Download Molecular Mechanisms in Exocytosis and Endocytosis

7th Junior Academics Meeting, an Independent Meeting held at University of Edinburgh, Edinburgh, U.K., 5–7 April 2009. Organized and Edited by Rolly
Wiegand (Edinburgh, U.K.).
Regulation of SNAP-25 trafficking and function by
palmitoylation
Jennifer Greaves1 , Gerald R. Prescott, Oforiwa A. Gorleku and Luke H. Chamberlain
Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, U.K.
Abstract
The SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein
SNAP-25 (25 kDa synaptosome-associated protein) is essential for regulated exocytosis in neuronal and
neuroendocrine cells. Whereas the majority of SNARE proteins contain transmembrane domains, SNAP-25
is instead anchored to membranes by the palmitoylation of a central cysteine-rich region. In this review,
we discuss the mechanisms of SNAP-25 palmitoylation and how this modification regulates the intracellular
trafficking and exocytotic function of this essential protein.
SNARE (soluble N-ethylmaleimidesensitive fusion protein-attachment
protein receptor) proteins and regulated
exocytosis
Exocytosis is the process whereby intracellular vesicles fuse
with the plasma membrane. This membrane fusion event
mediates the delivery of proteins and lipids to the plasma
membrane and the secretion of soluble vesicle cargo to the cell
exterior. Exocytosis occurs constitutively in all cells where it
is essential for the insertion of newly synthesized proteins
into the plasma membrane. In addition, a specialized form of
exocytosis, termed regulated exocytosis, occurs in certain cell
types and mediates the controlled secretion of physiologically
important molecules such as neurotransmitters, peptides
and hormones. The stimulus for regulated exocytosis in
neuronal and neuroendocrine cells is generally an increase
in cytoplasmic Ca2+ levels [1].
Exocytosis is dependent on a number of distinct and
overlapping protein–protein interactions [2]. Central to this
Key words: acylation, aspartate-histidine-histidine-cysteine (DHHC) palmitoyltransferase,
exocytosis, palmitoylation, 25 kDa synaptosome-associated protein (SNAP-25), soluble
N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE).
Abbreviations used: Akr1p, ankyrin repeat-containing protein 1; Apt1, acyl protein thioesterase
1; CRD, cysteine-rich domain; Erf2p (etc.), effector of Ras function protein 2 (etc.);
DHHC, aspartate-histidine-histidine-cysteine; HEK, human embryonic kidney; SNAP-25, 25 kDa
synaptosome-associated protein; SNARE, soluble N-ethylmaleimide-sensitive fusion proteinattachment protein receptor; VAMP2, vesicle-associated membrane protein 2.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2010) 38, 163–166; doi:10.1042/BST0380163
fusion event is the interaction between SNARE proteins
localized on the vesicle and plasma membranes. In neuronal
cells, the plasma membrane SNAREs SNAP-25 (25 kDa
synaptosome-associated protein) and syntaxin 1 interact with
the vesicle SNARE VAMP2 (vesicle-associated membrane
protein 2) to form a complex that is thought to drive
membrane fusion [3,4]. As SNARE proteins are essential
for regulated exocytosis there is considerable interest in how
these proteins are regulated by protein–protein and protein–
lipid interactions and by post-translational modifications
such as phosphorylation [2,5]. Interestingly, SNAP-25,
VAMP2 and syntaxin 1 have all been reported to be modified
by an additional type of post-translational modification,
namely palmitoylation [6–8].
Molecular Mechanisms in Exocytosis and Endocytosis
Molecular Mechanisms in Exocytosis
and Endocytosis
Protein palmitoylation
Palmitoylation refers to the post-translational addition of the
16-carbon saturated fatty acid palmitate to proteins in one of
two ways: by a cleavable thioester linkage to the thiol group
of cysteine residues (known as S-palmitoylation) or, less
frequently, by an amide linkage (known as N-palmitoylation)
[9,10].
Nearly all palmitoylated proteins are S-palmitoylated.
However, it is important to note that referring to a protein
as being ‘palmitoylated’ is historically based on the observed
incorporation of [3 H]palmitic acid into proteins. However,
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Biochemical Society Transactions (2010) Volume 38, part 1
when the lipid profile of palmitoylated proteins has been
examined using techniques such as MS, it is clear that other
medium- and long-chain fatty acids are also attached to
proteins through thioester linkages, such as the saturated
fatty acids myristate and stearate, and the unsaturated fatty
acids oleate or arachidonate [11,12]. Therefore, the term
S-acylation is more appropriate; however, since the majority of S-acylated proteins incorporate palmitate, the
term palmitoylation is generally used, and will be used
synonymously with acylation hereafter.
Protein palmitoylation has a primary function in allowing
otherwise soluble proteins to become stably membranebound by increasing the affinity of the modified protein
for the lipid bilayer. However, despite the important
role that palmitoylation plays in membrane binding, it is
becoming clear that palmitoylation does not always function
as a membrane anchor. This point is emphasized by the
demonstrated palmitoylation of a number of transmembrane
proteins, which clearly do not require palmitoylation for
membrane anchoring (e.g. VAMP2 and syntaxin 1). Indeed,
palmitoylation has now been shown to play diverse roles
in addition to membrane tethering: in the targeting of both
soluble and transmembrane proteins to distinct membrane
compartments and membrane microdomains [13–16]; in
promoting protein stability, for example by regulating protein
degradation or ensuring correct protein folding [17,18]; and
in regulating protein–protein interactions [19].
Palmitoylation is predominantly an enzyme-mediated
event, and the first bona fide palmitoyltransferases were
identified in studies in the budding yeast Saccharomyces
cerevisiae [20,21]. Two proteins were identified that were
required for Ras2p palmitoylation in yeast, Erf2p (effector
of Ras function protein 2) and Erf4p. Palmitoyltransferase
activity towards Ras2p requires both Erf2p and Erf4p, since
neither protein is capable of carrying out palmitoylation
reactions individually [22]. Concurrent with the discovery of
the Erf2p/Erf4p complex as a palmitoyltransferase, another
yeast protein, Akr1p (ankyrin repeat-containing protein 1),
was shown to mediate the S-palmitoylation of the type I
casein kinase Yck2p [23]. Unlike Erf2p, Akr1p does not
need to be part of a complex for palmitoyltransferase
activity, as it is able to work independently without
accessory subunits [23]. Both Erf2p and Akr1p share a
conserved ∼50 amino acid domain, known as a DHHC
(aspartate-histidine-histidine-cysteine) CRD (cysteine-rich
domain). Subsequent analysis of the yeast and mammalian
genomes has identified a family of seven DHHC-CRD
proteins in yeast; Akr1p, Akr2p, Erf2p, Pfa3p, Pfa4p, Pfa5p
and Swf1p; and 23 in mammals; DHHC1–DHHC23 [24].
Genetic and biochemical analyses have demonstrated the
palmitoyltransferase activity of several of these DHHCCRD containing proteins, and accordingly, many substrates
have been identified. Interestingly, DHHC proteins are
also palmitoylated and the DHHC domain is essential for
autopalmitoylation and also for palmitoyltransferase activity,
suggesting that palmitoylated DHHC is a key intermediate
in the palmitoylation reaction.
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Authors Journal compilation Figure 1 S-palmitoylation is a reversible process, which is
regulated by the opposing actions of DHHC palmitoyltransferases
and thioesterases such as Apt1
DHHC proteins are polytopic membrane proteins that
are localized on several intracellular compartments including
the Golgi, ER (endoplasmic reticulum), plasma membrane,
endosomes/vesicular compartments and the yeast vacuole
[25]. To date, it is not clear what elements present
within DHHC proteins dictate their respective intracellular
localizations.
The versatility of protein palmitoylation as a regulatory
module is enhanced by its reversibility, and a number of
proteins undergo dynamic cycles of palmitoylation and
depalmitoylation (see Figure 1). Compared with the DHHC
family of palmitoyltransferases, far less is known about the
proteins that catalyse depalmitoylation.
Palmitoylation of SNAP-25
Whereas syntaxin 1 and VAMP2 have membrane-spanning
domains, SNAP-25 is a soluble protein that requires
palmitoylation on a cluster of cysteine residues to facilitate
stable membrane attachment. Deletion of a 12 amino acid
region containing the four cysteine residues, or mutation
of the cysteine residues within the CRD of SNAP-25, both
render the protein cytosolic [6,26–28]. Since palmitoylation
of proteins is mediated by membrane-anchored DHHC
proteins, mechanisms that target SNAP-25 to its site of
palmitoylation must exist. Some studies have suggested
that initial membrane binding of SNAP-25 might be
mediated by interaction with its SNARE partner syntaxin 1
[26,29]. However, the minimum membrane-binding domain
of SNAP-25 has been mapped to amino acids 85–120, a
region containing the palmitoylated CRD plus the 28 amino
acids immediately downstream of this region [30]; this
domain is not thought to interact with syntaxin. Indeed,
we found that membrane binding of SNAP-25 was not
affected following siRNA (small interfering RNA)-mediated
knockdown of syntaxin 1A in PC12 cells [31]. Furthermore,
down-regulation of the SM (Sec1/Munc18) protein Munc18
prevents plasma membrane delivery of syntaxin in PC12 cells,
whereas the trafficking of SNAP-25 is unaffected [32], and
SNAP-25 was reported to traffic to the plasma membrane in
Molecular Mechanisms in Exocytosis and Endocytosis
non-neuronal cells that do not express syntaxin 1 [33]. Thus,
it appears that initial membrane targeting and palmitoylation
of SNAP-25 may occur independently of syntaxin.
We recently reported results of mutational analyses aimed
at enhancing our understanding of how SNAP-25 is targeted
to membranes and palmitoylated [31]. In this analysis, we
focused on the minimal membrane targeting sequence (amino
acids 85–120) present in full-length SNAP-25. Alaninescanning mutagenesis identified two sets of mutants that
blocked membrane association of SNAP-25 in PC12 cells:
cysteine mutants and mutations at a highly conserved
Gln116 /Pro117 motif downstream of the CRD.
Previous work reported that when individual cysteine
residues were mutated to alanine, serine or glycine, membrane
binding of SNAP-25 was significantly reduced [26–28]. Our
results confirmed that cysteine-to-alanine mutations inhibit
membrane binding of SNAP-25; interestingly, however, we
found that when individual cysteine residues were replaced
with the more hydrophobic leucine, membrane binding was
largely preserved. These results are consistent with the idea
that the hydrophobicity of the CRD, which has the sequence
Phe-Cys-Gly-Leu-Cys-Val-Cys-Pro-Cys, is important for
the initial membrane interaction of SNAP-25. We proposed
that the CRD facilitates a weak membrane interaction of
unpalmitoylated SNAP-25, thus allowing transient access
to membrane-anchored DHHC proteins. In this model,
palmitoylation at cellular membranes increases the membrane
affinity of SNAP-25, promoting stable membrane binding
and facilitating subsequent sorting; a similar mechanism of
palmitoylation and membrane binding has been proposed
previously for Ras proteins and for the molecular chaperone
cysteine-string protein [34–37]. In support of this model,
the inefficient membrane binding of SNAP-25 observed in
HEK (human embryonic kidney) -293 cells was significantly
enhanced by co-expression of either DHHC3, DHHC7
or DHHC17, proteins previously shown to palmitoylate
SNAP-25 [38]. Together these data suggest that SNAP-25
interacts autonomously with cell membranes and requires
only sufficient cellular expression of partner DHHC proteins
to mediate stable membrane association, allowing subsequent
intracellular sorting.
What then is the role of Gln116 -Pro117 identified in the
alanine scanning screen in PC12 cells? Further analyses in
HEK-293 cells found that these residues are essential for
interaction with DHHC17, but not DHHC3. Thus, the loss
of membrane binding observed with these mutants in PC12
cells is consistent with the conclusion that DHHC17 plays
a major role in palmitoylation and membrane binding of
SNAP-25 in this cell line; indeed work in Drosophila reported
that SNAP-25 was mislocalized in DHHC17 null mutants
[39,40].
Regulation of SNAP-25 function by
palmitoylation
There is considerable interest in the idea that cellular
membranes are composed of distinct microdomains that
differ in their lipid and protein content [41]. In particular,
the co-clustering of proteins at specific regions of the
plasma membrane may be important in ensuring efficient
protein–protein interaction. Numerous studies have reported
that palmitoylated proteins associate with cholesterol-rich
‘raft’ domains in the plasma membrane [42,43]. Indeed,
biochemical and cell imaging approaches have suggested
that SNAP-25 may also be partly localized in cholesterolrich microdomains. Immunofluorescence analyses of plasma
membrane fragments revealed that SNAP-25 is clustered in
a cholesterol-dependent manner [44], and work from our
group found that SNAP-25 co-purifies with cholesteroldependent membrane fractions recovered from detergentsolubilized cells [45]. Chemical depalmitoylation of SNAP25 reduced the interaction with detergent-resistant fractions,
demonstrating that palmitoylation of SNAP-25 is important
for association with these structures [46].
Further analyses revealed that the number of palmitoylation sites in SNAP-25 correlated with the level of association
with cholesterol-rich fractions. Introducing an additional
cysteine into SNAP-25 (F84C mutant) significantly increased
association of the protein with cholesterol-rich membranes
in vitro [46]. By examining the ability of the F84C SNAP-25
mutant to rescue exocytosis in PC12 cells expressing the light
chain of botulinum neurotoxin E, it was concluded that the
cysteine mutant had a reduced exocytotic activity [47]. Based
on these observations, we proposed that the palmitoylation
status of SNAP-25 regulates its association with cholesterolrich membrane domains, which in turn regulates its ability
to support efficient exocytosis. This model poses many
questions, which we are currently addressing: (i) how many
cysteine residues are actually palmitoylated in SNAP-25?
(ii) Is there heterogeneity in the palmitoylation of cellular
SNAP-25? (iii) Is SNAP-25 palmitoylation dynamically
regulated? (iv) Are there DHHC proteins localized to the
plasma membrane that modify SNAP-25? (v) Is DHHC
and/or Apt1 (acyl protein thioesterase 1) activity modulated
in response to exocytotic stimulation? (vi) What do highresolution approaches [e.g. STED (stimulated emission
depletion) microscopy, electron microscopy] tell us about
SNAP-25 membrane microlocalization? Answering these
questions will facilitate a more comprehensive understanding
of how the regulation of SNAP-25 palmitoylation is coupled
to efficient exocytosis.
Funding
Our work is funded by the Medical Research Council U.K.
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Received 5 April 2009
doi:10.1042/BST0380163