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COMMENTARY

Role of cholesterol in SNARE-mediated trafficking on intracellularmembranes

Carlos Enrich1,*, Carles Rentero1, Aitor Hierro2 and Thomas Grewal3,*

ABSTRACT

The cell surface delivery of extracellular matrix (ECM) and integrins

is fundamental for cell migration in wound healing and during cancer

cell metastasis. This process is not only driven by several soluble

NSF attachment protein (SNAP) receptor (SNARE) proteins,

which are key players in vesicle transport at the cell surface

and intracellular compartments, but is also tightly modulated by

cholesterol. Cholesterol-sensitive SNAREs at the cell surface are

relatively well characterized, but it is less well understood how

altered cholesterol levels in intracellular compartments impact on

SNARE localization and function. Recent insights from structural

biology, protein chemistry and cell microscopy have suggested

that a subset of the SNAREs engaged in exocytic and retrograde

pathways dynamically ‘sense’ cholesterol levels in the Golgi and

endosomal membranes. Hence, the transport routes that modulate

cellular cholesterol distribution appear to trigger not only a change in

the location and functioning of SNAREs at the cell surface but also

in endomembranes. In this Commentary, we will discuss how

disrupted cholesterol transport through the Golgi and endosomal

compartments ultimately controls SNARE-mediated delivery of

ECM and integrins to the cell surface and, consequently, cell

migration.

KEY WORDS: SNARE protein, Cholesterol, Trans-Golgi network,

Recycling endosome, Integrin trafficking, Cell migration

IntroductionCholesterol homeostasis is essential for the functional integrity of

the cell and requires a tight regulation of cholesterol levels inside

and outside of cells. Therefore, cholesterol-sensing regulatory

circuits communicate any alterations in cholesterol levels in order

to control sterol synthesis, uptake and transport (Ikonen, 2008;

Maxfield and van Meer, 2010).

Cholesterol is considered to be indispensable for cell migration.

A substantial body of literature suggests that specialized

cholesterol-rich microdomains and cholesterol-sensitive transport

pathways are required for the delivery of integrins and extracellular

matrix (ECM) to and from the cell surface. Yet, despite the now

well-recognized increased cholesterol demand in cancer cells, it

remains unclear how intracellular cholesterol transport routes are

linked with the molecular machinery that controls cell migration.

Here, we will first summarize our current knowledge of

cholesterol homeostasis, in particular the cellular distribution of

low-density lipoprotein (LDL), which is linked to events that are

highly relevant for cell migration. We will then focus on how

cholesterol transport along intracellular routes can modulate the

localization of several NSF attachment protein (SNAP) receptor

(SNARE) proteins, major players in membrane transport to the

cell surface along endocytic and exocytic pathways. Finally we

will discuss how localization of cholesterol-sensitive SNAREs, in

particular within Golgi–endosomal boundaries, determines the

efficacy of delivery of cell adhesion receptors and ECM, which

are key factors in cancer cell migration, to the cell surface.

Cholesterol has multiple features that enable interactionwith lipids and proteinsCholesterol is an amphipathic molecule with a hydrophilic

hydroxyl group and a hydrophobic section organized into four

rigid rings followed by a short branched hydrocarbon tail. Within

the membrane, the hydroxyl group of cholesterol interacts

with the polar head of phospholipids and sphingolipids,

whereas the hydrophobic part remains immersed in the

membrane alongside the hydrocarbon chains of the surrounding

lipids. Cholesterol is not uniformly distributed in the membrane,

and besides its interaction with membrane lipids, interaction with

proteins also have an important role in its distribution (Epand,

2006; Ikonen and Jansen, 2008). Consequently, there has been a

considerable interest on deciphering the molecular mechanisms

that allow these interactions. Indeed, several posttranslational

modifications appear to enable proteins to preferentially associate

with cholesterol-rich domains, including their myristoylation,

palmitoylation or glycosylphosphatidylinositol linkage. However,

for cholesterol to interact with transmembrane domains of

proteins, one would expect to find specific molecular features

at the lipid–water interface region of proteins such as defined

amino acid sequences or motifs. Indeed, several short cholesterol-

binding motifs that are located in the lipid–water interface region

favor interactions with cholesterol (Box 1). In some cases,

the precise positioning of a cholesterol-binding motif within

the lipid–water interface segment might be influenced by the

‘snorkelling’ effect seen for lysine and arginine residues with

long buried hydrophobic side-chains (Chamberlain et al., 2004;

Strandberg and Killian, 2003). However, how such interactions

contribute to maintaining a heterogeneous distribution of

cholesterol within the cell and to cholesterol movement

between cellular membranes is not fully understood.

Intracellular trafficking of cholesterolSeveral reviews have discussed the intracellular distribution of

cholesterol and cholesterol-rich membrane domains, pathways of

intracellular cholesterol movement and methods for studying

1Departament de Biologia Cellular, Immunologia i Neurociencies, Centre deRecerca Biomedica CELLEX, Institut d’Investigacions Biomediques August Pi iSunyer (IDIBAPS). Facultat de Medicina, Universitat de Barcelona, 08036-Barcelona, Spain. 2Structural Biology Unit, CIC bioGUNE, Bizkaia TechnologyPark, 48160 Derio; IKERBASQUE, Basque Foundation for Science, 48013 Bilbao,Spain. 3Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia.

*Authors for correspondence (enrich@ub.edu; thomas.grewal@sydney.edu.au)

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459

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sterol transport and distribution (Ikonen, 2008; Maxfield and vanMeer, 2010; Mesmin and Maxfield, 2009). In general, cells

acquire cholesterol through receptor-mediated endocytosis ofLDL or by de novo synthesis in the endoplasmic reticulum (ER)(Simons and Ikonen, 2000). After LDL internalization, esterifiedLDL-cholesterol traffics through the endocytic compartment to

be delivered to late endosomes and lysosomes, where it ishydrolyzed by lysosomal acid lipase to free the cholesterolmolecule. The majority of late-endosome-derived cholesterol is

then delivered to the plasma membrane, while some of it istransported to the ER to enable feedback control or undergoes

esterification for storage in lipid droplets in the form ofcholesteryl esters. The routes and mechanisms by which late

endosome-originating cholesterol reaches the plasma membraneare not clear, but as shown by our laboratory (Cubells et al., 2007;Reverter et al., 2014) and others (Kanerva et al., 2013; Uranoet al., 2008), might involve transport through the ER, the trans-

Golgi network (TGN) and recycling endosomes (Fig. 1A). Asoutlined below, although recycling endosomes and the TGNcontain much less cholesterol than the plasma membrane,

moderate changes in the levels of cholesterol that aretransported through these compartments appear to have drasticeffects on cellular behavior that is relevant in the context of

migration and invasion.The physiological importance of LDL uptake and lysosomal

processing is underscored by genetic defects that cause familial

hypercholesterolemia, as well as Niemann Pick type C (NPC)disease, a lethal condition that is characterized by intracellularaccumulation of unesterified cholesterol in late endosomes andlysosomes. Niemann Pick C1 protein (NPC1) and Niemann Pick

type C2 protein homolog (NPC2) bind to late-endosome-associatedcholesterol and facilitate its export from late endosomes (Ikonen,2006; Vance and Karten, 2014). Mutations in the genes encoding

NPC1 or NPC2 inhibit the egress of cholesterol from lateendosomes (Klein et al., 2006) and reduce its delivery to theGolgi, plasma membrane and recycling endosomes (Cubells et al.,

2007; Kanerva et al., 2013; Reverter et al., 2014; Urano et al.,2008). Together, this triggers a dysfunction of membranetrafficking, which is associated with cardiovascular, neurological

and lysosomal storage diseases (Cortes et al., 2013; De Matteis andLuini, 2011; Ikonen, 2006; Maxfield and Tabas, 2005). (Pre-)lysosomal cholesterol export most likely involves the transfer ofcholesterol from luminal NPC2 to membrane-associated NPC1,

followed by insertion of the aliphatic side chain of cholesterol intothe (pre-)lysosomal membrane (Infante et al., 2008; Wang et al.,2010). Although subsequent cholesterol transfer to other sites is not

fully understood, non-vesicular transport mediated by cytosoliccholesterol-binding proteins and vesicular pathways exist(Fig. 1B,C). In addition, membrane contact sites between late

endosome or lysosomes and other compartments, such as the ER,appear to enable cholesterol transfer (Alpy et al., 2013; Changet al., 2006; Ikonen, 2008; Rowland et al., 2014; van der Kant andNeefjes, 2014).

It should be noted that in some cell types, in particularhepatocytes and steroidogenic cells, uptake of cholesterol fromhigh-density lipoprotein (HDL) can significantly contribute to

cholesterol homeostasis. This is mediated by the scavengerreceptor class B member 1 (SRB1, also known as SR-BI), anintegral membrane protein that is highly expressed in liver and

steroidogenic tissues. SRB1 facilitates the incorporation of HDL-derived cholesteryl esters through a process called selectiveuptake (Leiva et al., 2011). This is not associated with HDL

particle internalization through endosomal or lysosomal pathways,but involves the binding of HDL to SRB1 at the cell surface,followed by passive diffusion of HDL-derived cholesteryl estersinto the plasma membrane. Internalized cholesteryl esters are

then rapidly hydrolyzed. Consequently, HDL-derived cholesterolquickly enters other sites, such as the ER and recycling endosomes,with kinetics that are much faster than for trafficking of LDL-

derived cholesterol (Heeren et al., 2006; Leiva et al., 2011).Therefore, cholesterol generated by this means can rapidlyaffect the distribution of cholesterol-sensing SNAREs in these

compartments (see below).

Box 1. Cholesterol-binding motifs in proteinsThe CRAC (cholesterol recognition/interaction amino acidconsensus) represents the best-characterized cholesterol-bindingmotif (Fantini and Barrantes, 2013; Li and Papadopoulos, 1998) andconforms to the pattern: L/V-X1–5-Y-X1–5-R/K (Fig. 2). Despitesome skepticism, CRAC is found in several cholesterol-bindingproteins, including caveolin-1 and the somatostatin receptor (Baieret al., 2011; Murata et al., 1995). In addition, in many cases theinteraction between cholesterol and CRAC has been confirmed byphysicochemical or mutagenic analysis. An inverted CRACcholesterol-binding motif, the CARC domain [K/R-X1-5-Y/F-X1–5-L/V], is found in some other proteins (Baier et al., 2011) (Fig. 2). Inaddition, the transmembrane domains of several proteins (i.e. TM4of the human b2-adrenergic receptor) contain a sequence with acombination of basic (R), aromatic (W) and aliphatic (L/V) residues(R-W-L, see Fig. 2B) that does not strictly fulfil the criteria of theCARC algorithm, but still binds to cholesterol (Hanson et al., 2008).Remarkably, the CRAC, CARC and R-W-L binding motifs exhibit acommon distribution of basic (K/R), aromatic (Y/F/W) and branchedaliphatic residues (L/V) that are important for cholesterol interaction.Furthermore, in a-synuclein, the Alzheimer’s b-amyloid peptide andthe N-terminal peptide of HIV-1 gp41, the cholesterol-binding sitesare located in tilted peptides, which lack the basic and aromaticresidues characteristic of the previous motifs (Charloteaux et al.,2006). The definition of tilted peptides is functional, not sequence-based, and collectively corresponds to short helical fragmentswith an asymmetric distribution of their hydrophobic residues thatdisturb the surrounding lipid organization and promote a tiltedorientation. Finally, another motif for cholesterol binding is thetetrapeptide YIYF (Epand, 2006; Kuwabara and Labouesse, 2002),which is frequently found near the end of transmembrane helicesof sterol-sensing domains. Sterol-sensing domains comprise fivetransmembrane helices and are found in several proteins involved incholesterol homeostasis, such as NPC1, HMG-CoA reductase andSCAP (Epand et al., 2010). The scheme below shows cholesterolembedded within the membrane phospholipid bilayer andneighboring a typical transmembrane helical domain of an integralmembrane protein (type II topology). The positions of potentialcholesterol binding motifs (CARC, R-W-L, CRAC, YIYF) withinSNAREs as listed in Fig. 2B are indicated. This representationassumes that cholesterol increases the relative thickness of thebilayer and its order (Sharpe et al., 2010).

Cholesterol

CRACYIYF

Cholesterol

CARCR-W-L

N-

-C

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SNARE proteins on endomembranes interact withcholesterolIn a recent comprehensive survey using photoreactive sterol

probes in combination with quantitative mass spectrometry,Hulce and co-workers identified over 250 cholesterol-bindingproteins, including several SNARE proteins (Hulce et al., 2013).

More than 60 SNAREs have been identified from yeast and

mammals, and they are known to be crucial components ofprotein complexes that drive membrane fusion in secretory andendocytic pathways (Jahn and Scheller, 2006). Site-specific

interaction and pairing of SNAREs on target membranes (t-SNAREs) with SNAREs on vesicles (v-SNAREs) facilitatestethering, docking and fusion of distinct vesicle-mediated

transport events. This complex and multifactorial processdetermines the efficiency and speed of delivery of secretoryvesicles. Although SNAREs were originally classified as v- and t-

SNAREs according to their localization, they can be structurallydistinguished as Q or R types (Hong, 2005; Hong and Lev, 2014;Sudhof and Rothman, 2009) and further segregated into foursubfamilies. These are the syntaxin subfamily (Qa), the S25N

(Qb) and S25C (Qc) subfamilies and members of the VAMP

subfamily, which are all R-type SNAREs. All syntaxins (exceptSTX11) and VAMPs are integral membrane proteins and containa transmembrane domain. SNAP23, SNAP25 and SNAP29 are all

members of both the Qb and Qc subfamilies and contain twotandem SNARE motifs, but lack a transmembrane.

Strikingly, from the 38 SNAREs found in humans (Hong andLev, 2014), 11 have been shown to interact with cholesterol

(Fig. 2A). However, little structural information on cholesterol–protein complexes is available, and despite the fact that crystalstructures of single or complexed SNAREs exist, information

regarding their interaction with cholesterol is scarce. Oneexception is the relatively well-characterized interaction betweenVAMP2 and cholesterol. Here, site-directed spin labeling,

continuous wave and pulsed electron paramagnetic resonancespectroscopy have shown that a significant conformational changetakes place in the transmembrane domain of VAMP2 upon

cholesterol binding, which is thought to support lipid mixing andeventually membrane fusion (Tong et al., 2009).

However, only one of the cholesterol-binding SNAREs isfound at the plasma membrane, the t-SNARE STX4 and its

partner SNAP23, which binds to cholesterol through

1

2 5

4

6

13 107

3

7

12

8

11

9

A

LDLC

ER

LDL HDLB

Clathrin

Cholesterol

MLN64

Hrs

ORP5

NPC1

NPC2

Rab7

Rab8

Rab9

AnxA6

STX6

chol-rich

Efflux

Lys

Degradation

LD

Storage

CEEsterification

LE/MVB

EE/SE/REUptake

Sorting

ER

Golgi

TGN

Biosynthesis

Nucleus

Caveolae ccp

LE

LE

Key

CE

CE

Fig. 1. Overview of cholesterol trafficking pathways. (A) Cellular cholesterol transport pathways. (1) Cholesterol is delivered to early (EE), sorting (SE) andrecycling endosomes (RE) by either endocytosis of LDL through clathrin coated pits (ccp), or selective cholesteryl ester (CE) uptake by SRB1 from HDL incholesterol-rich (chol-rich, e.g. caveolae) plasma membrane domains (shown in dark gray). (2) LDL is then transported to late endosomes/multivesicular bodies(LE/MVBs), where CEs are hydrolyzed. Free cholesterol is then distributed to other sites (see below), whereas the LDL particle is degraded in lysosomes (Lys)(3). In addition, recycling of cholesterol from EEs, SEs and REs (4) or the LE/MVB (5) to the plasma membrane, or its retrograde transport from EEs, SEs andREs to the TGN can occur (6). LDL-derived cholesterol in LE/MVBs is distributed to the ER for re-esterification and storage in lipid droplets (LD) (7), to the Golgiand TGN (8), is exported (efflux) to the plasma membrane (9) and sent to mitochondria (10). Cholesterol synthesized in the ER can be transported to the plasmamembrane directly (11) or through the Golgi (12, 13). (B,C) Vesicular and non-vesicular transport routes of LDL- and HDL-derived cholesterol through lateendosomes. NPC1 in the limiting membrane of late endosomes and lysosomes cooperates with multiple vesicular and non-vesicular cholesterol export pathways.NPC2 delivers cholesterol to NPC1, which inserts lumenal LDL-cholesterol to the limiting late endosomal and lysosomal membrane. (B) LDL-cholesterol, but onlyvery little HDL-derived cholesterol (indicated by the dotted line), is delivered to late endosomes. Cytoplasmic carrier proteins facilitate LDL-cholesterol exportfrom late endosomes and lysosomes. This includes oxysterol-binding protein-related protein 5 (ORP5), which possibly transfers cholesterol through membranecontact sites to the ER. Hrs/VPS27, a member of the ESCRT, might also be involved in NPC1-dependent late endosome-cholesterol export to the ER (dotted line)(Du et al., 2012). Transport to mitochondria involves MLN64 (Charman et al., 2010). (C) Alternatively, NPC1 might deliver cholesterol by vesicular membranetransport that requires Rab proteins (Rab7, Rab8, Rab9), SNAREs (STX6) and possibly annexin A6 (AnxA6) (see text for further details).

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palmitoylated cysteine residues located in the linker regionbetween the two SNARE domains (Salaun et al., 2005) (Table 1).Furthermore, several of the SNAREs that are capable of binding

to cholesterol are not known to function in a cholesterol-sensitivemanner, but represent SNAREs that are predominantly found onendomembranes, often shuttling between locations, such as late

endosomes and recycling endosomes, as well as the Golgi. Thus,the potential interaction between cholesterol and these SNAREson endomembranes could point to an exciting new molecular

pathway that couples local cholesterol levels in endosomalmembranes and the Golgi with dynamic changes in SNARElocation and thereby their function in cell dynamics and behavior.

In line with 70–80% of cellular cholesterol being found at the

plasma membrane, several studies have identified a crucial rolefor cholesterol in inducing and stabilizing the clustering ofSNAP23 and STX4, which is important for membrane fusion

(Chamberlain et al., 2001; Lang, 2007; Predescu et al., 2005; Puriand Roche, 2006). SNAP23 binds to STX4 in vivo (St-Deniset al., 1999). Accordingly, together these two SNAREs are

involved in the fusion of transferrin-containing recycling vesicles

with the basolateral membrane (Leung et al., 1998), as well assurface delivery of newly synthesized proteins from the Golgi tothe apical membrane in Madin–Darby canine kidney (MDCK)

cells (Lafont et al., 1999). Although we and others confirmed thatthese SNAREs, similar to STX1 or SNAP25, concentrate withinclusters at the plasma membrane (Reverter et al., 2011; Sieber

et al., 2006; Sieber et al., 2007), the underlying causes thatpromote STX4–SNAP23 clustering are not fully understood.

Given the high cholesterol content at the plasma membrane and

its membrane-condensing effect, which increases membraneorder and rigidity (Lingwood and Simons, 2010), thephysicochemical properties of cholesterol probably play a majorrole in the formation of SNARE microdomains at the plasma

membrane (van den Bogaart, 2013). In addition, factors thatpromote SNARE clustering at the plasma membrane includetheir partitioning into cholesterol-enriched membrane rafts,

competition with cholesterol for solvation by bulk lipids, andelectrostatic protein–lipid interactions. Furthermore, the clusteringof plasma-membrane-associated proteins, including the SNAREs

listed above, might be promoted by protein–protein interactions

STX4STX5STX7STX12STX18STX6STX8STX10VTI1BVAMP2VAMP3

297355261276335255236249232116100

Polybasic

SNARE domain Linker Transmembrane domainCN-

CARC motif R-W-L CRAC motif

STX18

STX5STX4

STX10

STX6

Ha Hb Hc

STX7

TMSNARE

STX12

0 50 100 150 200 250 300 350

STX8

CRAC motif CARC motif R-W-L motif Y/W in linker

VAMP2VAMP3

Amino acid

VTI1B

VesicleCholesterol ring-raft

SNAREs

A

C

B

Key

Fig. 2. Cholesterol-binding motifs intransmembrane and juxtamembrane domains ofcholesterol-binding SNARE proteins. (A) Schematicrepresentation of cholesterol-binding human SNAREproteins showing the position and type of cholesterol-binding motifs in the juxtamembrane or transmembranedomains (see Box 1). Two SNARE proteins that bind tocholesterol have not been included in this figure; the firstis Sec22a, an unusual t-(R)-SNARE protein with theyeast homolog being located at the ER–Golgi interfaceand containing a longin domain and a singletransmembrane domain similar to the human Sec22bisoform. In contrast, the human Sec22a isoformcontains four transmembrane domains with severalCRAC and CARC motifs, but lacks SNARE domainhomology, questioning its classification as a SNARE.The second is SNAP23, which binds to cholesterol bymeans of palmitoylation of cysteine residues in thelinker region between the two SNARE domains. TM,transmembrane domain. (B) Superposition of thejuxtamembrane regions showing parts of thetransmembrane, linker and SNARE domain of humancholesterol-binding SNAREs. The sequences (SNAREdatabase: http://bioinformatics.mpibpc.mpg.de/snare/)corresponding to the C-terminal region for each proteinreveal the presence of a basic cluster within the linkerregion that connects the SNARE and thetransmembrane domain, especially in STX4, STX7 andSTX12. The location of the potential cholesterol-bindingmotifs CRAC, CARC and R-W-L within SNAREs (seeFig. 2B) are indicated with red, orange and blue boxes,respectively. (C) Model depicting CARC, R-W-L andother cholesterol-binding motifs (see Box 1) thatcontribute to the formation of a cholesterol-enrichedring-raft in the outer leaflet of the fusion pore, which inturn would stabilize the local membrane curvature formembrane fusion.

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(Zilly et al., 2011), homotypic interactions between theSNARE motifs (Sieber et al., 2006), heterotypic protein–protein interactions (Yang et al., 2006) and anchoring to the

cortical cytoskeleton (Low et al., 2006; Torregrosa-Hetland et al.,2011).

Nevertheless, the extent to which cholesterol-mediated plasmamembrane clustering of SNAREs contributes to their overall

distribution remains unclear. The reported effects of cholesterolon SNARE clustering are predominantly based on extraction ofcholesterol from cells under non-physiological conditions with

methyl-b-cyclodextrin, which is known to cause a partial tocomplete disintegration of plasma membrane clusters that areenriched in STX1, SNAP25, STX4 and/or SNAP23 (Low et al.,

2006; Predescu et al., 2005; Reverter et al., 2011). Moreover,within a single cell type, cholesterol extraction can completelydisrupt plasma membrane clusters of a particular SNARE,

whereas other plasma-membrane-associated SNARE clustersremain unaffected, suggesting that the extent of cholesterol-mediated clustering differs among SNAREs and cell types. Theunderlying mechanisms have yet to be resolved, but certain

cholesterol pools located in specific microdomains might be moreaccessible and susceptible to depletion by experimental protocols,which could lead to a sequential disruption of membrane

microdomains with variations in their SNARE and cholesterolcontent.

The late endosomal compartment is linked to cell migrationThe late endosomal compartment is not only central to cholesterolhomeostasis (see above), but recent data are beginning to unravel

how late endosomes and lysosomes influence the dynamics ofadhesions between cells and the ECM and movement of cells inboth two- and three-dimensional microenvironments (Raineroand Norman, 2013). For instance, the endosomal sorting

complexes required for transport (ESCRT), which regulatedelivery of cargo and membrane transport along the endocyticpathway, control the transit of the tyrosine kinase Src from late

endosomes to focal adhesions, adhesion structures required forcell–ECM interactions, at the plasma membrane. At the cell

surface, Src is involved in mediating the disassembly andturnover of cell–ECM interactions, which are required for cellmigration (Tu et al., 2010). Particularly relevant for cancer

metastasis, oncogenic v-Src kinase is located in perinuclearstructures that resemble late endosomes, and it translocates to theplasma membrane to mediate cell transformation (Fincham et al.,1996). Along these lines, in pancreatic and ovarian cancers,

Rab25 together with the chloride intracellular channel 3 (CLIC3)drives invasiveness by recycling of a5b1 integrin from lateendosomes to the plasma membrane (Dozynkiewicz et al., 2012).

The protein machinery that controls the fusion of lateendosome and lysosomal compartments with the plasmamembrane is now relatively well documented and involves

syntaxin 7 (STX7), STX8, vesicle-associated membrane protein 7(VAMP7) and VAMP8, which are all members of the SNAREprotein family (Kent et al., 2012; Luzio et al., 2010; Luzio et al.,

2014; Pryor et al., 2004). Chavrier and co-workers haveestablished that VAMP7 and the exocyst complex deliver a lateendosomal pool of membrane type 1 metalloproteinase (MT1-MMP, also known as MMP14) to the cell surface for ECM

proteolysis, which is crucial for invasive migration (Steffen et al.,2008). Earlier steps in this pathway might involve CLIC3(Macpherson et al., 2014). Later trafficking events probably

require the actin cytoskeleton, as following its transit from Rab7-positive endosomes to the plasma membrane, MT1-MMP isretained in invasive protrusions by direct tethering to F-actin (Yu

et al., 2012).In addition, besides these SNARE-dependent trafficking events

that emanate from late endosome and drive cell migration,

distribution of LDL-derived cholesterol from lysosomes and lateendosome to other cellular sites has now been recognized to alsotrigger trafficking events that promote cell migration. Recently,Ikonen and co-workers identified the delivery of LDL-derived

cholesterol from late endosomes to focal adhesions (Kanervaet al., 2013). As outlined above, cholesterol is essential for thefunctioning of SNAREs at the plasma membrane and one could

envisage that the compromised motility and function of lateendosome and lysosomes in lysosomal storage diseases such as

Table 1. Distinctive properties of cholesterol-binding SNARE proteins

SNARE Type LocationCholesterolselectivea

Cholesterolsensitivea

Cholesterol-binding motifs

TMc ReferenceCRAC CARC R-W-L Otherb

STX4 t-(Qa) PM + 2 + 23 Chen and Scheller, 2001STX5 t-(Qa) Golgi 2 + + 17 Laufman et al., 2011STX7 t-(Qa) LE/EE + + * 23 Mullock et al., 2000STX12 t-(Qa) RE/EE + 2 + * 23 Prekeris et al., 1998STX18 t-(Qa) ER + + + + 17 Iinuma et al., 2009STX6 t-(Qc) TGN + 2 + 19 Bock et al., 1997STX8 t-(Qc) LE/EE + 2 + 19 Antonin et al., 2000STX10 t-(Qc) TGN/RE + 2 + 17 Ganley et al., 2008VIT1B v-(Qb) LE/EE 2 2 + 20 Pryor et al., 2004VAMP2 v-(R) SV + 2 + + 22 Chamberlain and Gould,

2002VAMP3 v-(R) RE/EE + 2 + * 20 McMahon et al., 1993

SNARE type [t-(Qa), t-(Qc), v-(Qb), v-(R)] and subcellular location are given. EE, LE, RE, early, late or recycling endosomes, PM (plasma membrane), SVsecretory vesicles. The presence of cholesterol-binding motifs (CRAC, CARC or R-W-L) in transmembrane (TM) domains are given. aCholesterol selectiveindicates whether proteins selectively bind a trans-sterol probe but not a non-steroidal neutral lipid probe; cholesterol sensitive indicates whether proteins showcompetition with unlabeled cholesterol (Hulce et al., 2013). b+, presence of undefined cholesterol-binding motif (i.e. tilted peptide); *, presence of aromatic (Y/W)residues in the linker region between the SNARE and transmembrane domains. cThe number of hydrophobic residues in the transmembrane domain accordingto: http://www.ch.embnet.org/software/TMPRED_form.html.

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NPC – which is caused by accumulation of cholesterol and/orsphingolipids (reviewed in detail in Fraldi et al., 2010; Ikonen and

Holtta-Vuori, 2004; Platt et al., 2012) – interferes with thefunctioning of those SNAREs that are localized in the lateendosome compartment and are relevant for cell migration(Ganley et al., 2008).

Loss of NPC1 interferes with cholesterol-sensitive vesicleformation in the GolgiBy using NPC1-mutant-like cell models that accumulatecholesterol in the late endosomes, by pharmacological means(treatment with U18666A) or annexin A6 overexpression, which

inhibits NPC1 activity possibly through direct protein–proteininteraction (Cubells et al., 2007), we initially showed thatblocking egress of cholesterol from late endosomes interfered

with cholesterol-dependent caveolin-1 transport from the Golgi tothe cell surface, thereby reducing the number of caveolae(Cubells et al., 2007). We speculated this to reflect earlier datafrom in vitro studies showing that precisely balanced cholesterol

levels are essential to drive vesicle formation from the Golgi(Stuven et al., 2003). Although cholesterol depletion inhibits theformation of secretory vesicles from the TGN (Wang et al.,

2000), elevated cellular cholesterol stimulates vesicle formationand the dispersal of Golgi-derived vesicles (Grimmer et al.,2005). Efforts to identify the cholesterol-sensitive factor that is

involved in this process found that cytoplasmic phospholipase A2(cPLA2) translocates to the Golgi upon elevation of cellularcholesterol (Grimmer et al., 2005). Based on these data and the

fact that cPLA2 converts phospholipids into lysophospholipidsthat have an inverted-cone shape, the authors hypothesized thatcPLA2 contributes to Golgi membrane deformations that arerequired for the formation of secretory vesicles, as

pharmacological inhibition of cPLA2 has been shown to inhibitcholesterol-induced vesiculation at the Golgi (Grimmer et al.,2005).

In further support of this hypothesis, our follow-up studiesrevealed that a decrease in the availability of cholesterol in theGolgi of NPC1-mutant-like cells upon using pharmacological

treatment with U18666A or annexin A6 overexpression perturbedthe translocation to and activity of cPLA2 at the Golgi (Cubellset al., 2008). We speculated that inhibition of cholesterol egressfrom late endosomes leads to a depletion of Golgi cholesterol,

therefore disrupting cholesterol-dependent cPLA2 recruitmentand vesiculation events at the Golgi (Cubells et al., 2008). Thiswould interfere with cholesterol delivery to the cell surface along

exocytic pathways that originate at the Golgi. As the plasmamembrane undergoes rapid and continuous turnover, the lack ofcholesterol replenishing the cell surface pool would be followed

by the disintegration of STX4 and SNAP23 clusters that form atcholesterol-enriched plasma membrane microdomains. Consistentwith this model, pharmacological inhibitors of cPLA2 strongly

reduced the localization of SNAP23 and STX4 at the plasmamembrane (Reverter et al., 2011). Moreover, depletion of thecPLA2 product, arachidonic acid, has been shown to inhibit STX4from forming SNARE complexes (Connell et al., 2007).

Arachidonic acid induces the so-called ‘syntaxin-opened’conformation that is required for SNARE assembly (Connellet al., 2007; Rickman and Davletov, 2005), further supporting the

involvement of cPLA2 in the modulation of syntaxins thatparticipate in SNARE trafficking and assembly.

Most strikingly, we found that although clusters of SNAP23 and

STX4 at the plasma membrane disintegrated in NPC1 mutant-like

cells, both SNAP23 and STX4 accumulated together with caveolin-1 in Golgi membranes, which as we shown, is associated with an

increased formation of SNAP23–STX4-containing t-SNAREcomplexes in the Golgi (Reverter et al., 2011). Consequently, theaccumulation and mislocalization of SNAP23–STX4 complexes inthe Golgi of these NPC1-mutant-like cells prevented SNAP23–

STX4 from promoting the secretion of fibronectin, an ECMcomponent that binds to cell adhesion receptors, at the plasmamembrane. Mislocalization and loss of SNAP23–STX4 function

upon cholesterol depletion might also occur in other STX4–SNAP23-dependent events that are relevant for cancer cellmigration, for instance the association of STX4–SNAP23 with

VAMP7, which is required to deliver MT1-MMP to invadopodiafor ECM degradation in invasive cancers (Williams et al., 2014).

Thus, the depletion of cholesterol from the Golgi and plasma

membrane owing to its accumulation in late endosomes not onlyinterferes with the localization and organization of someSNAREs at the plasma membrane, but also impacts on thedynamics of SNARE assembly and disassembly on intracellular

membranes, such as at the Golgi and transport pathwaysoriginating from the Golgi (Reverter et al., 2011; Reverteret al., 2014).

Golgi-localized cholesterol regulates SNARE-dependentintegrin traffickingBased on the studies discussed above, it is tempting to speculatethat the mislocalization of SNAP23–STX4 upon NPC1 inhibitionis only an example of the broader impact a cellular imbalance of

cholesterol has on endomembrane SNAREs, including thoseinvolved in post-Golgi exocytic pathways through the TGN andrecycling endosome compartments and those relevant for cellmigration.

Integrins are cell adhesion receptors; they are composed of aand b subunits that bind to the ECM and enable cells to migrate.To ensure forward movement, integrins are constantly

internalized and recycled for their redistribution at the leadingedge (Caswell and Norman, 2008; Jones et al., 2006; Pellinen andIvaska, 2006). Both cholesterol and several SNAREs have been

implicated in the molecular machinery that is responsible for theendocytic and exocytic trafficking of integrins. As such, thecholesterol content of the plasma membrane controls signalingevents that are mediated by aVb3 integrins (Green et al., 1999),

including focal adhesion kinase (FAK) and mitogen-activatedprotein kinase (MAPK) signaling pathways, and cell adhesionand migration on fibronectin-coated substrates (Ramprasad et al.,

2007). In this context, the ability of SNAREs to bind cholesterolin specific cellular sites could contribute to the establishment offunctional links between cholesterol and integrin localization and

function. These cholesterol-binding SNAREs include VAMP2,VAMP3 (also known as cellubrevin), STX3, STX4 and SNAP23,which all participate in the recycling of b1 integrin, and VAMP3

and STX6, which determine the levels of cell-surface-associateda5b1 integrin and of FAK (Hulce et al., 2013; Riggs et al., 2012;Tiwari et al., 2011) (see also Fig. 2A and Table 1).

Cholesterol-sensitive shuttling of STX6 between the TGN andrecycling endosomes determines integrin recycling andmigratory behaviour of cellsAmong the aforementioned v-SNAREs, VAMP3 contains acholesterol-interacting R-W-L motif (Fig. 2B). VAMP3 is highlysimilar to VAMP2 and has been shown to bind to cholesterol in

exocytic pathways (Table 1). Besides its similarity to VAMP2,

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VAMP3 also displays a significant sequence similarity toVAMP4, which might contribute to the ability of both VAMPs

to bind the t-SNAREs STX6, STX16 and VIT1A, which functionat the trafficking interface between the TGN, recycling endosomeand the plasma membrane. However, VAMP4 does not appear tobind cholesterol, but contains an E/DxxxLL motif that allows

interaction with adaptor protein-1 in the TGN (Peden et al.,2001). These differential binding properties might enableVAMP3 and VAMP4 to participate in different trafficking

pathways that intersect at the TGN, where they interact withtheir common t-SNARE partner STX6.

As discussed above, both VAMP3 and VAMP4 interact with

the t-SNARE STX6, which, at steady state, is predominantlylocalized at the TGN where it binds to resident molecules throughits SNARE motif (Bock et al., 1997; Wendler and Tooze, 2001)

and Habc domain (Abascal-Palacios et al., 2013; Perez-Victoriaet al., 2010). Besides VAMP4, this includes the Golgi SNAREprotein GS32 (also known as SNAP29) and the mammalian GolgiSec1p-like protein VPS45, both of which are known to be

involved in TGN trafficking (Watson and Pessin, 2000). STX6participates in various membrane fusion events with differentSNARE complexes. Upon completion of membrane fusion, a

tyrosine-based sorting motif (YGRL, position 140–143) locatedbetween the N-terminal domain and the SNARE motif isactivated, which results in its re-sorting to the TGN (Jahn and

Scheller, 2006; Jung et al., 2012). Importantly, STX6 binds tocholesterol (Hulce et al., 2013) and has been previouslyimplicated in cholesterol transport; it contributes to the delivery

of late-endosome-derived cholesterol to the ER via the TGN(Urano et al., 2008) and of lipids and proteins that are required forcaveolae endocytosis to the plasma membrane (Choudhury et al.,2006). This work has shown that inhibition of STX6 leads to a

reduction of caveolin-1 and caveolae at the cell surface, lendingfurther support for a model by which STX6 regulates secretorypathways in a cholesterol-sensitive manner. This ability of STX6

to regulate the transport of cholesterol-rich domains to and fromthe cell surface could determine the cell surface levels of a5b1integrin and FAK, and so modulate focal adhesion sites and

directional migration on fibronectin-coated substrates (Tiwariet al., 2011).

Taken together, cholesterol is linked to the localization andfunction of STX6 and other SNAREs that are predominantly

located at the TGN, the TGN–recycling-endosome boundariesand recycling endosomes. Using an array of NPC1 mutantmodels, we were able to provide novel insights into how

cholesterol pools at the Golgi–endosomal boundaries regulatecell migration, namely through altering the assembly anddisassembly of complexes between STX6 and VAMP3 or

VAMP4, respectively (Reverter et al., 2014). Mechanistically,we found that a decrease in the cholesterol level at the Golgiperturbed the trafficking between recycling endosomes and TGN

and triggered the accumulation of STX6 in VAMP3- and Rab11-containing recycling endosomes. This correlated with a reducedcell surface expression of integrins in NPC1 mutant models andresulted in impaired cell migration and invasion in two- and

three-dimensional environments. Support for endomembranecholesterol levels altering SNARE localization and function hasbeen provided by the fact that prolonged treatment of NPC1

mutant cells with LDL, which causes its overspill into lateendosomes, thereby enabling LDL-cholesterol to bypass theNPC1 mutation and to enter the TGN at later time points, induced

the targeting of STX6 back to the TGN. By contrast, incubation

of wild-type cells with HDL-cholesterol, which rapidly entersrecycling endosomes, but not the TGN, within 30 min after

binding of HDL to cell surface receptors, results in arelocalization of STX6 from the TGN to recycling endosomes(Reverter et al., 2014). Thus, the rapid and local elevation ofcholesterol levels in recycling endosomes of wild-type cells

observed after HDL incubation appears to increase the ability ofSTX6 to interact with VAMP3 in recycling endosomes.

Further support for a function of STX6 as a cholesterol-sensitive

endomembrane SNARE comes from its structure; this syntaxincontains an R-W-L motif (position 233–241) (Fig. 2B), as well as apolybasic cluster in the juxtamembrane region that has homology

to STX4, STX7 and STX12. This polybasic region is conservedand also found in STX1A, STX1B, STX2, STX3, STX17, STX20and STX16 (Murray and Tamm, 2011). In contrast, this positively

charged region appears to be less prevalent in syntaxins that arerequired for early steps in membrane trafficking, such as STX18 inthe ER or STX5 in the Golgi. Interestingly, the syntaxins with themost positive charges localize to the plasma membrane in

microdomains that are characterized by a high degree of negativecharge density and are enriched in cholesterol (Murray and Tamm,2011). Thus, the spatiotemporal localization and function of the

syntaxins that have cholesterol-binding motifs and polybasicclusters, such as STX6, STX4, STX7 and STX12, might behighly responsive to fluctuations in cholesterol levels at either the

plasma membrane or intracellular compartments.The drastic changes in STX6 localization we observed in

NPC1-mutant cells and upon incubation with lipoproteins suggest

that STX6 trafficking between TGN and recycling endosome andits compartment-specific interaction with the v-SNARESVAMP3 and VAMP4 is controlled by its ability to sensecholesterol levels in the TGN and/or recycling endosomes,

thereby possibly regulating cell migration through STX6-dependent trafficking of the integrins aVb3 and a5b1. NPC1-mediated reduction of cholesterol in TGN membranes or selective

elevation of recycling endosome-localized cholesterol couldpromote the translocation of STX6 to these membranes and soincrease its ability to interact with VAMP3 (Reverter et al.,

2014). In fact, recycling endosomes are the main intracellularcholesterol repository compartments in a number of cell types,including Chinese hamster ovary and non-polarized hepatomaHepG2 cells, fibroblasts and human B lymphocytes (Hao et al.,

2002; Hsu and Prekeris, 2010; Maxfield and McGraw, 2004).Although other alternative pathways might exist, our findings

point to a link between cholesterol-sensitive SNAREs and the

final steps in integrin recycling relevant for wound healing andcancer cell migration (Fig. 3). Earlier work has implicatedcholesterol in the formation of signaling complexes that contain

aVb3, CD47 and G-proteins (Green et al., 1999) and in thecontrol of cell adhesion and migration onto fibronectin. Thispossibly requires Rab11, which modulates cholesterol transport

and homeostasis (Holtta-Vuori et al., 2002), and facilitates therecycling of b1 integrin (Powelka et al., 2004). Further supportfor this notion comes from an increased cholesterol requirementfor invasion in MDA-MB-231 breast cancer and A431

epidermoid carcinoma cells (Freed-Pastor et al., 2012); bothcell models are highly relevant in this context as enhancedintegrin recycling therein drives aggressive cancer cell behavior

(Muller et al., 2009). It has yet to be demonstrated whether theseobservations are based on some SNAREs functioning ascholesterol sensors in cancer. Indeed, STX6 overexpression

increases cell migration, and its expression is elevated in breast,

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liver and prostate cancers (Riggs et al., 2012), which all have

established links to cholesterol homeostasis.Furthermore, in breast cancer cells, SNAP23 and STX12, which

also bind to cholesterol in the plasma membrane and recycling

endosomes, are required for the delivery of Src, epidermal growthfactor receptor (EGFR) and b1 integrin to cholesterol-richinvadopodia for cell invasion. Interestingly, b1 integrin affects

the interaction between SNAP23 and STX12 and the formation ofthe SNARE-dependent Src–EGFR–b1-integrin complex (Williamsand Coppolino, 2014). In this context, cholesterol-dependenttrafficking mechanisms appear to be essential (Caldieri and

Buccione, 2010), and cholesterol-binding proteins such ascaveolin-1 (Murata et al., 1995) actively participate in thetransport of cholesterol, as well as facilitating its interaction with

integrins and their activation (Parton and del Pozo, 2013).

Conclusions and perspectivesAs discussed above, cholesterol levels at late endosomes and theGolgi modulate the localization and activity of a subset ofcholesterol-sensing SNARE proteins that regulate fibronectin

secretion, integrin recycling and cell migration.The underlying molecular mechanisms await further

investigation. Cholesterol levels in the TGN and recyclingendosome are much lower than those in the plasma membrane.

Together with the drastic experimental procedures (e.g. treatmentwith methyl-b-cyclodextrin) required to reduce membrane orderat the plasma membrane (Reverter et al., 2014), this might

indicate that moderate changes in cholesterol levels areinsufficient to significantly alter membrane fluidity within theTGN or recycling endosome subcompartments to affect the

affinity of SNAREs towards membranes and thus their

distribution. Instead, we envisage that cholesterol transport is

targeted directly to or into the vicinity of SNARE complexes,possibly by the docking of cholesterol carriers into closeproximity or directly onto endomembrane SNAREs. This could

enable SNARE–cholesterol interaction to impact on the stabilityof assembled SNARE complexes within a certain location,providing the opportunity for these complexes to dissociate and

move along transport pathways to find other interaction partnerselsewhere. However, it is still unknown whether changes incholesterol levels affect the three-dimensional structure andconformation of specific SNARE proteins (as postulated for

VAMP2) (Tong et al., 2009), or whether cholesterol levels andthe lipid environment could interfere with the affinity orcapability of certain SNAREs to cluster and/or remain in a

particular membrane.Alternatively, rather than directly targeting endomembrane

SNAREs, cholesterol could modulate the activity of Golgi-

tethering factors that are required for SNARE assembly. Forinstance, Golgi-associated retrograde protein (GARP) andconserved oligomeric Golgi (COG) complex regulate the fusion

of endosome-derived, retrograde transport carriers to the TGN.This process requires the assembly of the t- and v-SNAREs (STX6,STX16, VIT1A and VAMP4) to facilitate the final steps in theretrograde transport of recycled receptors or integrins, which enter

the trans-Golgi for re-sialylation, or transport of Golgi-residentproteins (i.e. TGN38) to TGN–endosome boundaries (Bonifacinoand Hierro, 2011). The ability of GARP, COG and other Golgi-

tethering complexes to promote or prevent the assembly of the v-and t-SNAREs mentioned above or possibly the pairing of otherspecific v- and t-SNAREs might depend on their spatiotemporal

localization and ability to interact with other cellular SNAREs.

Fibronectin secretionCaveolae

RE

LE

EE

Golgi complex

Rab4

Clathrin- coated pit

VAMP8SNAP23

STX6STX4Rab5

Rab7

Rab9

Rab11

VAMP7STX8

STX7VTI1B

VAMP8 VAMP4VTI1A

STX6STX16

VAMP3VTI1A

STX6STX16

STX12VAMP3

Clathrin Cholesterol NPC1 NPC2 Integrin α5β Integrin αVβ3

1

22

Key

Fig. 3. Model for how cholesterol-sensitive SNARE complexes in endocytic and exocytic trafficking pathways regulate cell migration. Loss of NPC1function blocks the exit of cholesterol from the late endosome (LE), which leads to reduced cholesterol levels at the Golgi complex and the plasma membrane. Thistriggers the mislocalization of the t-SNARE STX6 from the Golgi complex to recycling endosomes (RE) (1). Increased assembly of the STX6–VAMP3 complex inrecycling endosomes reduces recycling and cell surface expression of aVb3 and a5b1 integrins (2), which negatively impacts on cell migration. Orange arrowsindicate trafficking routes of cholesterol. Blue arrows show integrin trafficking routes. Dotted lines represent cholesterol trafficking routes that are impaired due to lossof NPC1 function. Black arrows indicate vesicular transport routes from the Golgi to recycling endosomes and the plasma membrane. Cholesterol-rich membranedomains (dark gray), endocytic routes through clathrin coated pits or caveolae, and several Rab proteins are indicated. SNARE complexes within a particularcompartment are indicated in boxes; the cholesterol-sensitive SNAREs in each compartment are highlighted in orange. EE, early endosomes.

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Besides the affinity of tethers to their respective SNAREs, theavidity of binding, post-translational modifications or as

highlighted here, the amount of cholesterol can influence theassembly or disassembly of SNAREs. The identified role forcholesterol in controlling integrin trafficking through SNAREscould not only be relevant for the ability of cancer cells to move

and spread, but is also important for wound healing in diabetes.Undoubtedly, further in vivo and in vitro studies will shed morelight on the complex role cholesterol plays in modulating the

molecular mechanisms that enable SNAREs to control membranetrafficking and fusion events that are relevant for cell migration inboth physiological and pathological contexts.

AcknowledgementsWe would like to thank all members of our laboratories, past and present, for theirinvaluable contributions and apologize to all those researchers whose work couldnot be discussed owing to space limitations.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work was supported by the Ministerio de Economıa y Competitividad(MINECO) [grant numbers BFU2012-36272 and CSD2009-00016] and FundacioMarato TV3 [grant number PI042182] (Spain) to C.E.; and The University ofSydney, Australia [grant numbers U1758, U7007] to T.G., C.R. is thankful toCONSOLIDER-INGENIO (MINECO) research program for post-doctoral fellowship[grant number CSD2009-00016]. A.H. is grateful to Carlos III Health Institute [grantnumber PI11/00121] and Basque Government [grant number PI2011-26].

ReferencesAbascal-Palacios, G., Schindler, C., Rojas, A. L., Bonifacino, J. S. and Hierro,A. (2013). Structural basis for the interaction of the Golgi-associated retrogradeprotein complex with the t-SNARE Syntaxin 6. Structure 21, 1698-1706.

Alpy, F., Rousseau, A., Schwab, Y., Legueux, F., Stoll, I., Wendling, C.,Spiegelhalter, C., Kessler, P., Mathelin, C., Rio, M. C. et al. (2013). STARD3or STARD3NL and VAP form a novel molecular tether between late endosomesand the ER. J. Cell Sci. 126, 5500-5512.

Antonin, W., Holroyd, C., Tikkanen, R., Honing, S. and Jahn, R. (2000). The R-SNARE endobrevin/VAMP-8 mediates homotypic fusion of early endosomesand late endosomes. Mol. Biol. Cell 11, 3289-3298.

Baier, C. J., Fantini, J. and Barrantes, F. J. (2011). Disclosure of cholesterolrecognition motifs in transmembrane domains of the human nicotinicacetylcholine receptor. Sci. Rep. 1, 69.

Bock, J. B., Klumperman, J., Davanger, S. and Scheller, R. H. (1997). Syntaxin6 functions in trans-Golgi network vesicle trafficking. Mol. Biol. Cell 8, 1261-1271.

Bonifacino, J. S. and Hierro, A. (2011). Transport according to GARP: receivingretrograde cargo at the trans-Golgi network. Trends Cell Biol. 21, 159-167.

Caldieri, G. and Buccione, R. (2010). Aiming for invadopodia: organizingpolarized delivery at sites of invasion. Trends Cell Biol. 20, 64-70.

Caswell, P. and Norman, J. (2008). Endocytic transport of integrins during cellmigration and invasion. Trends Cell Biol. 18, 257-263.

Chamberlain, L. H. and Gould, G. W. (2002). The vesicle- and target-SNAREproteins that mediate Glut4 vesicle fusion are localized in detergent-insolublelipid rafts present on distinct intracellular membranes. J. Biol. Chem. 277,49750-49754.

Chamberlain, L. H., Burgoyne, R. D. and Gould, G. W. (2001). SNARE proteinsare highly enriched in lipid rafts in PC12 cells: implications for the spatial controlof exocytosis. Proc. Natl. Acad. Sci. USA 98, 5619-5624.

Chamberlain, A. K., Lee, Y., Kim, S. and Bowie, J. U. (2004). Snorkelingpreferences foster an amino acid composition bias in transmembrane helices.J. Mol. Biol. 339, 471-479.

Chang, T. Y., Chang, C. C., Ohgami, N. and Yamauchi, Y. (2006). Cholesterolsensing, trafficking, and esterification. Annu. Rev. Cell Dev. Biol. 22, 129-157.

Charloteaux, B., Lorin, A., Crowet, J. M., Stroobant, V., Lins, L., Thomas, A.and Brasseur, R. (2006). The N-terminal 12 residue long peptide of HIV gp41 isthe minimal peptide sufficient to induce significant T-cell-like membranedestabilization in vitro. J. Mol. Biol. 359, 597-609.

Charman, M., Kennedy, B. E., Osborne, N. and Karten, B. (2010). MLN64mediates egress of cholesterol from endosomes to mitochondria in the absenceof functional Niemann-Pick type C1 protein. J. Lipid Res., 51, 1023-1034.

Chen, Y. A. and Scheller, R. H. (2001). SNARE-mediated membrane fusion. Nat.Rev. Mol. Cell Biol. 2, 98-106.

Choudhury, A., Marks, D. L., Proctor, K. M., Gould, G. W. and Pagano, R. E.(2006). Regulation of caveolar endocytosis by syntaxin 6-dependent delivery ofmembrane components to the cell surface. Nat. Cell Biol. 8, 317-328.

Connell, E., Darios, F., Broersen, K., Gatsby, N., Peak-Chew, S. Y., Rickman,C. and Davletov, B. (2007). Mechanism of arachidonic acid action on syntaxin-Munc18. EMBO Rep. 8, 414-419.

Cortes, V. A., Busso, D., Mardones, P., Maiz, A., Arteaga, A., Nervi, F. andRigotti, A. (2013). Advances in the physiological and pathological implicationsof cholesterol. Biol. Rev. Camb. Philos. Soc. 88, 825-843.

Cubells, L., Vila de Muga, S., Tebar, F., Wood, P., Evans, R., Ingelmo-Torres,M., Calvo, M., Gaus, K., Pol, A., Grewal, T. et al. (2007). Annexin A6-inducedalterations in cholesterol transport and caveolin export from the Golgi complex.Traffic 8, 1568-1589.

Cubells, L., Vila de Muga, S., Tebar, F., Bonventre, J. V., Balsinde, J., Pol, A.,Grewal, T. and Enrich, C. (2008). Annexin A6-induced inhibition of cytoplasmicphospholipase A2 is linked to caveolin-1 export from the Golgi. J. Biol. Chem.283, 10174-10183.

De Matteis, M. A. and Luini, A. (2011). Mendelian disorders of membranetrafficking. N. Engl. J. Med. 365, 927-938.

Dozynkiewicz, M. A., Jamieson, N. B., Macpherson, I., Grindlay, J., van denBerghe, P. V., von Thun, A., Morton, J. P., Gourley, C., Timpson, P., Nixon, C.et al. (2012). Rab25 and CLIC3 collaborate to promote integrin recycling fromlate endosomes/lysosomes and drive cancer progression. Dev. Cell 22, 131-145.

Du, X., Kazim, A. S., Brown, A. J. and Yang, H. (2012). An essential role of Hrs/Vps27 in endosomal cholesterol trafficking. Cell Rep., 1, 29-35.

Epand, R. M. (2006). Cholesterol and the interaction of proteins with membranedomains. Prog. Lipid Res. 45, 279-294.

Epand, R. M., Thomas, A., Brasseur, R. and Epand, R. F. (2010). Cholesterolinteraction with proteins that partition into membrane domains: an overview.Subcell. Biochem. 51, 253-278.

Fantini, J. and Barrantes, F. J. (2013). How cholesterol interacts with membraneproteins: an exploration of cholesterol-binding sites including CRAC, CARC, andtilted domains. Front. Physiol. 4, 31.

Fincham, V. J., Unlu, M., Brunton, V. G., Pitts, J. D., Wyke, J. A. and Frame,M. C. (1996). Translocation of Src kinase to the cell periphery is mediated by theactin cytoskeleton under the control of the Rho family of small G proteins. J. CellBiol. 135, 1551-1564.

Fraldi, A., Annunziata, F., Lombardi, A., Kaiser, H. J., Medina, D. L.,Spampanato, C., Fedele, A. O., Polishchuk, R., Sorrentino, N. C., Simons,K. et al. (2010). Lysosomal fusion andSNARE function are impaired by cholesterolaccumulation in lysosomal storage disorders. EMBO J. 29, 3607-3620.

Freed-Pastor, W. A., Mizuno, H., Zhao, X., Langerød, A., Moon, S. H.,Rodriguez-Barrueco, R., Barsotti, A., Chicas, A., Li, W., Polotskaia, A. et al.(2012). Mutant p53 disrupts mammary tissue architecture via the mevalonatepathway. Cell 148, 244-258.

Ganley, I. G., Espinosa, E. and Pfeffer, S. R. (2008). A syntaxin 10-SNAREcomplex distinguishes two distinct transport routes from endosomes to thetrans-Golgi in human cells. J. Cell Biol. 180, 159-172.

Green, J. M., Zhelesnyak, A., Chung, J., Lindberg, F. P., Sarfati, M., Frazier,W. A. and Brown, E. J. (1999). Role of cholesterol in formation and function of asignaling complex involving alphavbeta3, integrin-associated protein (CD47),and heterotrimeric G proteins. J. Cell Biol. 146, 673-682.

Grimmer, S., Ying, M., Walchli, S., van Deurs, B. and Sandvig, K. (2005). Golgivesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism. Traffic 6, 144-156.

Hanson, M. A., Cherezov, V., Griffith, M. T., Roth, C. B., Jaakola, V. P., Chien,E. Y., Velasquez, J., Kuhn, P. and Stevens, R. C. (2008). A specific cholesterolbinding site is established by the 2.8 A structure of the human beta2-adrenergicreceptor. Structure 16, 897-905.

Hao, M., Lin, S. X., Karylowski, O. J., Wustner, D., McGraw, T. E. and Maxfield,F. R. (2002). Vesicular and non-vesicular sterol transport in living cells. Theendocytic recycling compartment is a major sterol storage organelle. J. Biol.Chem. 277, 609-617.

Heeren, J., Beisiegel, U. and Grewal, T. (2006). Apolipoprotein E recycling:implications for dyslipidemia and atherosclerosis. Arterioscler. Thromb. Vasc.Biol. 26, 442-448.

Holtta-Vuori, M., Tanhuanpaa, K., Mobius, W., Somerharju, P. and Ikonen, E.(2002). Modulation of cellular cholesterol transport and homeostasis by Rab11.Mol. Biol. Cell 13, 3107-3122.

Hong, W. (2005). SNAREs and traffic. Biochim. Biophys. Acta 1744, 493-517.

Hong, W. and Lev, S. (2014). Tethering the assembly of SNARE complexes.Trends Cell Biol. 24, 35-43.

Hsu, V. W. and Prekeris, R. (2010). Transport at the recycling endosome. Curr.Opin. Cell Biol. 22, 528-534.

Hulce, J. J., Cognetta, A. B., Niphakis, M. J., Tully, S. E. and Cravatt, B. F.(2013). Proteome-wide mapping of cholesterol-interacting proteins inmammalian cells. Nat. Methods 10, 259-264.

Iinuma, T., Aoki, T., Arasaki, K., Hirose, H., Yamamoto, A., Samata, R., Hauri,H. P., Arimitsu, N., Tagaya, M. and Tani, K. (2009). Role of syntaxin 18 in theorganization of endoplasmic reticulum subdomains. J. Cell Sci. 122, 1680-1690.

Ikonen, E. (2006). Mechanisms for cellular cholesterol transport: defects andhuman disease. Physiol. Rev. 86, 1237-1261.

Ikonen, E. (2008). Cellular cholesterol trafficking and compartmentalization. Nat.Rev. Mol. Cell Biol. 9, 125-138.

Ikonen, E. and Holtta-Vuori, M. (2004). Cellular pathology of Niemann-Pick typeC disease. Semin. Cell Dev. Biol. 15, 445-454.

COMMENTARY Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459

1079

Jour

nal o

f Cel

l Sci

ence

Ikonen, E. and Jansen, M. (2008). Cellular sterol trafficking and metabolism:spotlight on structure. Curr. Opin. Cell Biol. 20, 371-377.

Infante, R. E., Wang, M. L., Radhakrishnan, A., Kwon, H. J., Brown, M. S. andGoldstein, J. L. (2008). NPC2 facilitates bidirectional transfer of cholesterolbetween NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes.Proc. Natl. Acad. Sci. USA 105, 15287-15292.

Jahn, R. and Scheller, R. H. (2006). SNAREs – engines for membrane fusion.Nat. Rev. Mol. Cell Biol. 7, 631-643.

Jones, M. C., Caswell, P. T. and Norman, J. C. (2006). Endocytic recyclingpathways: emerging regulators of cell migration. Curr. Opin. Cell Biol. 18, 549-557.

Jung, J. J., Inamdar, S. M., Tiwari, A. and Choudhury, A. (2012). Regulation ofintracellular membrane trafficking and cell dynamics by syntaxin-6. Biosci. Rep.32, 383-391.

Kanerva, K., Uronen, R. L., Blom, T., Li, S., Bittman, R., Lappalainen, P.,Peranen, J., Raposo, G. and Ikonen, E. (2013). LDL cholesterol recycles to theplasma membrane via a Rab8a-Myosin5b-actin-dependent membrane transportroute. Dev. Cell 27, 249-262.

Kent, H. M., Evans, P. R., Schafer, I. B., Gray, S. R., Sanderson, C. M., Luzio, J. P.,Peden, A. A. and Owen, D. J. (2012). Structural basis of the intracellular sorting ofthe SNARE VAMP7 by the AP3 adaptor complex. Dev. Cell 22, 979-988.

Klein, A., Amigo, L., Retamal, M. J., Morales, M. G., Miquel, J. F., Rigotti, A.and Zanlungo, S. (2006). NPC2 is expressed in human and murine liver andsecreted into bile: potential implications for body cholesterol homeostasis.Hepatology 43, 126-133.

Kuwabara, P. E. and Labouesse, M. (2002). The sterol-sensing domain: multiplefamilies, a unique role? Trends Genet. 18, 193-201.

Lafont, F., Verkade, P., Galli, T., Wimmer, C., Louvard, D. and Simons, K.(1999). Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells. Proc. Natl. Acad. Sci. USA 96, 3734-3738.

Lang, T. (2007). SNARE proteins and ‘membrane rafts’. J. Physiol. 585, 693-698.Laufman, O., Hong, W. and Lev, S. (2011). The COG complex interacts directlywith Syntaxin 6 and positively regulates endosome-to-TGN retrograde transport.J. Cell Biol. 194, 459-472.

Leiva, A., Verdejo, H., Benıtez, M. L., Martınez, A., Busso, D. and Rigotti, A.(2011). Mechanisms regulating hepatic SR-BI expression and their impact onHDL metabolism. Atherosclerosis 217, 299-307.

Leung, S. M., Chen, D., DasGupta, B. R., Whiteheart, S. W. and Apodaca, G.(1998). SNAP-23 requirement for transferrin recycling in Streptolysin-O-permeabilized Madin-Darby canine kidney cells. J. Biol. Chem. 273, 17732-17741.

Li, H. and Papadopoulos, V. (1998). Peripheral-type benzodiazepine receptorfunction in cholesterol transport. Identification of a putative cholesterolrecognition/interaction amino acid sequence and consensus pattern.Endocrinology 139, 4991-4997.

Lingwood, D. and Simons, K. (2010). Lipid rafts as a membrane-organizingprinciple. Science 327, 46-50.

Low, S. H., Vasanji, A., Nanduri, J., He, M., Sharma, N., Koo, M., Drazba, J. andWeimbs, T. (2006). Syntaxins 3 and 4 are concentrated in separate clusters onthe plasma membrane before the establishment of cell polarity. Mol. Biol. Cell17, 977-989.

Luzio, J. P., Gray, S. R. and Bright, N. A. (2010). Endosome-lysosome fusion.Biochem. Soc. Trans. 38, 1413-1416.

Luzio, J. P., Hackmann, Y., Dieckmann, N. M. and Griffiths, G. M. (2014). Thebiogenesis of lysosomes and lysosome-related organelles. Cold Spring Harb.Perspect. Biol. 6, a016840.

Macpherson, I. R., Rainero, E., Mitchell, L. E., van den Berghe, P. V., Speirs,C., Dozynkiewicz, M. A., Chaudhary, S., Kalna, G., Edwards, J., Timpson, P.et al. (2014). CLIC3 controls recycling of late endosomal MT1-MMP anddictates invasion and metastasis in breast cancer. J. Cell Sci. 127, 3893-3901.

Maxfield, F. R. and McGraw, T. E. (2004). Endocytic recycling. Nat. Rev. Mol. CellBiol. 5, 121-132.

Maxfield, F. R. and Tabas, I. (2005). Role of cholesterol and lipid organization indisease. Nature 438, 612-621.

Maxfield, F. R. and van Meer, G. (2010). Cholesterol, the central lipid ofmammalian cells. Curr. Opin. Cell Biol. 22, 422-429.

McMahon, H. T., Ushkaryov, Y. A., Edelmann, L., Link, E., Binz, T., Niemann,H., Jahn, R. and Sudhof, T. C. (1993). Cellubrevin is a ubiquitous tetanus-toxinsubstrate homologous to a putative synaptic vesicle fusion protein. Nature 364,346-349.

Mesmin, B. and Maxfield, F. R. (2009). Intracellular sterol dynamics. Biochim.Biophys. Acta 1791, 636-645.

Muller, P. A., Caswell, P. T., Doyle, B., Iwanicki, M. P., Tan, E. H., Karim, S.,Lukashchuk, N., Gillespie, D. A., Ludwig, R. L., Gosselin, P. et al. (2009).Mutant p53 drives invasion by promoting integrin recycling. Cell 139, 1327-1341.

Mullock, B. M., Smith, C. W., Ihrke, G., Bright, N. A., Lindsay, M., Parkinson, E.J., Brooks, D. A., Parton, R. G., James, D. E., Luzio, J. P. et al. (2000). Syntaxin7 is localized to late endosome compartments, associates with Vamp 8, and Isrequired for late endosome-lysosome fusion. Mol. Biol. Cell 11, 3137-3153.

Murata, M., Peranen, J., Schreiner, R., Wieland, F., Kurzchalia, T. V. andSimons, K. (1995). VIP21/caveolin is a cholesterol-binding protein. Proc. Natl.Acad. Sci. USA 92, 10339-10343.

Murray, D. H. and Tamm, L. K. (2011). Molecular mechanism of cholesterol- andpolyphosphoinositide-mediated syntaxin clustering. Biochemistry 50, 9014-9022.

Parton, R. G. and del Pozo, M. A. (2013). Caveolae as plasma membranesensors, protectors and organizers. Nat. Rev. Mol. Cell Biol. 14, 98-112.

Peden, A. A., Park, G. Y. and Scheller, R. H. (2001). The Di-leucine motif ofvesicle-associated membrane protein 4 is required for its localization and AP-1binding. J. Biol. Chem. 276, 49183-49187.

Pellinen, T. and Ivaska, J. (2006). Integrin traffic. J. Cell Sci. 119, 3723-3731.Perez-Victoria, F. J., Schindler, C., Magadan, J. G., Mardones, G. A.,Delevoye, C., Romao, M., Raposo, G. and Bonifacino, J. S. (2010). Ang2/fat-free is a conserved subunit of the Golgi-associated retrograde proteincomplex. Mol. Biol. Cell 21, 3386-3395.

Platt, F. M., Boland, B. and van der Spoel, A. C. (2012). The cell biology ofdisease: lysosomal storage disorders: the cellular impact of lysosomaldysfunction. J. Cell Biol. 199, 723-734.

Powelka, A. M., Sun, J., Li, J., Gao, M., Shaw, L. M., Sonnenberg, A. and Hsu,V. W. (2004). Stimulation-dependent recycling of integrin beta1 regulated byARF6 and Rab11. Traffic 5, 20-36.

Predescu, S. A., Predescu, D. N., Shimizu, K., Klein, I. K. and Malik, A. B.(2005). Cholesterol-dependent syntaxin-4 and SNAP-23 clustering regulatescaveolar fusion with the endothelial plasma membrane. J. Biol. Chem. 280,37130-37138.

Prekeris, R., Klumperman, J., Chen, Y. A. and Scheller, R. H. (1998). Syntaxin13 mediates cycling of plasma membrane proteins via tubulovesicular recyclingendosomes. J. Cell Biol. 143, 957-971.

Pryor, P. R., Mullock, B. M., Bright, N. A., Lindsay, M. R., Gray, S. R.,Richardson, S. C., Stewart, A., James, D. E., Piper, R. C. and Luzio, J. P.(2004). Combinatorial SNARE complexes with VAMP7 or VAMP8 definedifferent late endocytic fusion events. EMBO Rep. 5, 590-595.

Puri, N. and Roche, P. A. (2006). Ternary SNARE complexes are enriched in lipidrafts during mast cell exocytosis. Traffic 7, 1482-1494.

Rainero, E. and Norman, J. C. (2013). Late endosomal and lysosomal traffickingduring integrin-mediated cell migration and invasion: cell matrix receptors aretrafficked through the late endosomal pathway in a way that dictates how cellsmigrate. BioEssays 35, 523-532.

Ramprasad, O. G., Srinivas, G., Rao, K. S., Joshi, P., Thiery, J. P., Dufour, S.and Pande, G. (2007). Changes in cholesterol levels in the plasma membranemodulate cell signaling and regulate cell adhesion and migration on fibronectin.Cell Motil. Cytoskeleton 64, 199-216.

Reverter, M., Rentero, C., de Muga, S. V., Alvarez-Guaita, A., Mulay, V., Cairns,R., Wood, P., Monastyrskaya, K., Pol, A., Tebar, F. et al. (2011). Cholesteroltransport from late endosomes to the Golgi regulates t-SNARE trafficking,assembly, and function. Mol. Biol. Cell 22, 4108-4123.

Reverter, M., Rentero, C., Garcia-Melero, A., Hoque, M., Vila de Muga, S.,Alvarez-Guaita, A., Conway, J. R., Wood, P., Cairns, R., Lykopoulou, L. et al.(2014). Cholesterol regulates Syntaxin 6 trafficking at trans-Golgi networkendosomal boundaries. Cell Reports 7, 883-897.

Rickman, C. and Davletov, B. (2005). Arachidonic acid allows SNARE complexformation in the presence of Munc18. Chem. Biol. 12, 545-553.

Riggs, K. A., Hasan, N., Humphrey, D., Raleigh, C., Nevitt, C., Corbin, D.and Hu, C. (2012). Regulation of integrin endocytic recycling and chemotacticcell migration by syntaxin 6 and VAMP3 interaction. J. Cell Sci. 125, 3827-3839.

Rowland, A. A., Chitwood, P. J., Phillips,M. J. and Voeltz, G. K. (2014). ER contactsites define the position and timing of endosome fission. Cell 159, 1027-1041.

Salaun, C., Gould, G. W. and Chamberlain, L. H. (2005). The SNARE proteinsSNAP-25 and SNAP-23 display different affinities for lipid rafts in PC12 cells.Regulation by distinct cysteine-rich domains. J. Biol. Chem. 280, 1236-1240.

Sharpe, H. J., Stevens, T. J. and Munro, S. (2010). A comprehensivecomparison of transmembrane domains reveals organelle-specific properties.Cell 142, 158-169.

Sieber, J. J., Willig, K. I., Heintzmann, R., Hell, S. W. and Lang, T. (2006). TheSNARE motif is essential for the formation of syntaxin clusters in the plasmamembrane. Biophys. J. 90, 2843-2851.

Sieber, J. J., Willig, K. I., Kutzner, C., Gerding-Reimers, C., Harke, B., Donnert,G., Rammner, B., Eggeling, C., Hell, S. W., Grubmuller, H. et al. (2007).Anatomy and dynamics of a supramolecular membrane protein cluster. Science317, 1072-1076.

Simons, K. and Ikonen, E. (2000). How cells handle cholesterol. Science 290,1721-1726.

St-Denis, J. F., Cabaniols, J. P., Cushman, S. W. and Roche, P. A. (1999).SNAP-23 participates in SNARE complex assembly in rat adipose cells.Biochem. J. 338, 709-715.

Steffen, A., Le Dez, G., Poincloux, R., Recchi, C., Nassoy, P., Rottner, K., Galli,T. and Chavrier, P. (2008). MT1-MMP-dependent invasion is regulated by TI-VAMP/VAMP7. Curr. Biol. 18, 926-931.

Strandberg, E. and Killian, J. A. (2003). Snorkeling of lysine side chains intransmembrane helices: how easy can it get? FEBS Lett. 544, 69-73.

Stuven, E., Porat, A., Shimron, F., Fass, E., Kaloyanova, D., Brugger, B.,Wieland, F. T., Elazar, Z. and Helms, J. B. (2003). Intra-Golgi protein transportdepends on a cholesterol balance in the lipid membrane. J. Biol. Chem. 278,53112-53122.

Sudhof, T. C. and Rothman, J. E. (2009). Membrane fusion: grappling withSNARE and SM proteins. Science 323, 474-477.

Tiwari, A., Jung, J. J., Inamdar, S. M., Brown, C. O., Goel, A. and Choudhury,A. (2011). Endothelial cell migration on fibronectin is regulated by syntaxin6-mediated alpha5beta1 integrin recycling. J. Biol. Chem. 286, 36749-36761.

COMMENTARY Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459

1080

Jour

nal o

f Cel

l Sci

ence

Tong, J., Borbat, P. P., Freed, J. H. and Shin, Y. K. (2009). A scissors mechanismfor stimulation of SNARE-mediated lipid mixing by cholesterol. Proc. Natl. Acad.Sci. USA 106, 5141-5146.

Torregrosa-Hetland, C. J., Villanueva, J., Giner, D., Lopez-Font, I., Nadal, A.,Quesada, I., Viniegra, S., Exposito-Romero, G., Gil, A., Gonzalez-Velez, V.et al. (2011). The F-actin cortical network is a major factor influencing theorganization of the secretory machinery in chromaffin cells. J. Cell Sci. 124, 727-734.

Tu, C., Ortega-Cava, C. F., Winograd, P., Stanton, M. J., Reddi, A. L., Dodge, I.,Arya, R., Dimri, M., Clubb, R. J., Naramura, M. et al. (2010). Endosomal-sorting complexes required for transport (ESCRT) pathway-dependentendosomal traffic regulates the localization of active Src at focal adhesions.Proc. Natl. Acad. Sci. USA 107, 16107-16112.

Urano, Y., Watanabe, H., Murphy, S. R., Shibuya, Y., Geng, Y., Peden, A. A.,Chang, C. C. and Chang, T. Y. (2008). Transport of LDL-derived cholesterolfrom the NPC1 compartment to the ER involves the trans-Golgi network and theSNARE protein complex. Proc. Natl. Acad. Sci. USA 105, 16513-16518.

van den Bogaart, G., Lang, T. and Jahn, R. (2013). Microdomains of SNAREproteins in the plasma membrane. Curr. Top Membr. 72, 193-230.

van der Kant, R. and Neefjes, J. (2014). Small regulators, major consequences –Ca2+ and cholesterol at the endosome-ER interface. J. Cell Sci. 127, 929-938.

Vance, J. E. and Karten, B. (2014). Niemann-Pick C Disease and mobilization oflysosomal cholesterol by cyclodextrin. J. Lipid Res. 55, 1609-1621.

Wang, Y., Thiele, C. and Huttner, W. B. (2000). Cholesterol is required for theformation of regulated and constitutive secretory vesicles from the trans-Golginetwork. Traffic 1, 952-962.

Wang, M. L., Motamed, M., Infante, R. E., Abi-Mosleh, L., Kwon, H. J., Brown,M. S. and Goldstein, J. L. (2010). Identification of surface residues onNiemann-Pick C2 essential for hydrophobic handoff of cholesterol to NPC1 inlysosomes. Cell Metab. 12, 166-173.

Watson, R. T. and Pessin, J. E. (2000). Functional cooperation of twoindependent targeting domains in syntaxin 6 is required for its efficientlocalization in the trans-golgi network of 3T3L1 adipocytes. J. Biol. Chem.275, 1261-1268.

Wendler, F. and Tooze, S. (2001). Syntaxin 6: the promiscuous behaviour of aSNARE protein. Traffic 2, 606-611.

Williams, K. C. and Coppolino, M. G. (2014). SNARE-dependent interaction ofSrc, EGFR and b1 integrin regulates invadopodia formation and tumor cellinvasion. J. Cell Sci. 127, 1712-1725.

Williams, K. C., McNeilly, R. E. and Coppolino, M. G. (2014). SNAP23,Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) mediatetrafficking of membrane type 1-matrix metalloproteinase (MT1-MMP) duringinvadopodium formation and tumor cell invasion. Mol. Biol. Cell 25, 2061-2070.

Yang, X., Xu, P., Xiao, Y., Xiong, X. and Xu, T. (2006). Domain requirement forthe membrane trafficking and targeting of syntaxin 1A. J. Biol. Chem. 281,15457-15463.

Yu, X., Zech, T., McDonald, L., Gonzalez, E. G., Li, A., Macpherson, I.,Schwarz, J. P., Spence, H., Futo, K., Timpson, P. et al. (2012). N-WASPcoordinates the delivery and F-actin-mediated capture of MT1-MMP at invasivepseudopods. J. Cell Biol. 199, 527-544.

Zilly, F. E., Halemani, N. D., Walrafen, D., Spitta, L., Schreiber, A., Jahn, R. andLang, T. (2011). Ca2+ induces clustering of membrane proteins in the plasmamembrane via electrostatic interactions. EMBO J. 30, 1209-1220.

COMMENTARY Journal of Cell Science (2015) 128, 1071–1081 doi:10.1242/jcs.164459

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