MINIREVIEW

What does S-palmitoylation do to membrane proteins?

Sanja Blaskovic, Mathieu Blanc and F. Gisou van der Goot
de
Global Health Institute, Ecole Polytechnique Fe rale de Lausanne, Switzerland

Keywords S-palmitoylation is post-translational modification, which consists in the

DHHC; membrane proteins; palmitoylation; addition of a C16 acyl chain to cytosolic cysteines and which is unique
protein complexes; protein conformation;
amongst lipid modifications in that it is reversible. It can thus, like phos-
rafts
phorylation or ubiquitination, act as a switch. While palmitoylation of
Correspondence soluble proteins allows them to interact with membranes, the conse-
F. G. van der Goot, Ecole Polytechnique quences of palmitoylation for transmembrane proteins are more enig-
 de
Fe rale de Lausanne, Institute of Global matic. We briefly review the current knowledge regarding the enzymes
Health, Station 15, 1015 Lausanne, responsible for palmitate addition and removal. We then describe various
Switzerland observed consequences of membrane protein palmitoylation. We propose
Fax: +41 021 693 9538
that the direct effects of palmitoylation on transmembrane proteins, how-
Tel: +41 021 693 1791
ever, might be limited to four non-mutually exclusive mechanistic conse-
E-mail: [email protected]
quences: alterations in the conformation of transmembrane domains,
(Received 8 February 2013, revised 20 association with specific membrane domains, controlled interactions with
March 2013, accepted 25 March 2013) other proteins and controlled interplay with other post-translational modi-
fications.
doi:10.1111/febs.12263

Introduction
S-palmitoylation is a post-translational modification of mitochondria [6]. Palmitoylation, however, also occurs
proteins which consists in the attachment of a satu- on transmembrane proteins, which will be the focus of
rated C16 fatty acid chain to specific cysteines via a this review. These proteins are by definition membrane
thioester bond [1]. As opposed to other lipid modifica- anchored and thus the effect of attaching an acyl chain
tions, S-palmitoylation is reversible. Since S-palmitoy- to them is less intuitive, especially considering that
lation impacts on protein function, as discussed below, acylation frequently occurs on cysteines adjacent to, or
its reversibility allows regulation, or fine-tuning, of even within, the transmembrane domain (TMD). The
protein function, in a manner similar to protein phos- cytosolic domains of membrane proteins can also
phorylation or ubiquitination. undergo S-palmitoylation.
Recent large-scale profiling studies indicated that the The list of membrane proteins that undergo palmi-
number of S-palmitoylated proteins varies from ~ 50 toylation is ever growing and spans a bewildering vari-
in yeast [2] to several hundreds in mammals [3,4]. ety of key cellular functions. Amongst the studied
Palmitoylation of soluble proteins allows their con- palmitoylated proteins are cell adhesion molecules [7]
trolled association with membranes, trafficking and such as integrins [8] and claudins [9], tetraspanins such
domain localization, as shown for the small rat sar- as CD81 [10], CD36 [11] and CD151 [12], many
coma (Ras) GTPase at the cell surface [5] or phospho- G-protein-coupled receptors (GPCRs) such as rhodop-
lipid scramblase 3 mediated apoptosis at the sin [13], a great variety of channels and transporters

Abbreviations
APT, acyl protein thioesterase; ER, endoplasmic reticulum; GPCR, G-protein-coupled receptor; LRP6, lipoprotein-receptor-related protein 6;
PK, protein kinase; Ras, rat sarcoma; SNARE, SNAP receptor; TEM8, tumor endothelial marker 8; TMD, transmembrane domain; b-2AR,
b-2-adrenergic receptor.

2766 FEBS Journal 280 (2013) 2766–2774 ª 2013 The Authors Journal compilation ª 2013 FEBS
S. Blaskovic et al. Palmitoylation of membrane proteins

(epithelial sodium channel [14], aquaporins [15], BK identify. Four consequences that we review here
channel [16], dopamine or glutamate transporters etc.), through selected examples, however, and which could
multiple receptors (acetylcholine, cannabinoid, opiod, be either direct or early events, have emerged over
b-adrenergic, dopamine, prostacyclin, estrogen, proges- time and are not mutually exclusive: alterations in pro-
terone, androgen, NMDA, AMPA receptors etc. [17– tein conformation, association with specific membrane
21]), receptor ligands such as Fas ligand [22], but also domains, controlled interactions with other proteins
enzymes such as b- and c-secretases [23,24], the E3 and controlled interplay with other post-translational
ligase gp78 [25], the protein disulfide isomerase TMX modifications (Fig. 1).
[26], chaperones (calnexin [27]) or numerous viral gly-
coproteins (influenza hemaglutinin, vesicular stomatitis
Palmitoyltransferases and
virus G protein, SARS CoV S protein etc. For a
thioesterases
review see [28]). In each study documenting protein
palmitoylation, a cellular and/or functional conse- Palmitate is available in the cell in the form of palmi-
quence was observed such as altered signaling capacity toyl-CoA at nanomolar concentrations [33]. Unless
[6,8,20], reduced activity [29], modified trafficking palmitoyl-CoA is enriched in specific membrane micr-
[30,31] and differential stability [13,32]. Many of the odomains [34], cellular concentrations are insufficient
reported effects, however, are likely to be of secondary for spontaneous palmitoylation of proteins to occur.
consequence. The direct, mechanistic, consequences of S-palmitoylation is thus mostly enzymatic and medi-
palmitoylation of membrane proteins are difficult to ated by a family of DHHC (Asp-His-His-Cys) motif

NON-PALMITOYLATED PALMITOYLATED

PAT

1. Initiation of
Ex.: LRP6
conformational changes
C
APT

PAT
2. Regulation of Ex.: Neurotensin receptor,

membrane domain association gp78, TMX

C
APT
Fig. 1. Consequences of the
palmitoylation of membrane proteins.
Palmitoylation of proteins in the proximity
of their transmembrane domain has been
proposed (1) to affect the conformation of PAT
their transmembrane domain such as Ex.: tetraspanins,
3. Regulation of protein
tilting it, (2) to regulate in a positive, but integrins, calnexin
complex formation
also possibly negative, manner their APT C
association with lipid rafts, (3) to promote
the formation of protein complexes and (4)
to prevent ubiquitination of lysines in
proximity to the palmitoylation site. These
various effects are not mutually exclusive
and might actually explain one another, PAT
e.g. tilting of the transmembrane domain 4. Interplay with other
Ex.: LRP6, TEM8,
could promote its association with a post-translational
BK channels
modifications C
specific domain which could sequester it APT
away from its E3 ubiquitin ligase or/and K- Ub
promote its association with proteins
present in this domain.

FEBS Journal 280 (2013) 2766–2774 ª 2013 The Authors Journal compilation ª 2013 FEBS 2767
Palmitoylation of membrane proteins S. Blaskovic et al.

containing palmitoyltransferases, of which there are 23 ing that a large-scale proteomic analysis of palmitoy-
in humans [1], some 20 in worms and flies and seven lated proteins suggested that DHHC5, DHHC6 and
in yeast [2]. It is at present largely unclear why cells DHHC8 are themselves palmitoylated [4] but these hits
express multiple DHHC proteins and what the sub- were not further validated, nor were the consequences
strate specificity of these enzymes is [35,36]. So far no of palmitoylation addressed.
real consensus sequence surrounding the cysteine resi- In the cytosol, S-palmitoylation is reversed by acyl
dues has been identified [2] even though some rela- protein thioesterases (APTs) of which three have so
tively successful prediction algorithms are available far been identified, APT1, APT2 and APT1-like
(e.g. http://csspalm.biocuckoo.org/). [45,46]. Only a handful of studies have addressed their
DHHC proteins are membrane proteins that span implication in the palmitoylation–depalmitoylation
the bilayer at least four times and harbor the con- cycles of proteins. APT1 was shown to depalmitoylate
served DHHC cysteine-rich catalytic domain on the Ga proteins [47,48], APT2 to depalmitoylate growth-
cytosolic face. They differ in their subcellular localiza- associated protein-43 [46,49], both were found to
tion: many localize to the early secretory pathway depalmitoylate Ras [49], and APT1 and APT1-like
[endoplasmic reticulum (ER) and Golgi] while others were found to depalmitoylate the calcium-activated
can be found at the plasma membrane or in endocytic large potassium channel (BK) [50].
vesicles [37]. DHHC proteins greatly vary in their Altering the activity or expression levels of APTs
N-terminal and C-terminal cytosolic domains which clearly constitutes an effective method of regulating
may carry protein–protein interaction domains such as protein palmitoylation levels. For example, based on a
ankyrin repeats in DHHC13 and DHHC17 or a pre- functional screen to identify microRNAs involved in
dicted src homology 3 domain in DHHC6. These dendritic spine morphogenesis, microRNA-138 was
extensions are probably involved in their localization found to be highly enriched and to regulate expression
and in substrate specificity [38,39] since the DHHC- of APT1 in dendrites, thereby controlling palmitoyla-
containing catalytic domain is not sufficient to convey tion of Ga13 [48].
specificity [39,40].
What determines the specificity of a given DHHC
Effects of palmitoylation on the
protein for its substrates is currently unclear. In yeast,
conformation of transmembrane
it was proposed that certain DHHC proteins preferen-
proteins
tially modify soluble proteins, while others appear to
require an additional modification, such as myristoyla- S-palmitoylation frequently occurs on cysteines adja-
tion or prenylation [2,41]. Interestingly, Swf1 showed cent to TMDs. Since this clearly does not have a mem-
preference towards transmembrane proteins and cyste- brane anchoring function, it was proposed early on
ines that are in close proximity to TMDs [2]. In that palmitoylation of juxtamembranous cysteines
humans, it was found that certain substrates can be could induce tilting of TMDs [51]. Joseph and Nagaraj
modified by a restricted set of DHHC proteins, e.g. showed that palmitoylation of a hydrophobic synthetic
the SNAP receptor (SNARE) protein SNAP25 can be peptide reconstituted into liposomes affected the orien-
modified on its four cysteines by DHHC3, DHHC7 tation of the TMD. This concept has remained attrac-
and DHHC17 [42]. In contrast the transmembrane ER tive and has been utilized to explain the effect of
chaperone calnexin is exclusively palmitoylated on its palmitoylation on the behavior of several membrane
juxtamembranous double cysteine motif by DHHC6 proteins [52–55]. The effect of palmitoylation on the
[27], while integrin a6b4 is specifically modified by conformation of a transmembrane protein was not
DHHC3 [8]. explicitly addressed, however. We have approached the
So far little information is available regarding the issue when studying the palmitoylation of lipoprotein-
regulation of DHHC proteins, in terms of both expres- receptor-related protein 6 (LRP6) [30]. LRP6 has a
sion and activity. It has been shown, in yeast and in well-established role in canonical Wnt signaling, a key
mammals, that palmitoylation of Ras requires the for- pathway in early embryonic development as well as
mation of a complex between the palmitoyltransferase cancer. Upon binding of Wnt proteins to their recep-
Erf2 (DHHC9 in mammals) and the interacting pro- tors of the Frizzled family, a ternary Wnt-Frizzled-
tein Erf4 (GCP16 in mammals) [43,44]. Interaction of LRP6 is formed and transmits the signal across the
DHHC proteins with cofactors might be more wide- membrane. LRP6 is a type I membrane protein with
spread. Also they might undergo specific post-transla- two juxtamembrane palmitoylation sites [30]. Upon
tional modifications in response to specific stimuli or mutation of the palmitoylation sites, LRP6 is recog-
stress conditions. In this context, it is worth mention- nized by the ER quality control and retained. LRP6

2768 FEBS Journal 280 (2013) 2766–2774 ª 2013 The Authors Journal compilation ª 2013 FEBS
S. Blaskovic et al. Palmitoylation of membrane proteins

has a 23-residue-long TMD, i.e. two residues longer lipid rafts, however. It was indeed found that palmi-
than the vast majority of single-pass membrane pro- toylation of the chaperone calnexin and the thioredox-
teins. Since the ER membrane has been proposed to in-related transmembrane protein TMX, two
be thinner than the plasma membrane, a long TMD transmembrane proteins of the ER, is required for
might lead to a hydrophobic mismatch. Tilting of the their targeting to specific ER domains, the ER-mito-
TMD through palmitoylation, as proposed by Joseph chondrial interaction sites [26]. Palmitoylation of caln-
and Nagaraj [51], would reduce the effective hydro- exin also appears to be necessary for its localization to
phobic length. To test this possibility, we shortened the nuclear envelope [27]. Palmitoylation of the ER E3
the TMD of LRP6. In the absence of palmitoylation ligase gp78, involved in particular in the downregula-
shortening relieved the ER retention, whereas in the tion of HMGCoA reductase, was recently found to
presence of palmitoylation shortening led to ER reten- regulate the localization of the protein to the periph-
tion, consistent with an effect of palmitoylation on the eral ER [25]. Although detergent-resistant ER domains
conformation of the TMD [30]. have been observed [61], the very low cholesterol con-
Interestingly, ER retention of palmitoylation-defi- tent of this compartment suggests that these domains
cient LRP6 was due to ubiquitination of a lysine resi- must have different assembly mechanisms and thus do
due in close proximity to the TMD [30]. Mutation of not fulfill the definition of lipid rafts.
this lysine relieved ER retention. The protein was then By controlling the association of membrane proteins
properly targeted to the plasma membrane and compe- with specific membrane domains/compartments, palmi-
tent for Wnt signaling. Intriguingly, however, mutation toylation can bring together, or alternatively segregate,
of the juxtamembranous lysine on the wild-type back- proteins that have the ability to interact under specific
ground, while not affecting plasma membrane target- circumstances. For example, neurotensin receptor-1, a
ing, drastically reduced Wnt signaling [30]. Thus both GPCR implicated in breast cancer progression, is
in the ER and at the plasma membrane there appears palmitoylated, allowing it to interact with cholesterol-
to be cross-talk between palmitoylation and ubiquiti- rich domains where its downstream Ga protein resides
nation of LRP6 (see below). [62]. Thus in the absence of palmitoylation signaling is
reduced. The opposite situation was observed for the
anthrax toxin receptor TEM8 where palmitoylation
Association with membrane domains
negatively regulates raft association, thus sequestering
A major focus of studies on the palmitoylation of the receptor away from its E3 ubiquitin ligase Cbl [32].
membrane proteins has been the role of this modifica- In the absence of palmitoylation, TEM8 is prematurely
tion in promoting their association with lipid rafts – ubiquitinated at the cell surface and targeted to lyso-
cholesterol- and sphingolipid-rich membrane domains somes.
[56]. Palmitoylation of many proteins will indeed pro- How palmitoylation affects raft association of pro-
mote their association with lipid rafts (for a review see teins is unclear. It could be due to the affinity of palmi-
[57]). A recent example is the death receptor 4 that tate for specific lipids or lipid domains. This is
undergoes DHHC3-mediated palmitoylation [58] on suggested by studies of different isoforms of Ras that
three cytosolic cysteines, thus allowing its proper tar- are or are not palmitoylated [63]. Palmitoylation was
geting to the plasma membrane and its association shown to promote association with cholesterol-rich na-
with lipid rafts, which was found to be required for noclusters, while non-palmitoylated Ras was excluded
ligand-induced cell death [59]. Palmitoylation-depen- from these domains [64]. In the case of transmembrane
dent raft association has also been shown recently for proteins, however, modified raft association could also
b-secretase BACE1 [23], cannabinoid receptor [19], be due to the effect of palmitoylation on the conforma-
influenza virus M2 protein [60] and close homolog of tion of TMDs. Indeed, since cholesterol-rich domains
adhesion molecule L1 (CHL1) [50]. Exceptions do are thought to be thicker than other parts of the mem-
exist, however, such as the G protein of vesicular brane due to the presence of cholesterol, modification
stomatitis virus and the transferrin receptor which are of TMD tilting through palmitoylation might affect the
both palmitoylated on three sites yet localize to non- effective length of the hydrophobic segments leading to
raft domains (for a review see [57]), or the anthrax protein partitioning through the membrane thickness.
toxin receptor tumor endothelial marker 8 (TEM8),
for which palmitoylation was actually found to
Effect on protein–protein association
negatively regulate raft association [32].
The ability of palmitoylation to target proteins to Palmitoylation of membrane proteins also appears to
specific membrane domains might not be restricted to affect their ability to associate with other, soluble or

FEBS Journal 280 (2013) 2766–2774 ª 2013 The Authors Journal compilation ª 2013 FEBS 2769
Palmitoylation of membrane proteins S. Blaskovic et al.

transmembrane, proteins. One striking example is pro- adenosine monophosphate (cAMP) and activation of
vided by the so-called tetraspanin webs, large protein protein kinase A (PKA). In the late 1980s it was
complexes that include multiple palmitoylated tetra- reported that b-2AR is palmitoylated and that this is
spanin proteins [12,65–67] as well as palmitoylated important to prevent uncontrolled receptor desensitiza-
integrin subunits such as a3, a6 and b4 [8,68,69]. tion and thereby loss of signaling [75]. Recently it was
Palmitoylation may occur on multiple sites as shown demonstrated that palmitoylation enables b-2AR to
for KAI1, a member of the tetraspanin family that interact with cAMP degradation enzyme phosphodies-
inhibits migration and invasion of metastatic prostate terase 4D as well as with the adaptor protein b-arres-
cancer cells and is palmitoylated on five cysteines [65]. tin 2, which is required for agonist-induced receptor
Palmitoylation regulates interactions between tetraspa- internalization [18]. Thus by interfering with desensiti-
nins and between tetraspanins and integrins, and this zation the lack of b-2AR palmitoylation leads to
can occur in rather sophisticated ways as recently enhanced receptor-induced PKA activities in the cyto-
shown for CD151 for which palmitoylation differen- plasm. Consistent with these conclusions, palmitoyla-
tially affects its interaction with a3b1 versus a6b4 inte- tion-deficient b-2AR was found to be phosphorylated
grins [12]. even in the absence of stimulus [76,77]. Interestingly,
We have recently found that palmitoylation of the stimulation with the agonist has also been proposed to
ER chaperone calnexin is required for the formation induce depalmitoylation of the receptor [78]. A full
of a super-complex that includes the ER translocon, understanding of how cycles of palmitoylation–de-
the ribosome, the oligosaccharyltransferase and calnex- palmitoylation regulate b-2AR signaling will require
in and is stabilized by polymerized actin [27]. This further inquiry.
complex allows newly synthesized proteins to be
sequentially handled by the enzymes and chaperones
Interplay between palmitoylation and
that allow it to reach its native state.
other post-translational modifications
Palmitoylation, however, can also negatively affect
protein multimerization as was shown for the neural As just illustrated by b-2AR, there are interplays
cell adhesion molecule 140 (NCAM140) that is essen- between palmitoylation and other post-translational
tial for axonal growth. DHHC7-dependent palmitoyla- modifications on the same protein, generally in close
tion of NCAM140 [70] leads to raft association but proximity to the palmitoylation sites. As mentioned
negatively regulates the hemophilic dimerization of earlier, we found that palmitoylation-deficient LRP6
extracellular NCAM domains in cis [71]. Interestingly, undergoes ubiquitination in the ER of juxtamembra-
neuritogenesis requires interplay between NCAMs and nous lysine residues [30]. Similarly we found that
fibroblast growth factor receptor signaling, and fibro- palmitoylation-deficient TEM8 undergoes ubiquitina-
blast growth factor leads to the activation of DHHC7 tion at the cell surface, again in the vicinity of the
that in turn enhances NCAM palmitoylation [70], fur- palmitoylation sites, resulting in destabilization of
ther promoting axonal growth [72]. TEM8 and its premature degradation [32]. Premature
Palmitoylation may also control the interaction of degradation of palmitoylation-deficient mutants has
membrane proteins with cytoplasmic proteins as been observed for a variety of proteins both in tissue
shown for the cation-dependent mannose-6-phosphate culture cells such as for CCR5 [79,80] but also in
receptor (CD-M6PR), a type I transmembrane protein, knock-in mice for rhodopsin [13]. Interestingly, rho-
responsible for sorting of newly synthesized acid-hy- dopsin destabilization was not apparent in tissue cul-
drolases from the trans-Golgi network to endosomes. ture studies but clearly evident in mice. Also in yeast,
Once the M6PR has delivered its hydrolases to late palmitoylation of the SNARE protein (see below) Tlg1
endosomes, it is recycled back to the Golgi for re-utili- was found to prevent ubiquitination and degradation
zation. This step depends on palmitoylation of M6PR [52]. Although palmitoylation is generally found to sta-
[73] by DHHC15 [74]. More specifically it was found bilize proteins, the reverse has also been observed as
that palmitoylation allows the interaction of the cyto- for the E3 ubiquitin ER ligase gp78 [25].
solic tail of M6PR with the retromer complex [74]. Interplay also occurs between palmitoylation and
Palmitoylation is also important for the function post-translational modifications other than ubiquitina-
of a wide array of GPCRs, one of the largest classes tion. This is the case for the large conductance cal-
of transmembrane proteins, involved in a variety of cium-activated potassium (BK) channels and especially
signaling pathways. A well-characterized example is those containing the stress-regulated exon (STREX)
the b-2-adrenergic receptor (b-2AR) which triggers splice variant [16,81,82]. BKs are six-spanning mem-
activation of an adenylate cyclase, production of cyclic brane proteins with a long cytosolic tail, corresponding

2770 FEBS Journal 280 (2013) 2766–2774 ª 2013 The Authors Journal compilation ª 2013 FEBS
S. Blaskovic et al. Palmitoylation of membrane proteins

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proofreading the manuscript. This work was supported
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by the Swiss National Science Foundation and the
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reveals distinct requirements for tumor cell behaviors
MB is a recipient of a long-term EMBO fellowship.

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