.devcel.2023.06.001
SUMMARY
Membranes are essential for life. They act as semi-permeable boundaries that define cells and organelles.
In addition, their surfaces actively participate in biochemical reaction networks, where they confine pro-
teins, align reaction partners, and directly control enzymatic activities. Membrane-localized reactions
shape cellular membranes, define the identity of organelles, compartmentalize biochemical processes,
and can even be the source of signaling gradients that originate at the plasma membrane and reach
into the cytoplasm and nucleus. The membrane surface is, therefore, an essential platform upon which
myriad cellular processes are scaffolded. In this review, we summarize our current understanding of
the biophysics and biochemistry of membrane-localized reactions with particular focus on insights
derived from reconstituted and cellular systems. We discuss how the interplay of cellular factors results
in their self-organization, condensation, assembly, and activity, and the emergent properties derived
from them.
INTRODUCTION HOW DOES CONFINEMENT TO A TWO-DIMENSIONAL

SURFACE INFLUENCE BIOCHEMICAL REACTIONS?
Every cell is separated from the extracellular space by the
plasma membrane. In addition, membranes encapsulate Enzymes facilitate chemical reactions that, uncatalyzed, would
numerous subcellular compartments to separate biochemical have kinetics incompatible with life. Membranes influence the
reactions with semi-permeable boundaries while also providing rates and specificity of these reactions by confining them within
extensive surfaces for proteins to bind to. For example, a typical a defined volume (volume-confined reactions) or on a surface (sur-
hepatocyte cell has a volume of about 5,000 mm3 and contains face-confined reactions). Eukaryotic cells are characterized by
membranes with a total surface area of approximately numerous membrane-encapsulated compartments in which vol-
110,000 mm2.1 It is, therefore, not surprising that a plethora of ume-confined reactions take place under conditions distinct
biochemical reactions, assemblies, and processes are spatially from the cytosolic milieu. The influence of volume confinement
and temporally coordinated on a membrane surface. The mem- on biochemical reactions has been extensively reviewed else-
brane accommodates signaling molecules that are permanently where2 and will not be the subject of this article. Here, we
embedded in membranes, such as transmembrane proteins, focus on how the membrane influences reactions confined to its
and transiently associated, membrane-binding proteins that, surface.
together, permit both intercellular and intracellular communica- Binding of proteins to membranes affects the thermody-
tion. As such, membrane-localized reactions act as signal pro- namics and kinetics of biochemical interactions in fundamental
cessing and molecular assembly hubs where information can ways (Figure 1). Assuming a cuboidal hepatocyte with a side
be written, read, and erased with high fidelity in both space length of 20 mm,4 binding to the inner leaflet of the plasma mem-
and time. brane confines a protein to an area of around 2,400 mm2 and into
The membrane surface enables protein interactions that a thin layer of about 5 nm below the membrane. The accessible
would otherwise be impossible in solution. In this review, we volume is, therefore, reduced from 8,000 mm3 to only 12 mm3,
will first explain how the membrane functions as an active increasing the effective local concentration of reactants by a fac-
signaling scaffold that can initiate and control biochemical reac- tor of more than 600 (Figure 1A). The presence of diffusion bar-
tions. In the following sections, we will use examples to illustrate riers or the formation of protein assemblies can laterally confine
how membrane-localized reactions orchestrate complex cellular the reaction partners further. The implications for protein-protein
behavior by applying a paradigm of writers, readers, and erasers interactions are significant. In solution, reactants are often
to help conceptualize what is arising at the membrane; namely, present only at nanomolar concentrations, whereas their disso-
that enzymes pattern the membrane by adding, detecting, and ciation constants (KD) are in the micromolar range. Confining sol-
removing marks on its surface. uble proteins in such a small volume dramatically increases the
Developmental Cell 58, August 7, 2023 ª 2023 The Author(s). Published by Elsevier Inc. 1315
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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OPEN ACCESS Perspective
A B
C D
Figure 1. How does confinement to a surface influence biochemical reactions?

(A) By confining a protein into a thin layer 5 nm from the surface, membrane binding dramatically increases the concentration of reactants.
(B) Although the characteristic diffusion coefficient of GFP in the eukaryotic cytoplasm is approximately 30 mm2/s,3 it is reduced by 100-fold on the membrane.
(C) Membrane binding not only reduces lateral mobility, but also limits the rotational degrees of freedom.
(D) The combination of spatial and rotational confinements helps to align reaction partners and, thereby, lowers the entropic cost of their interactions.
apparent affinity and specificity, enabling protein interactions vating proteins (GAPs). As these enzymes can be retained on
impossible in solution.5 the membrane, the outcome of the biochemical reaction can
The reduction of dimensionality can speed up biochemical re- significantly change. First, localization to a 2D surface can facil-
actions not only due to an increase in the thermodynamic stabil- itate processive enzymatic activity, where an individual enzyme
ity of protein complexes, but also because the encounter prob- catalyzes the modification of many substrates.10–12 Second, en-
ability between proteins is density dependent, which can be zymes might be recruited by their membrane-bound product via
significantly enhanced on a membrane surface (Figure 1B).6 In a secondary binding site. In this case, membrane recruitment not
fact, there is a chance that mobile reactants never run into only promotes processivity but also gives rise to a positive feed-
each other in three dimensions, whereas they will eventually back loop that promotes ultrasensitive behavior.10,13,14 Here,
encounter each other if bound to the same two-dimensional small changes in enzyme concentration can give rise to a dra-
(2D) surface (unless they are separated by physical barriers).7,8 matic, switch-like transition between two different activity states.
At the same time, membrane binding typically slows down pro- For example, a biochemical system composed of a reversibly
tein diffusion by a factor of 100, reducing encounter rates. In binding protein and its regulatory enzymes can collectively
extreme cases, reactants could even be effectively immobilized switch between its off-state, where most proteins are in solution,
on the membrane and the reaction rate will tend to zero.5 An and its on-state, where the activating enzyme dominates the
enhancement of the encounter rate is, therefore, unlikely to be system and most proteins are membrane bound. Strong, non-
the only reason for faster reactions. In addition to increased linear positive feedback can also give rise to bi-stability, in which
local concentrations, membrane confinement also accelerates the system has two stable steady states and shows hysteretic
biochemical reactions by aligning reaction partners.6 As the rota- behavior (see glossary in Supplemental Information). In extreme
tional motion of a protein is significantly restricted to the axis cases, the biochemical network can give rise to irreversible tran-
perpendicular to the membrane, the entropic cost of protein in- sitions, where the system is trapped in one activity state.10,15–17
teractions is significantly lowered (Figures 1C and 1D). In this case, the positive feedback is so strong that it cannot be
If reaction partners reversibly bind to the membrane, the kinetic outcompeted by the enzyme that catalyzes the reverse reac-
enhancement also depends on the time the proteins spend on the tion.18 These kind of emergent properties of biochemical net-
membrane: for fast binding rates, the combination of dimension- works on membrane surfaces likely play an important role in
ality reduction and an increase in effective concentration allows determining the directionality of vesicle trafficking pathways.19
proteins to react more quickly, whereas the rate of membrane as- By contrast, in the case of multi-step phosphorylation
sociation can become rate limiting for slow binding reactions.9 cascades, membrane localization normally suppresses bi-sta-
Membrane binding also affects the emergent properties of bility.20–24 The decreased mobility and high density of the sub-
biochemical networks, in which many proteins interact to exhibit strate on the membrane facilitate enzyme rebinding, promoting
collective behavior. Examples of soluble enzymes acting on a processive phosphorylation mechanism, which is incompat-
membrane-bound substrates include kinases, phosphatases, ible with bi-stability.25 However, as suggested for the multi-
guanine nucleotide exchange factors (GEFs), and GTPase-acti- site phosphorylation of the CD3x subunit of the T cell receptor
1316 Developmental Cell 58, August 7, 2023

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Perspective OPEN ACCESS
Box 1. The Writer/Reader/Eraser paradigm for signaling systems
A B
(A) In signaling systems, writers add marks to substrates, such as other proteins or lipids. These post-translational modifica-
tions are recognized by reader proteins and can be subsequently removed by erasers. The opposing activities of writers
and erasers can generate spatially defined and time-limited biochemical patterns on the cell membrane that are recognized
and interpreted by readers. In GTPase signaling, writers, readers, and erasers have also been referred to as activators, effec-
tors, and inactivators, respectively.
(B) The writer, reader, and eraser activities are localized to individual protein domains, which can be combined together in a
particular protein or within a protein complex. Functional coupling of activities can result in complicated biochemical networks
with myriad inputs and feedback control mechanisms.
(TCR) by the Src family kinase Lck, a switch-like transition in referred to as readers. Finally, enzymes that remove membrane
signaling cascades is still possible on membrane surfaces.26 surface marks will be considered as erasers. Under certain cir-
This theoretical study has proposed that a short refractory cumstances, erasers that generate a new mark by removing
period of the enzyme after substrate phosphorylation could another mark could also be considered as writers. The counter-
maintain the distributed, ultrasensitive mechanism. How mem- acting activities of writers and erasers control membrane sur-
brane binding actually affects the emerging properties of face-based marks in a time- and space-dependent manner.
signaling networks must, therefore, be tested empirically. Readers then recognize and interpret these time-limited and
Spatiotemporal protein patterns commonly emerge from spatially defined marks on the membrane surface. These func-
the highly cooperative binding of proteins and their reduced tions can be part of the same protein or of a protein complex,
mobility on the membrane surface. For example, the bacterial allowing for the functional coupling of enzymatic activities
cell division proteins MinD and MinE self-organize into (Box 1B).
protein oscillations in vivo27,28 and traveling waves in vitro.27
These spatiotemporal patterns can only emerge because WRITERS: HOW MEMBRANES ARE PATTERNED
the mobility of membrane-bound proteins is sufficiently IN CELLS
decreased. By contrast, their fast diffusion in solution rapidly
disperses the unbound proteins.27,28 Similarly, the polarized The membrane surfaces of individual intracellular compartments
distribution of activated Cdc42 necessitates slow diffusion comprise unique combinations of lipids and proteins. These
on the plasma membrane to constrain proteins within a small biochemical identities, or ‘zip codes,’ are generated by writers
surface area.29 whose activity in space and time is precisely controlled. With
more than 1,000 lipid species and nearly 24,000 proteins, of
THE READER/WRITER/ERASER PARADIGM IN THE which approximately 6,500 are integral membrane proteins33,34
CONTEXT OF MEMBRANE-LOCALIZED PROTEIN and a further 3,500 predicted to bind membranes,35 human cells
INTERACTIONS have evolved an enormous arsenal of tools to provide unique
biochemical identities.
Basic cellular processes depend on the precise execution of In the cell, the activities of different writers are often function-
molecular programs in space and time. The fundamental logic ally coupled in a precise and highly dynamic manner.36 For
of these reactions can be understood by using the writer/ example, phosphoinositide kinases phosphorylate the hydroxyl
reader/eraser paradigm (Box 1A). Originally introduced to char- groups of the inositol ring of phosphatidylinositols (PIs) at
acterize the histone code,30 this paradigm has been success- specific positions (Figure 2A). The dynamic interconversion of
fully applied to many other cellular signaling networks.31,32 phosphoinositides (PIPs) controls membrane trafficking in the
Accordingly, enzymes that modify the membrane surface by endolysosomal pathway37 as well as cell polarity.38 For a
adding marks will be referred to as writers, whereas proteins comprehensive overview of the roles played by PIPs in scaf-
that detect and bind to membrane surface marks will be folding biological processes at discrete locations in the cell
Developmental Cell 58, August 7, 2023 1317

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A B
C D E
F G

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with high temporal resolution, we refer readers to an excellent together, enhance their activity toward Rab7.46 The subsequent
review.39 down-regulation of PI(3)P synthesis by WDR91, a Rab7 effector,
In addition to lipid kinases, lipases can pattern membranes is essential for endolysosomal trafficking and neuronal develop-
by specifically removing lipid headgroups. For example, ment in mice.47 During polarized cargo trafficking from the Golgi,
phospholipase C (PLC) hydrolyses the headgroup of the generation of GTP-bound ARF6 drives the recruitment of the
phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), generating lipid kinase phosphatidylinositol 4-phosphate 5-kinase type-1
diacylglycerol (DAG). For an in-depth treatment of signal inte- gamma (PIP5K1C), which mediates the conversion of phosphati-
gration, transduction, and regulation of the six mammalian dylinositol 4-phosphate (PI(4)P) into PI(4,5)P2 and the consequent
PLC families, we refer the reader to an excellent review.40 tethering of cargo vesicles to the plasma membrane by the exocyst
Furthermore, the membrane can be rendered more prone to complex.48
binding by proteins that partially insert into the bilayer by the Protein myristoylation, prenylation, or palmitoylation is yet
action of lipid desaturases (Figure 2A). another way in which the surfaces of membranes can be
Together with PIPs, membranes are also identified by their patterned49 (Figure 2C). For the GTPases ARF6 and Ras, the dif-
protein content. For example, protein tyrosine kinases mark re- ferential intracellular distribution of modifying enzymes can
ceptor tails with phosphotyrosine, creating docking motifs for a establish a spatial organizing system that contributes to a
plethora of adaptor proteins and effector kinases. The activity defined distribution of lipidated proteins on different organ-
of GEFs leads to the membrane association of small GTPases elles.50,51 In addition, the ubiquitin-like protein ATG8 is conju-
of the Rab family due to their post-translational lipid modifica- gated to phosphatidylethanolamine (PE), a reaction that is
tions. These molecular switches can bind reversibly to the mem- referred to as lipidation and is accomplished by a complex of
brane and act as important markers of endomembranous cell E1-, E2-, and E3-like enzymes, similar to ubiquitylation.52 The
compartments (Figures 2B and 2C). The compartment specificity recruitment of the ATG12-ATG5-ATG16L1 E3-like complex to
of these GTPases is largely governed by their cognate GEFs the nascent phagophore during macro-autophagy is mediated
(Figure 2B).41 All known GEFs are peripheral membrane proteins by a subset of the PI(3)P-binding b-propeller WIPI proteins of
themselves42 with ancillary reader domains that often recognize the PROPPIN family,53 connecting the class III phosphatidylino-
molecular marks or interaction partners that regulate their nucle- sitide 3-kinase (PI3K)-mediated synthesis of PI(3)P, which itself
otide exchange activity via the allosteric control of autoinhibi- is dependent on WIPI2 in a positive feedback loop,54 to the lipi-
tion.43 These complex biochemical networks can also include dation of ATG8 with PE.55 ATG8 can also undergo conjugation to
non-linear feedback controls.19 Indeed, reconstitution experi- phosphatidylserine (PS) in pathways that are different from
ments with Rab5, its exchange factor Rabex5, and effector Ra- macro-autophagy.56 The marking of the nascent phagophore
baptin5, on supported lipid bilayers showed that this minimal with ATG8 provides a template for the interaction of cargo
protein system includes a positive feedback loop that gives adapters and receptors and eventual encapsulation of the cargo
rise to an ultrasensitive, collective activation and spatial for targeting to the lysosome. The ATG8-interacting motif, also
patterning of Rab5 on the membrane.17,44 referred to as the LC3-interacting region (LIR), drives the recruit-
The activity of writers can also be functionally coupled to give ment of interaction partners to ATG8-positive membranes.57
rise to signaling cascades. For example, during the maturation of Recently, it was shown that ubiquitin as well as the ubiquitin-
early endosomes along the endolysosomal pathway, the activity related proteins NEDD8 and ISG15 can also be directly conju-
of Rabex5:Rabaptin5 is followed by that of the Rab7 GEF complex gated to the headgroup of PE.58
Mon1-Ccz1.45 As a result, Rab5 is replaced by Rab7 in a process Although vesicular trafficking inevitably leads to the redistribu-
termed Rab conversion.45 In vitro experiments have demonstrated tion of lipids within cells, the majority of lipids reach their destina-
that Mon1-Ccz1 recognizes a combination of membrane-bound tion via lipid transfer proteins (Figure 2D).59 Patterning of cellular
Rab5-GTP and phosphatidylinositol 3-phosphate (PI(3)P), which, membranes can also be accomplished by integral membrane
Figure 2. Writing, reading, and erasing the membrane code

Writers
(A) Lipid-based membrane patterning can consist of the kinase-mediated phosphorylation of the headgroup of phosphatidylinositol (PI); the lipase-mediated
hydrolysis of headgroups, for example, to generate diacylglycerol (DAG); or the desaturation of lipids to increase inter-lipid spacing.
(B) Protein tyrosine kinases catalyze the phosphorylation of specific tyrosine residues in the cytoplasmic tails of receptors, creating docking sites for adaptor and
effector proteins. GEFs catalyze the exchange of GDP for GTP in Arf, Ras, and Rab family GTPases, thereby promoting their membrane binding.
(C) Myristoyl-, palmitoyl-, farnesyl-, and geranylgeranyltransferases modify proteins such as Arf, Ras, and Rab family GTPases, allowing them to bind to specific
domains in membranes. E1, E2, and E3(-like) enzymes mediate the conjugation of ubiquitin(-like) enzymes to lipid headgroups.
(D) Lipid transfer proteins transport lipids between different membranes and can, thereby, add or remove signaling lipids.
(E) Flippases, floppases, and scramblases facilitate the inter-leaflet transport of lipids and can, therefore, change the chemical nature of the membrane surface.
Readers
(F) Signaling lipids such as phosphorylated forms of PI are recognized by PH, PX, and FYVE domains or the PROPPINs, whereas C1 domains bind to DAG. C2
domains recognize phosphatidylserine (PS)-rich membranes, frequently in a Ca2+-dependent manner (orange dots indicate Ca2+ ions).
(G) Amphipathic lipid packing sensors (ALPS) sense hydrophobic defects in the membrane, some C2 domains are able to sense membrane tension, and different
degrees and types of membrane curvature can be recognized by F-, N-, or I-BAR domains.
(H) Frequently, marks on the membrane are read out in a combinatorial manner by coincidence detection, where the simultaneous presence of two marks results
in more efficient recruitment of a protein to the membrane (i). A variation of this principle is the detection of a single signal by multivalent interactions (ii).
Erasers
(I) Membrane marks can be removed by erasers such as phosphoinositide phosphatases, which dephosphorylate PIPs, by GTPase-activating proteins such as
Arf and Rab GAPs, which convert these GTPases to their GDP-bound state and, thereby, facilitate their dissociation from the membrane, as well as by proteins
having deubiquitinase(-like) activities, which release ubiquitin family proteins from the membrane.

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proteins that facilitate the intra-bilayer reorganization of lipids The encapsulation of biochemical processes requires mem-
and their derivatives (Figure 2E). These proteins include the branes of varying degrees of curvature, from enveloped viruses
following: flippases, which drive the ATP-dependent movement and endocytic vesicles with very high degrees of curvature to
of specific phospholipids from the extracellular leaflet of the Golgi membranes that are composed of flattened stacks of
plasma membrane to the cytoplasmic face; floppases, which membranes with highly curved tips. Curvature is, therefore,
catalyze phospholipid movement in the opposite direction; and a common topological feature of membranes that can be
scramblases, which randomize the distribution of lipids in the recognized or induced by proteins (Figure 2G). The first curva-
bilayer in an ATP-independent manner.60 ture sensors identified were those of the F-, N- and I-BAR family
of proteins,72 which recognize different degrees and types of
READERS: HOW ARE MEMBRANE SURFACES AND membrane curvature.61 The fusion of synaptic vesicles with the
PATTERNS RECOGNIZED? plasma membrane during the exocytosis of neurotransmitter is
driven by C2 domains in synaptotagmin-1, which induce a high
The membrane surface is recognized by readers, proteins that degree of membrane curvature and, thereby, lower the energy
bind to specific lipids and proteins or their combinations. barrier to membrane fusion.73,74 The PI(4,5)P2-mediated adsorp-
Readers, therefore, decode the biochemical identity of mem- tion of the caveolae coat protein, Cavin1, and subsequent mem-
branes and localize enzymatic activities in a time- and space- brane penetration of its trimeric helical region 1 (HR1) domain
dependent manner. has also been proposed to potentiate membrane curvature gen-
The arrangement of chemically diverse lipids with dynamically eration and drive the recruitment of Caveolin1.75
adjusted stoichiometries in a membrane provides innumerable Membrane marks are often read out in a combinatorial manner,
features that can be read out by the protein machinery of the and the coincident detection of two or more marks increases the
cell. At the simplest level, membrane-binding domains bind to specificity and affinity of interactions (Figure 2H). For example, if a
lipid headgroups in a stereospecific manner (Figure 2). The binding partner interacts with two membrane-localized signals
known repertoire of membrane-binding domains that recognize with a KD of 10 mM each, then the overall KD of its interaction
lipid headgroups include the PH, PX, and FYVE domains as well with the membrane is, in theory, 0.1 nM76 (in practice this value
as the PROPPINs that typically bind phosphorylated PIPs and is usually higher due to steric and entropic constraints). The bind-
C1 and C2 domains that recognize DAG and PS, respectively61 ing of homo-oligomers to homotypic surface patterns is a special
(Figure 2F). case of coincident detection in which a single signal is detected
The presentation of the lipid headgroup on a crowded sur- with high affinity by multivalent interactions. For example, the
face confers additional binding specificity. As such, mem- early endosomal autoantigen 1 (EEA1) uses its homodimeric
brane-binding domains that bind to the same headgroup can FYVE domains to tether PI(3)P-positive early endosomes to
localize to distinct membranes within the cell. This is particu- Rab5-positive endocytic vesicles.77 Multivalent homotypic and
larly evident for the C1 domains of protein kinase C (PKC). heterotypic binding interactions can also exhibit cooperativity in
Conventional PKCs typically recognize DAG in the plasma their binding.78 Importantly, the requirement of coincident signals
membrane,62 whereas the C1 domains of the novel PKCs for productive interactions is akin to logical AND gates. In elec-
and the PKDs localize to DAG pools in the trans-Golgi network tronic circuits, an AND gate generates an output only when all in-
(TGN).63,64 The specific subcellular localization of DAG-bind- puts are detected. In the biological context, the protein will only be
ing C1 domains is likely driven by a combination of protein- weakly activated by individual inputs, whereas the integration of
protein interactions; coincident headgroup recognition in multiple inputs leads to a strong response.78,79
the interfacial region65; and the specific molecular surface of
the domain, which penetrates the hydrophobic interior of the ERASERS: HOW MARKS ARE REMOVED FROM THE
bilayer.66 MEMBRANE
Lipid packing impacts the presentation of headgroups and,
consequently, membrane surface topology (Figure 2G). So- In order to terminate biochemical signals on the membrane sur-
called packing defects can be recognized by specific protein face, cells must be able to erase the marks generated by the
motifs, including the ALPS (amphiphathic lipid packing sensor) writers (Figure 2I). Eraser proteins, therefore, allow membrane-
motif. The 40-amino-acid-long ALPS motif is unstructured in based processes to be specific, reversible, and processive. In
solution but folds into a stable amphipathic helix (AH), which in- combination with writers and readers, the counteracting
serts hydrophobic residues into one face of highly curved mem- biochemical activities of eraser proteins also allow for a rapid
branes.67 Curvature-sensing AH motifs have later been identified response of the biochemical systems upon the removal of a
in the vesicle-tethering Golgin protein GMPA210, the nucleo- signal.80
porin Nup133, and the Osh4p sterol transport protein.68 An AH In endocytosis, phosphatases erase the signals of various
in kinesin-1 has recently been proposed to drive the coincident PIPs, including PI(4,5)P2, PI(3,4)P2, and PI(4)P, which is required
detection of anionic lipids and cargo adaptors to drive lysosome to allow for membrane budding, scission, uncoating, and fusion
positioning,69 whereas an AH in the transcriptional repressor with endosomes.39 In the attenuation of growth factor signaling,
Opi1, which orchestrates lipid metabolism, recognizes phospha- the phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) signal
tidic acid (PA) in the ER.70 Recent work has also implicated the is reversed to PI(4,5)P2 by the phosphatase and tensin homolog
role of membrane tension in modulating the membrane binding (PTEN).81 Different Rab proteins are sequentially activated and
of individual C2 domains,71 adding yet another layer of control inactivated to allow the regulated maturation of organelles.19
(Figure 2G). For instance, Gyp1, a GAP for the ER-localized Rab Ypt1, is an

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effector of the downstream GTPase Ypt32.82 A similar principle cruited to activated receptors, phosphorylate PI(4,5)P2 to
may be applied to the conversion of Rab5 to Rab7.19,83,84 PI(3,4,5)P3, thereby writing a new mark on the membrane. At
Another example is provided by Arf GTPases during COPI the apex of the PI3K-elicited signaling cascade is activation of
vesicle formation. Here, Arf1 has to be inactivated and removed the ‘master’ kinase, phosphoinositide-dependent kinase 1
from the membrane to allow the vesicle’s uncoating, transport, (PDK1), by PI(3,4,5)P3. PDK1 is maintained in an inactive confor-
and subsequent fusion with its target compartment.85 The tar- mation in the cytosol in which its PI(3,4,5)P3-binding (reader) PH
geting of ArfGAP is coupled with vesicle biogenesis via its domain is sequestered in an autoinhibitory interaction with its ki-
curvature-sensitive binding to the membrane.85 The dynamic nase domain.93 PI(3,4,5)P3-binding elicits not only the displace-
localization of the neuronal protein ADAP1 is controlled by ment of the PH domain from the kinase domain but also licenses
PI(3,4,5)P3 in concert with the motor protein KIF13B. PI(3,4,5) the dimerization and trans-autophosphorylation of its kinase do-
P3 binding to ADAP1 in the tip of the growing axon both dissoci- mains (Figure 3B). Sequestration of the dimerization interface of
ates ADAP1 from KIF13B and activates its GTPase activity PDK1 ensures that promiscuous autoactivation is unlikely to
against ARF6, thereby locally inactivating it and promoting occur in the absence of PI(3,4,5)P3. Simultaneous sequestration
dendrite branching.86 of the lipid-binding pocket also establishes a competition be-
On a more fundamental level, the activity of erasers is needed tween the membrane and the kinase domain for binding, thereby
to counteract the activities of writers to prevent the spread of setting a threshold PI(3,4,5)P3 concentration required for activa-
membrane marks throughout the cell, which would render the tion. Full-length PDK1, therefore, exhibits strong positive cooper-
signal non-specific. This is illustrated in cells in which Atg4, the ativity in binding to PI(3,4,5)P3, leading to switch-like activation.93
DUB that removes lipidated Atg8 from the membrane, is deleted. Trans-autophosphorylation alone, however, is insufficient to
In these cells, Atg8 proteins eventually mark multiple membrane convert PDK1 into an active conformation,93 which depends
compartments in addition to dedicated autophagic mem- explicitly on PI(3,4,5)P3 or PI(3,4)P2. In this sense, the membrane
branes.87 The DUB Doa4 removes the ubiquitin conjugated to and signaling lipid are the ultimate regulators of activity.
PE in late endosomes.58 Similarly, for the reconstituted Rab5- Together with PDK1, the pro-growth and survival kinases Akt
Rabex5:Rabaptin5 activation network, it was found that the and Sgk3 are the primary effectors of PI3K signaling. Similar to
GAP RabGAP-5 suppresses homogeneous Rab5:GTP accumu- PDK1, Akt and Sgk3 are characterized by a membrane-binding
lation on the membrane surface. Instead, accelerated GTP hy- reader domain followed by a writer kinase domain. Although
drolysis limits Rab5 spreading and activation waves.17 In a nega- Akt is allosterically activated by both PI(3,4,5)P3 and PI(3,4)
tive feedback reaction, phospholipase C beta (PLCb) limits its P294–96 (Figure 3C), serum- and glucocorticoid-regulated kinase
own activity at the plasma membrane by stimulating GTP hydro- 3 (Sgk3) is activated exclusively by the endosomal lipid PI(3)P in
lysis in the heterotrimeric G-protein subunit Gaq, thereby trig- an analogous manner.97 A large proportion of endosomal PI(3)P
gering its own dissociation from its membrane anchor.88 originates from the membrane-localized lipid phosphatases
SH2-domain-containing 5-phosphatase 2 (SHIP2) and inositol
HOW DO MEMBRANES INFLUENCE THE BIOCHEMICAL polyphosphate 4-phosphatase (INPP4), erasers that double as
ACTIVITY OF PROTEINS? writers, sequentially converting class-I-PI3K-derived PI(3,4,5)
P3 into the new marks PI(3,4)P2 and PI(3)P.98–100
Every biochemical reaction is fundamentally limited by the Other regulatory principles are exemplified by the Tec kinases,
intrinsic rate of the reaction under physiological conditions at critical regulators of immune cell signaling. Bruton’s tyrosine ki-
saturating substrate concentration. The turnover numbers of en- nase (Btk) serves to illustrate the concept of coincidence detec-
zymes can vary greatly, from 104 to 106 molecules min1 for the tion. In addition to a PI(3,4,5)P3-sensitive PH domain, Btk con-
fastest known enzymes to just 30 molecules min1 for some of tains a conserved module of Src homology 3 (SH3), Src
the slowest.89 Signaling enzymes, such as kinases and small homology 2 (SH2), and kinase domain (for a recent review of
GTPases, typically exhibit turnover rates orders of magnitude the ancient Src-like module, we refer readers to Shah et al.101).
lower than diffusion-limited enzymes and are often regulated In the example of Btk, kinase activation depends not only on
allosterically. The acquisition of the correct geometry for phos- PI(3,4,5)P3 but also on the sequential recognition of phosphotyr-
phorylation90 and the local concentration of reaction partners osine in the cytoplasmic tail of the receptor by its SH2 domain
are, therefore, essential for catalysis. and a polyproline motif by its SH3 domain (Figure 3D). Similar
The activity of many protein kinases is acutely regulated on to PDK1, lipid-mediated Btk dimerization on the membrane sur-
membranes (Figure 3). At the simplest level, the membrane face has been proposed to facilitate trans-autophosphoryla-
provides a platform upon which biochemical reactions are tion.102 Perturbations of the Btk gene, which is located on the
restricted to a fixed distance from the membrane. This is perhaps X chromosome, are the cause of X-linked agammaglobulinemia
best exemplified by the myotonic dystrophy protein kinases (XLA), a disease characterized by the absence of circulating an-
(DMPKs) that regulate actomyosin contraction in the cell cortex, tibodies and a susceptibility to infection.103,104 Not surprisingly,
in which the catalytic kinase domains are separated from the all of the regulatory mechanisms described above have been
membrane-binding domains by a coiled-coil domain evolution- observed to be perturbed by XLA-associated mutations.105
arily conserved in length (Figure 3A).91,92 Specific, lipid-mediated allosteric activation of protein
The generation and turnover of lipid second messengers in the kinases can also be accompanied by their recruitment by small
membrane can offer an additional, allosteric layer of regulation. GTPases. Son-of-sevenless (SOS)-dependent activation of
The PI3K pathway is directly coupled with the activation of growth Ras, for example, creates a membrane-anchored mark for the
factor receptors in the plasma membrane. Class I PI3Ks, re- recruitment of the effector kinase Raf. In the absence of activated

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A B C D
E F G
Figure 3. How do membranes influence the biochemical activity of proteins?

(A) Recruitment to the membrane surface can position a catalytic activity of an enzyme, here shown for the ROCK kinase, at a fixed distance. This distance is
controlled by the length of a semi-rigid coiled-coil domain connecting the kinase (green) and membrane-binding domains.
(B) Membrane binding can release intramolecular inhibitory interactions by reorienting the membrane-binding domain such as in Akt.
(C and D) (C) Allosteric activation by the release of autoinhibition can be coupled with the promotion of dimerization and transactivation by the clustering of
signaling lipids on the surface, as exemplified by the PDK1 kinase. (D) An extension of this principle is the additional coincidence detection of a signaling lipid and
the phosphorylated cytoplasmic tails of a transmembrane receptor shown here for the kinase BTK.
(E) The membrane surface can also serve to orient reaction partners relative to each other as shown for the Raf kinase, recruited by Ras (orange), and its substrate
kinase MEK.
(F) Membrane-localized reactions can be laterally confined by diffusion barriers, as for example by the actin cytoskeleton.
(G) At high densities, membrane marks such as ATG8 family proteins can exclude other molecules and template macromolecular assemblies.
Ras, Raf exists as an inactive monomer.106,107 Displacement of suggests that Ras itself may dimerize on the membrane,108–112
its membrane-binding cysteine-rich domain (CRD) upon Ras although this is still actively debated in the field.113 Presumably,
binding to the Ras-binding domain (RBD) leads to the assembly Ras dimerization or clustering would stabilize Raf binding to the
of an active, back-to-back dimer of the Raf kinase domain,106 membrane through avidity effects. In summary, the active confor-
which then propagates the signal by phosphorylating the next ki- mation of Ras is intimately tied to its membrane localization and,
nase in the pathway, MEK (Figure 3E). Accumulating evidence thereby, its capacity to engage downstream effectors.

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A B
C D
Figure 4. Membrane-membrane and membrane-condensate contact sites

(A) For proteins having two different binding sites for membrane marks, their recruitment to membrane contact sites is facilitated when the two marks are present
on the two different membranes. In case multiple membrane-binding domains are present, or upon multimerization, these interactions can be further stabilized.
(B) At extended membrane contact sites, the crowding of membrane marks and membrane tethers can result in stable assemblies with low diffusion rates.
(C) Similar principles of protein targeting to membrane contact sites are also applicable at membrane-condensate contact sites where multivalent interactions
can closely align the membrane and condensate surfaces.
(D) The membrane can additionally induce the assembly of condensates by the recruitment and clustering of proteins, which harbor binding sites for other
multivalent macromolecules (schematically exemplified here with the recruitment of cargo receptors and ubiquitinated proteins in autophagy).
Reactions on the membrane can be further controlled by bar- These examples demonstrate how specific, membrane-
riers that laterally confine the diffusion of lipids and proteins licensed interactions serve to template the assembly of supra-
(Figure 3F). The meshwork of the actin cortex, for example, limits molecular complexes at discrete locations. The membrane
not only 2D diffusion in the plane of the membrane but also three- locally concentrates, activates, spatially confines, and organizes
dimensional (3D) diffusion of components within reaction com- reaction partners to drive efficient catalysis. In doing so, the
partments just beneath it.114 Actin polymerization dynamics membrane ensures high fidelity and low noise.
and the activity of motor proteins can generate cortical flows
on the membrane surface, which strongly affects the steady- MEMBRANE-MEMBRANE AND MEMBRANE-
state distribution of lipids and membrane-anchored proteins.115 CONDENSATE CONTACT SITES
The restricted organization of receptors in the membrane that
arises from intercellular communication and the consequent So far, we have outlined how the membrane can act as an active
physical barriers to receptor transport has also been shown to scaffold to template the assembly of macromolecular com-
govern the output of cellular signaling pathways.116 plexes and control their catalytic activities. In this section, we
The marking of membranes with proteins can template macro- discuss special cases in which two membranes come into con-
molecular assemblies such as the ATG8-labeled nascent auto- tact with each other or in which membranes come into contact
phagosome, which recruits proteins containing the LIR motif, with membrane-less biomolecular condensates.
via a direct interaction with ATG8 in a polyvalent manner Membrane contact sites are defined by the tethered proximity
(Figure 3G). Finally, recent technological advances, particularly of two organelles.118 The mechanisms by which reader proteins
in mass spectrometry, have also revealed the roles played by are specifically targeted to membrane contact sites are manifold
membrane lipids in modulating the structure, conformation, (Figures 4A and 4B). It is becoming clear that the unique proper-
and function of many membrane-embedded proteins.117 ties of two membrane surfaces can be exploited to recruit

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proteins to their contact site. For example, the lipid transfer pro- The process of membrane-induced condensate formation is
tein OSBP is a parallel dimer containing a PI(4)P- and ARF1- even better understood for plasma-membrane-localized recep-
GTP-binding PH domain in its N terminus and a central FFAT tor signaling.135 For example, upon TCR activation, the writer ki-
motif, which binds to VAP proteins.119–121 The PI(4)P and nase Lck phosphorylates the cytoplasmic tail of CD3z, which is
ARF1-GTP are localized in membranes of the TGN, whereas read out by the ZAP70 kinase. ZAP70, in turn, writes a phosphor-
the VAP proteins reside in the ER membrane.120,121 The dimeric ylation code on the adaptor protein LAT, which is subsequently
OSBPs, therefore, prefer to bind to membranes where PI(4)P/ read by various proteins, resulting in condensate formation and
ARF1-GTP and VAP proteins are concentrated, respectively. At downstream signaling at the membrane. Apart from activating
TGN-ER contact sites, these two membranes are available for Ras via Grb2 and SOS, the condensates include N-WASP and
binding at the same time; therefore, in total, six binding sites ARP2/3, triggering actin polymerization.136–138 Similarly, the
are present for the OSBP readers. Another example is the phosphorylated form of the cytoplasmic tail of the Nephrin cell
Num1 protein, which anchors mitochondria to the plasma mem- adhesion protein is able to localize 2D condensates of NCK
brane within a larger structure called the MECA (mitochondria- and N-WASP to the membrane.139,140 In TCR signaling, the con-
ER cortex anchor). Num1 harbors a C-terminal PI(4,5)P2-binding densates exclude the CD45 protein phosphatase, thus spatially
PH domain. The N terminus contains a dimeric coiled-coil separating the eraser from the writers and readers in the
domain, which also interacts with the outer mitochondrial mem- signaling condensate on the membrane.136 At the same time,
brane.122 Since the plasma membrane is enriched in the PI(4,5) the actin-containing condensates feed back to the initial writers
P2 mark, the Num1 reader is preferentially recruited to mitochon- by clustering the receptors on the membrane.139
drial sites close to the plasma membrane where four binding
sites for Num1 are available. Here, homotypic and heterotypic INTEGRATION OF WRITER, READER, AND ERASER
coincidence detections are combined to drive the localization ACTIVITIES
of Num1.
In recent years, the presence of membrane-less organelles, The dynamic writing, reading, and erasing of the membrane
also referred to as biomolecular condensates, has attracted code permits the assembly of complex biochemical circuits. A
considerable attention. These structures are macroscopically crucial property of such circuits, which are based on a network
stable, but individual components frequently show high of many low-affinity interactions, is that the overall assembly
mobility, both within the condensates and in exchange with a can be very stable while individual interaction partners exhibit
cytosolic pool. There is extensive crosstalk between mem- high exchange rates. This allows other factors to rapidly outcom-
brane-bound and membrane-less organelles (Figures 4C and pete specific interactions to modify the entire assembly dynam-
4D). Some insights regarding the principles of these interac- ically over time. For example, in clathrin-mediated endocytosis,
tions have been obtained from the study of cargo-driven selec- a network of low-affinity interactions changes over time, culmi-
tive autophagy in yeast and human cells. Here, the prApe1- nating in the assembly of a clathrin cage, which is actively disas-
(yeast) and p62- (mammals) containing cargo material forms sembled only at the very end of the process.141 During bacterial
condensates, which initiate the formation of membrane contact cell division, the tubulin homolog FtsZ and its membrane anchor
sites that connect the ER and ATG9 scramblase-positive vesi- and actin homolog FtsA form treadmilling filaments on the inner
cles via the lipid transfer protein ATG2.123–130 This results in the leaflet of the cytoplasmic membrane. These filaments organize
induction of autophagosome biogenesis where the PI(3)P mark into the so-called Z-ring that rotates within the cell circumfer-
on the growing phagophore membrane generated by the writer ence, whereas the dynamic recruitment of transmembrane pro-
class III PI3K is detected by readers of the PROPPIN family, teins assembles the cell division machinery.142 Such transient
which, in turn, recruit the ATG12-ATG5-ATG16L1 writer com- protein assemblies with a high turnover of components are
plex to mark the membrane with ATG8 proteins.55 During generally favorable for dynamic cellular processes as they do
engulfment, cargo receptors in the condensates, such as not require a dedicated protein machinery to disassemble stable
p62, interact with ATG8 family proteins on the growing phago- complexes.143
phore and, thereby, closely align the membrane to the cargo, a In this section, we focus on the signaling downstream of the
process also referred to as wetting.131 The individual interac- epidermal growth factor receptor (EGFR) to illustrate the integra-
tions between the receptors in the condensates and the mem- tion of writers, readers, and erasers to drive signaling in the cell
brane can be very weak because both binding partners are pre- via two alternate pathways (Figure 5). The membrane serves to
sent at high local concentration on the surface of the two pre-organize and locally concentrate inactive EGFR. EGF bind-
structures. At least in vitro, even low-affinity, low-specificity in- ing to the extracellular domain of the EGFR relieves autoinhibi-
teractions between phase-separating polymers with a naked tion and induces receptor-mediated dimerization.144,145 These
membrane can result in membrane alignment and bending conformational changes are transmitted to the intracellular tyro-
around the condensate.131 Vice versa, during starvation- sine kinase domain, thereby promoting autophosphorylation.146
induced autophagy, the nascent ATG8-protein-marked phago- Autophosphorylation of the C-terminal, cytoplasmic tail of EGFR,
phore may be read out by polymeric cargo receptors recruiting as well as trans-phosphorylation by other kinases in the cell, cre-
it to the membrane. The cargo receptors, in turn, recruit poly- ates a scaffold for the assembly of a number of supramolecular
ubiquitinated proteins (Figure 4D). Upon the completion of au- complexes at the plasma membrane. Two major intracellular
tophagosome biogenesis, the PI(3)P and ATG8 marks are pathways that are activated by EGFR are the Ras-Raf-MEK-
erased by the ATG4 proteins132,133 and PI(3)P-specific phos- ERK pathway and the Ras-PI3K-Akt pathway, which promote
phatases,134 respectively. cell growth, proliferation, and survival.147,148

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Figure 5. Integration of writer, reader, and eraser activities

Schematic of EGFR signaling to illustrate how the activities of writers, readers, and erasers are integrated at the membrane surface. Upon the activation and
subsequent phosphorylation of the cytoplasmic tails of EGFR, the Ras GEF SOS is recruited to EGFR by Grb2. SOS coincidentally binds to PIP2 via its PH domain,
which, in turn, releases its autoinhibition, allowing it to activate Ras. In a positive feedback loop, SOS binds activated, membrane-localized Ras, thereby further
increasing its GEF activity. The simultaneous binding to phosphorylated EGFR via Grb2, PI(4,5)P2, and active Ras increases the residence time and catalytic
activity of SOS, resulting in a feedforward loop for Ras activation. Active Ras recruits Raf, which additionally binds lipids in the membrane. These two interactions
release Raf autoinhibition, allowing it to dimerize and activate the downstream kinase MEK. Upon activation, Ras also recruits and activates the lipid kinase PI3K,
which, in turn, produces PI(3,4,5)P3 to recruit Akt to the membrane, whereupon the PH-domain-mediated autoinhibition of Akt is released. The eraser NF1 re-
moves the Ras signal from the membrane by activating its GTPase activity. The activity of NF1 may be sterically excluded from activated EGFR because SOS is
stably bound to the receptor via Grb2. The eraser for PI3K signaling, PTEN, is recruited to the membrane via its own product, PI(4,5)P2, resulting in a positive
feedback loop. In the scheme, the marks are depicted in orange, the readers in purple, the writers in green and the erasers in blue. Note that this is a simplification,
as many writers and erasers frequently have reader domains, and the eraser of one signal can at the same time be the writer of another signal.
EGF-mediated trans-autophosphorylation leads to the recruit- Grb2 is unlikely to lead to SOS activation.11,138 Interestingly, acti-
ment of the reader protein Grb2 via its SH2 domain.149 Grb2, in vated Ras is barely found in endocytic vesicles containing EGFR
turn, recruits the Ras GEF SOS with its SH3 domain.150 In the and Grb2 following receptor recycling at the plasma mem-
absence of activated receptors, SOS is autoinhibited by its brane,157,158 suggesting that endocytosis may terminate
membrane-binding PH and histone domains, which block the growth-factor-dependent Ras signaling but not the plasma
allosteric binding site for Ras-GTP,151 thereby preventing pro- membrane-resident Ras-Raf-MEK-ERK pathway.
miscuous Ras activation. Recruitment of SOS by Grb2 results The activated Ras mark on the membrane also stimulates the
in an increase in the local concentration of SOS at the mem- PI3K-Akt pathway. Downstream of the EGFR, the class I PI3K
brane. Lipid marks such as PI(4,5)P2 and PA promote the alpha (PI3Ka) plays the predominant role in signaling.159 The
relief of SOS autoinhibition by disengaging its regulatory do- structure of p110g bound to H-Ras has revealed the molecular
mains.152,153 Reconstitution of phosphorylated fibroblast growth basis of Ras binding,160 whereas single molecule studies show
factor receptors (FGFRs) tails, Grb2, and SOS on SLBs has how synergistic binding to both the receptor and Ras marks ac-
recently revealed that activated FGFRs undergo phase separa- tivates the writer function of p110a to drive PI(3,4,5)P3 syn-
tion, which enhances the catalytic efficiency of SOS toward thesis.161,162
Ras.154 Similar phase transitions of receptors and cytosolic EGFR signaling is antagonized by the erasers for PI(3,4,5)P3
adaptor proteins have been reported in the context of TCR and and Ras signaling, PTEN, and neurofibromin (NF1), respectively.
FGFR2 signaling.136,155,156 Interestingly, the multivalent recruit- Both PTEN and NF1 are tumor suppressors, and their loss or
ment and activation of SOS appears to be sufficient to retain it mutation is also common in cancer and overgrowth disor-
on the membrane in an active conformation for as long as it takes ders.81,163–167 The PTEN eraser is a lipid phosphatase with spec-
to inactivate it by endocytosis.156 Binding of activated Ras to an ificity for the 30 phosphate of the inositol ring. PTEN is mainly
allosteric site in SOS further enhances its GEF activity16 in a associated with the dephosphorylation of the PI(3,4,5)P3 mark
feedforward loop that can drive the processive activation of on the plasma membrane, although recent studies have also
thousands of Ras molecules at a time.156 SOS activation has implicated it in the termination of PI(3,4)P2 signaling on endo-
been proposed to involve condensates containing receptors, somes.168,169 A PI(4,5)P2-binding motif in the disordered N termi-
Grb2, and SOS.11 These condensates would permit the iterative nus promotes membrane binding and increased phosphatase
sampling of the membrane in which the probability of SOS auto- activity,170 demonstrating that the product of the reaction drives
inhibition being released is increased. The relatively slow release a positive feedback loop, similar to the effect activated Ras ex-
of autoinhibition acts as a kinetic ‘‘proof reading’’ such that tran- erts on SOS.16,151 The C2 domain of PTEN is tightly associated
sient, monovalent recruitment of SOS to activated receptors by with the phosphatase domain and is critical for membrane

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binding and the orientation of the catalytic site for substrate electron microscopy,188,189 and super-resolution micro-
engagement.171 Membrane binding, however, is acutely regu- scopy,190,191 must be applied to image cellular processes in
lated by the multi-site phosphorylation of the C-terminal 47 situ. In particular, cryoelectron tomography is beginning to yield
amino acids of PTEN, which drives specific interactions of the unprecedented snapshots of membrane-based processes at
tail with the C2 and phosphatase domains that maintain PTEN near atomic resolution.192 Combining structural information
in an autoinhibited conformation and weaken its membrane as- from various sources with mass-spectrometry-based, label-
sociation.171–174 free interactomics is necessary to annotate 3D snapshots of
Recently determined structures of the eraser NF1, a near-native cellular assemblies.193,194 Machine learning is
RasGAP, have revealed a putative autoinhibited conformation improving the sensitivity and dynamic range of proteomics appli-
in which the catalytic GAP domain is sequestered, along with cations.195 Endogenous tagging of proteins using genome edit-
the membrane-binding surface of the SEC-PH domain, in intra- ing technology is already proving to be a powerful tool in our
molecular interactions with the central scaffold domains of the arsenal, as it allows the dynamics of proteins and protein com-
molecule.175,176 These findings imply that significant conforma- plexes to be visualized by live-cell microscopy while maintaining
tional changes must occur upon membrane recruitment to the correct stoichiometries.196 In addition, when combined with
permit the engagement of GTP-bound Ras. The obligate homo- proximity labeling techniques coupled with mass spectrom-
dimer of NF1 also implies that Ras dimers108–111,177 could be etry,197,198 endogenous tagging permits the detection of tran-
the cellular substrates of NF1. Ras interaction with NF1 is sient interactions at biologically relevant expression levels. Along
antagonized by A-Raf binding,178 illustrating the mutual exclu- the same lines, the quantification of the abundance of molecules
sivity of the interaction between the activated Ras mark and in the cell is providing essential numbers for concentration and
its reader (Raf) or eraser (NF1). Finally, proximity labeling ap- stoichiometry,196,199 which are ultimately needed for both recon-
proaches have been employed to map the time-resolved prox- stitution experiments and mathematical modeling. In vitro,
imity proteome of EGFR from ligand-mediated activation biochemical reconstitutions of increasing complexity that reca-
through receptor internalization and recycling, revealing the pitulate the boundary conditions present in cells are needed to
changing landscape of signaling protein interactions in both test what is necessary and sufficient for the assembly of large
space and time.179 macromolecular machines, signal propagation, or the biogen-
In summary, the example of EGFR signaling illustrates how the esis of subcellular organelles. New tools for the detection of sin-
generation of signaling lipids and proteins by writers, the coinci- gle molecules on surfaces can, in principle, be used to rebuild
dent detection of multiple signals by readers, and constrained complex membrane-scaffolded assemblies and monitor their
diffusion in the plane of the membrane pattern both the plasma dynamics.200
membrane and endomembranous compartments. In combina-
tion with erasers, which counteract these events, the membrane SUPPLEMENTAL INFORMATION
can scaffold biochemical pathways to drive signal transduction
with high fidelity and spatiotemporal resolution. Supplemental information can be found online at https://doi.org/10.1016/j.
devcel.2023.06.001.
OUTLOOK
ACKNOWLEDGMENTS
The compartmentalization of biochemical reactions is essential

We acknowledge funding from the Austrian Science Fund (FWF F79, P32814-
in the crowded environment of the cell. By analogy to the histone B, and P35061-B to S.M.; P34607-B to M.L.; and P30584-B and P33066-B to
code, the dynamic patterning and remodeling of membrane sur- T.A.L.) and the European Research Council (ERC) under the European Union’s
faces by writers and erasers facilitate the acute spatial and Horizon 2020 research and innovation program (grant agreement no.
101045340 to M.L.). We are grateful for comments on the manuscript by Jus-
temporal control of molecular interactions (readers). Local con- tyna Sawa-Makarska, Verena Baumann, Marko Kojic, Philipp Radler, Ronja
centration and membrane-licensed interactions of reaction part- Reinhardt, and Sumire Antonioli.
ners as well as the coincident detection of multiple signals can be
exploited to drive cooperative, switch-like behavior and high-fi- DECLARATION OF INTERESTS
delity transmission of information. The individual interactions,
however, that drive the assembly of supramolecular complexes S.M. is a member of the scientific advisory board of Casma Therapeutics.
on the membrane are necessarily weak. This presents obvious
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*Correspondence: thomas.leonard@meduniwien.ac.at (T.A.L.), martin.loose@ist.ac.at (M.L.), sascha.martens@univie.ac.at (S.M.)

INTRODUCTION HOW DOES CONFINEMENT TO A TWO-DIMENSIONAL

Figure 1. How does confinement to a surface influence biochemical reactions?

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Box 1. The Writer/Reader/Eraser paradigm for signaling systems

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Figure 2. Writing, reading, and erasing the membrane code

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Figure 3. How do membranes influence the biochemical activity of proteins?

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Figure 4. Membrane-membrane and membrane-condensate contact sites

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Figure 5. Integration of writer, reader, and eraser activities

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The compartmentalization of biochemical reactions is essential

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