Download SNAREs

SNAREs
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SNAREs
David K. Banfield and Wanjin Hong
Introduction
Eukaryotic cells contain multiple membrane-bound compartments between
which proteins and lipid molecules are continually shuttled via membranebound vesicular carriers. Despite the constant flux of proteins and lipid
through these compartments their functional and composition integrity is
maintained. While the molecular machinery involved in vesicle recognition
and fusion can often be transport-step/fusion-event specific, one group of
proteins – the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) play a common and central role in this process.
Transport-step-specific combinations of SNARE proteins, localized to the
vesicle and the target organelle, form complexes that facilitate the final
step leading to the fusion of vesicles with their cognate target organelles.
In general, the role of SNAREs appears to be conserved irrespective of their
location of function in the cell, and much of what has been established for
SNAREs in a particular trafficking pathway or organelle, is broadly applicable to SNAREs that function in the Golgi. Here we review Golgi SNAREs and
the role they play in membrane and protein trafficking in the Golgi
apparatus with, a particular emphasis on their functions in yeast and
human cells.
General features of Golgi SNAREs
The majority of SNARE proteins that function in the Golgi are type II integral
membrane proteins anchored in the lipid bilayer by virtue of their single
C-terminal transmembrane domain (TMD) see Fig. 1. The TMDs of SNAREs are
presumably crucial for the stable association of SNARE proteins with membranes, but also play a role in establishing the steady-state distribution of
SNAREs in the Golgi (Banfield et al. 1994; Rayner and Pelham 1997; Watson
and Pessin 2001). In addition, in vitro fusion assays have established that the
transmembrane domains of v-SNAREs (Xu et al. 2005) and of Qa-SNAREs
(Han et al. 2004) are important for the formation of the hemi-fusion intermediates that precede membrane fusion and vesicle content mixing with the
target compartment.
Adjacent to the TMD is a short stretch of amimo acids (10 in length)
referred to as the membrane proximal region (MPR). The amino acid sequence and length of this region is not evolutionarily conserved. The MPR
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D. K. Banfield and W. Hong
Figure 1. General features of Golgi SNAREs. Based on their structural and functional features
Golgi SNAREs are grouped into four categories. Category I is comprised of the Qa-SNAREs
and some Qb- and Qc-SNAREs. Category II is mainly comprised of the Qc-SNAREs. Category III
the R-SNARE Sec22p/Sec22b and category IV the R-SNARE Ykt6. The filled rectangles denote
the location of the membrane proximal region (MPR) see Table 1 and the text for further
details.
serves to separate the TMD from the SNARE-motif, which precedes it. The
length of the MPR appears to be important for the function of some SNAREs,
at least in vitro (McNew et al. 1999, 2000; Melia et al. 2002) however,
whether these observations extend to Golgi-localized SNAREs is presently
not known.
The SNARE-motif is comprised of a number of heptad-repeats, typically
7–8, which are responsible for the formation of the amphipathic helical
bundles characteristic of SNARE complexes. An evolutionarily conserved
amino acid residue that occupies a central position in the SNARE-motif, and
which contributes to the zero ionic layer of SNARE complexes, is the basis of a
SNARE protein family classification scheme (see below).
In addition to the so-called SNARE-motif or core domain, SNARE proteins
also contain N-terminal extensions (N-terminal domain (NTD)) of varying
length and folds (see Figs. 1, 2 and Table 1). Golgi Qa- and Qb-SNAREs
contain a domain which adopts a three-helix fold, termed an Habc domain,
whereas Golgi R-SNAREs, with the exception of VAMP4, contain a longin
fold. Golgi Qc-SNAREs typically contain short N-terminal regions that are
predicted to be unstructured, although the NTDs of the Qc-SNAREs Tlg1p
and Syntaxin 6, likely adopt an Habc fold. The longin fold, found in Golgi
R-SNAREs, is also present in several sub-units of the Golgi-localized vesicle
tethering complexes TRAPPI and TRAPPII (Kim et al. 2006) and is predicted to
be present in two sub-units of the Golgi vesicle coat complex – coatomer,
although the significance of this is not presently understood (Schlenker
et al. 2006).
In some cases the N-terminal domains of Golgi SNAREs are capable of
binding to their respective SNARE-motif, in which case the SNARE is said to
adopt a closed or folded-back conformation. Folded-back conformations are
known to occur for the R-SNAREs Ykt6p (Tochio et al. 2001) and Sec22p
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Figure 2. The N-terminal domain folds of Golgi SNAREs. (A) Cartoon representation of the
crystral structure of the human Vti1b Habc domain (Miller et al. 2007; pdb accession
number 2qyw) viewed from the side. The three helices of the domain are labelled from N –
to C a, b and c. (B) The same structure is in (A) but viewed from the N-terminus down the
three helix bundle. (C) The NMR-derived solution structure of the longin domain of yeast
Ykt6p (Tochio et al. 2001; pdb accession number 1h8m). The cartoons represent 180
rotations of one another.
(Mancias and Goldberg 2007). For, Ykt6p this conformation appears to be
important for the protein’s stability and likely plays a key role in the targeting
of this protein by regulating the association of the cytoplasmic prenylated
form of Ykt6 with membranes (Tochio et al. 2001; Fukasawa et al. 2004;
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Table 1. Yeast and human Golgi-resident SNAREs
Type
Human
Yeast
homolog
TMD
N-terminal
extention
N-terminal
fold
Qa
Syntaxin 5
Sed5p
Yes
Yes
Habc
Syntaxin 16
Tlg2p
Yes
Yes
Habc
(predicted)
Syntaxin 10
–
Yes
Yes
Habc
(predicted)
Syntaxin 11
–
No
Yes
Habc
(predicted)
GS27
(membrin,
GOS-27)
Bos1p
Yes
Yes
Habc
(predicted)
Vti1a
(Vti1-rp2)
Vti1p
Yes
Yes
Habc
GS28
(GOS-28)
Gos1p
Yes
Yes
Habc
(predicted)
Syntaxin 6
Tlg1p
Yes
Yes
Habc
(predicted)
Bet1
Bet1p
Yes
No
Random coil
GS15
Sft1p
Yes
No
Random coil
Sec22b
(ERS-24)
Sec22p
Yes
Yes
Longin
Ykt6
Ykt6p
No (prenyl)
Yes
Longin
VAMP4
–
Yes
Yes
Unstructured
SNAP-29
(GS32)
–
No
No
–
Qb
Qc
R
Qb þ Qc
Hasegawa et al. 2004). For Sec22p, a folded-back conformation appears to be
a prerequisite for this SNARE’s efficient incorporation into COPII-coated
vesicles (Liu et al. 2004; Mancias and Goldberg 2007). The N-terminal domain
of the Golgi syntaxins Sed5p (yeast)/Syn5p (mammals) are known to bind to
the Sec1–Munc18 (SM) family member protein Sly1 (Yamaguchi et al. 2002;
Dulubova et al. 2003; Arac et al. 2005). The association of Sly1p with Sed5p is
important for the specificity of Golgi SNARE complex assembly (Peng and
Gallwitz 2002) whereas the association of Sly1 with Syntaxin 5 is important for
ER–Golgi transport (Williams et al. 2004). A folded-back conformation of
Sed5p may be involved in the efficient packaging of this SNARE into COPIIcoated vesicles (Mossessova et al. 2003). Apparently, COPII preferentially
binds Sed5p when the protein is part of the Sed5p–Bos1p–Sec22p SNARE
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Figure 3. The topological arrangements of v- and t-SNAREs.
complex because t-SNARE assembly presumably removes the auto-inhibitory
contacts of the closed conformation of the protein, exposing its COPII sorting
signal (Mossessova et al. 2003).
SNARE protein classification and nomenclature
Functionally, SNAREs can be classified as either v-SNAREs or t-SNAREs.
v-SNAREs are localized to the transport vesicle, whereas t-SNAREs are
predominantly localized to the vesicle’s target compartment. Currently,
the generally accepted view is that a single membrane-anchored v-SNARE
forms a SNARE-complex in trans with a heterotrimeric t-SNARE. See Fig. 3
and Table 1 for a description of yeast and human Golgi-resident SNARE
proteins.
SNARE proteins can be further sub-divided based on their amino acid
sequence similarities and the position their homologs occupy in SNARE
complex macromolecular structures (Fasshauer et al. 1998; Bock et al.
2001). The macromolecular structures of the exocytic and endocytic SNARE
complexes revealed that they are parallel four-helical bundles (Sutton et al.
1998; Antonin et al. 2002). In the case of the endocytic SNARE complex, four
different SNARE proteins contribute a single helix each to the complex (Fig. 4),
this arrangement is also very likely to be the case for Golgi SNARE-complexes.
Thus, the syntaxin sub-family has been termed the Qa-SNAREs whereas
SNAREs that share the greatest degree of amino acid similarity with the Nterminal SNARE-motif of SNAP-25 (SNAP-25N) are referred to as Qb-SNAREs.
Similarly, SNAREs that are most similar to the C-terminal SNARE-motif of
SNAP-25 (SNAP-25C) are referred to as Qc-SNAREs. Members of the so-called
VAMP family are collectively referred to as R-SNAREs. The Qa-, Qb-, Qc- and
R-SNARE nomenclature refers to the presence of a highly evolutionarily
conserved amino acid residue at the so-called zero ionic layer of the
four-helical bundle – a glutamine for the Q-SNAREs and an arginine for the
R-SNAREs (Fig. 4). The general expectation is that members of each family
will occupy the equivalent position in their respective SNARE complexes as
the corresponding SNARE in the exocytic and endocytic SNARE complexes –
adopting a Qa:Qb:Qc:R stoichiometry often referred to as the 3Q:1R rule.
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Figure 4. The SNARE complex is a four-helical bundle. (A) An elongated side view cartoon
representation of the macromolecular structure of the endocytic SNARE complex (Zwilling et al.
2007). The position of the zero ionic layer is indicated by the arrow. Syntaxin 6 is represented by
the yellow helix, whereas Syntaxin 13, Vti1a and VAMP4 are represented by the blue, magenta
and green helices, respectively. (B) An enlarged and skewed side view of the SNARE complex
cartoon. The colour scheme used is as in (A). (C) A view down the helical bundle of a cartoon
representation of the SNARE complex in which the amino acid residue side-chains defining the
zero ionic layer are indicated. The colour scheme used is as in (A). (D) The zero ionic layer residues
of the endocytic SNARE complex. Note that while Vti1a is classified as a Q-SNARE, it contributes
an aspartic acid, rather than glutamine to the layer. Cartoons where generated using MacPyMOL
and the pdb file 2nps.
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Although the 3Q:1R rule is crucial for the formation of properly functioning
SNARE complexes, amino acid substitution experiments have shown that it is
not necessary, for example, that arginine be contributed by an R-SNARE per se
(Katz and Brennwald 2000; Graf et al. 2005). However, exceptions to these
general rules exist. For example, Sft1p and Bet1p, two yeast Golgi resident
Qc-SNAREs, contain an aspartic acid and a serine (respectively) at the zero
layer position, however the biological significance of this variability is
presently unknown.
The general mode of SNARE protein function
It is now generally accepted that the predominate function of SNARE proteins
is to act as facilitators of intra-cellular membrane fusion events within the
endomembrane system, through the formation of complexes between
SNAREs on vesicles and SNAREs on organellar membranes. This association
of SNAREs in trans is thought to be important in bringing the vesicle and
organellar membranes close enough together to facilitate membrane fusion.
In general, the SNARE complex that forms conforms to the 3Q:1R rule (Katz
and Brennwald 2000).
How are the individual SNARE proteins contributed to the SNARE complex? In vitro fusion assays with yeast Golgi SNAREs revealed that the
heterotrimeric t-SNARE is comprised of a heavy chain – a Qa-SNARE (a
syntaxin such as Sed5p or Syn5) and two different SNAREs which comprise
the two t-SNARE light chains. Thus the t-SNARE consists of one Qa-SNARE
together with either a Qb þ Qc, Qb þ R or Qc þ R pair defining the t-SNARE
light chains. Employing this scheme the v-SNARE would be contributed by the
remaining SNARE, i.e., either a Qb-, Qc- or R-SNARE, depending on the
composition of the t-SNARE complex (Fig. 2). The v-SNARE is often an RSNARE, but this may not be so for SNAREs in the Golgi, as liposome fusion
assays have established that the Qc-SNAREs, Bet1p and Sft1p, function as vSNAREs in this context (McNew et al. 2000; Parlati et al. 2002). However, an in
vitro transport employing yeast Golgi SNAREs revealed that, in addition to
Bet1p, the Qb-SNARE Bos1p and the R-SNARE Sec22p may also function as vSNAREs in transport between the ER and Golgi (Spang and Schekman 1998).
Although the composition of the t-SNARE complex and it’s respective vSNARE appears to be quite rigid in vitro (Parlati et al. 2000, 2002) it seems
likely that greater compositional flexibility exists in cells (Tsui and Banfield
2000; Tsui et al. 2001; Banfield 2001). In yeast, for example, some Golgi
SNAREs interact with Qa-SNAREs other than Sed5p and in so doing participate
in multiple transport steps (e.g. Vti1p, Ykt6p and Tlg1p). In addition, several
yeast Golgi SNAREs are not essential for yeast cell growth. Given the importance of SNAREs in membrane fusion these observations have been viewed
as being consistent with a functionally redundant role of SNAREs and reflexing a lack of selectivity in the composition of SNARE complexes. Despite
the apparent flexibility in SNARE pairing interactions in cells, adherence to
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Figure 5. The Golgi SNARE cycle.
the 3Q:1R rule remains important. This requirement has been successfully
exploited as a means to identify functionally interacting SNARE complexes
(Graf et al. 2005). While the importance of residues in the immediate vicinity
of the zero ionic layer have been documented (Stone et al. 1997; Graf et al.
2005) a comprehensive examination of the relative importance of other
regions of the SNARE-motif in Golgi SNARE function is lacking.
What directs the specificity of SNARE complex formation? Prior to SNARE
complex formation, the v-SNARE and t-SNAREs encounter each other in cis
(Fig. 5) and SNARE complex formation appears to proceed from the N- to Cterminus (Sorensen et al. 2006; Pobbati et al. 2006). The close opposition of
the v- and t-SNAREs is mediated by a variety of factors, so-called tethering
factors, which presumably function to ensure that only the correct SNAREs
form biologically meaningful trans-complexes. The formation of cognate,
fusogenic SNARE complexes between opposing membranes drives fusion.
Although cartoons, such as the one depicted in Fig. 5, often show the
formation of trans-SNARE complexes comprised of 1–2 complexes (for the
sake of simplicity) the average number of complexes participating in one
fusion reaction, based on studies on the neuronal exocytic SNARE complex, is
likely to be on the order of 3–8 (Han et al. 2004; Rickman et al. 2005;
Montecucco et al. 2005). The rosette-like structures that are observed to
form from the association of multiple SNARE complexes may be important for
mediating membrane fusion, perhaps via the transmembrane domain of
SNAREs (Han et al. 2004).
Following fusion of the vesicle with the Golgi, SNAREs remain bound to
one another in trans. Trans-SNARE complexes are dissociated through the
combined actions of a-SNAP/Sec17p and NSF/Sec18p after which, SNAREs are
free to be recycled and reused in another round of vesicular transport and
membrane fusion. Several Golgi SNAREs have been shown to cycle between
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the Golgi and the ER (Ballensiefen et al. 1998; Wooding and Pelham 1998;
Ossipov et al. 1999; Cosson et al. 2004), reflecting the requirement for SNAREs
in anterograde as well as retrograde vesicle-mediated transport (Spang and
Schekman 1998). In addition, apart from the requirement that cells reuse
SNARE proteins in successive rounds of transport, it seems likely that this
recycling process is intimately linked to the establishment and dynamic nature
of the Golgi apparatus itself (Cosson et al. 2004).
The specificity of SNARE complex formation
Table 2 lists SNARE complexes known to function in transport to the Golgi in
yeast and mammalian cells. The complexes that mediate such traffic in
mammalian cells have predominantly been identified by co-immune precipitation experiments. In contrast, in budding yeast this information has been
obtained from a variety of approaches, including co-immune precipitation,
genetic studies and in vitro mixing and fusion assays. An observation that has
Table 2. SNARE complexes known to function in transport to the Golgi
Mammals
Yeast
Complex
Transport step (s)
Complex
Transport step (s)
Syntaxin 5 (Qa)
GS28 (Qb)
GS15 (Qc)
Ykt6 (R)
Recyling endosome–TGN
Sed5p
Gos1p
Sft1p
Ykt6p
Intra-Golgi
Syntaxin 5
GS28
Bet1
Ykt6
ERGIC – Golgi
Sed5p
Bos1p
Bet1p
Sec22p
ER–Golgi
Syntaxin 5
GS27
Bet1
Sec22p
ER–ERGIC
Sed5p
Bos1p
Bet1p
Ykt6pa
ER–Golgi
Syntaxin 16
Vti1a
Syn6
VAMP4
Early endosome–TGN
Sed5p
Gos1p
Bet1pa
Ykt6p
Intra-Golgi
Syntaxin 16
Vti1a
Syntaxin 10
VAMP3
Late endosome–TGN
a
Assumed on the basis of over-expression experiments in sec22D (Liu and Barlowe
2004) and sft1D cells (Tsui et al. 2001).
SNAREs in bold, italicized font are encoded by non-essential genes. ERGIC (ER-Golgi
intermediate compartment).
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dogged the role of SNAREs in the specificity of membrane fusion events has
been the apparent lack of specificity among many SNARE–SNARE associations. This lack of specificity is particularly apparent in in vitro mixing
experiments using bacterially expressed mammalian as well as yeast SNARE
proteins (Yang et al. 1999; Fasshauser et al. 1999; Tsui and Banfield 2000).
Recent evidence suggests that non-cognate SNARE complexes form in cells,
but that cells have some, as yet to be identified mechanism, which selects only
the physiologically relevant complexes for use in membrane fusion reactions
(Bethani et al. 2007). Thus, the identification of SNARE–SNARE interactions by
co-immune precipitation may not be sufficient to assign particular SNAREs to
a functional complex (Bethani et al. 2007).
In in vitro fusion assays using the theoretical maximum tetrameirc combinations of SNAREs encoded by the yeast genome, only 9/275 were found to be
fusogenic (McNew et al. 2000; Parlati et al. 2000, 2002; Paumet et al. 2004).
Two of the nine fusogenic complexes contained the Golgi Qa-SNARE Sed5p:
Sed5p/Bos1p/Sec22p (t-SNARE) þ Bet1p (v-SNARE) and Sed5p/Gos1p/Ykt6p
(t-SNARE) þ Sft1p (v-SNARE), complexes which mediate fusion of vesicles
with the cis- and trans-Golgi, respectively. These two complexes correspond
to the mammalian Syntaxin 5-containing complexes: Syntaxin 5/membrin/
Sec22b (t-SNARE) þ Bet1 (v-SNARE) (Hay et al. 1998), although some studies
suggest that the v-SNARE may be Sec22b (Xu et al. 2000; Joglekar et al. 2003)
and to Syntaxin 5/GS28/Ykt6 (t-SNARE) þ GS15 (v-SNARE) (Xu et al. 2002).
These in vitro fusion assay data suggest that SNARE proteins encode the
necessary information to direct the formation of fusogenic SNARE complexes. In yeast, Sed5p is the only syntaxin required for transport through
the Golgi, however, Sec22p and Gos1p are encoded by non-essential genes.
Thus in cells lacking either the SEC22 or GOS1 genes (presumably) only a
single Sed5p-containing SNARE complex would remain. Additional Sed5pcontaining fusogenic SNARE complexes have been proposed to form in cells on
the basis of biochemical as well as genetic studies (Tsui and Banfield 2000;
Liu and Barlowe 2002) see Table 2. In cells, Ykt6p appears to be able to
substitute for Sec22p (Liu and Barlowe 2002). Similarly, under conditions when
the Qc-SNARE Bet1p is ectopically over-expressed, cells can survive without the
Qc-SNARE Sft1p (Tsui and Banfield 2000). Thus, with the exception of the QaSNARE, yeast Golgi Qb-, Qc- and R-SNAREs display varying degrees of presumptive functional redundancy. Whether these additional complexes constitute
functionally overlapping SNARE complexes, redundant complexes or complexes that form as a result of the absence of the cognate SNARE, requires
further investigation.
The observation that some Sed5p interacting SNAREs also form complexes
with other Qa-SNAREs functioning on other organelles suggests that a single
SNARE is likely to be insufficient to direct complex specificity. For example,
Vti1p (Lupashin et al. 1997; Von Mollard et al. 1997) and Ykt6p (Kweon et al.
2003) bind to multiple Qa-SNAREs and function in multiple transport pathways.
Combinatorial binding interactions may therefore influence the specificity of
SNAREs
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SNARE complex formation (Banfield 2001) in vivo. Finally, the extent to which
regions of the SNARE-motif, other than the zero ionic layer, contribute to the
specificity of Golgi SNARE complex assembly is an important issue which
remains to be addressed.
i-SNAREs
In vitro mixing studies with the soluble forms of Sed5p and its Golgi SNARE
binding partners revealed far more ternary complexes than were identified
on the basis of SNARE-mediated liposome fusion assays (Tsui et al. 2001;
McNew et al. 2000; Parlati et al. 2002). The presence of more than two fusion
competent Sed5p-containing SNARE complexes would help to reconcile
conceptual problems arising from the fact that one SNARE from each of
these complexes is non-essential – Sec22p and Gos1p, respectively (see Table
2) (McNew et al. 2000; Parlati et al. 2002). However, another explanation has
been proposed to account for the additional Sed5p-containing SNARE complexes observed in in vitro mixing studies with soluble SNAREs. Using their
well established SNARE-mediated liposome fusion assay Varlamov et al.
(2004) established that certain sub-units of the cis-Golgi SNARE complex
could inhibit fusion mediated by the trans-Golgi SNARE complex and vice
versa (Varlamov et al. 2004) – the authors termed these SNAREs as i-SNAREs.
While the opposing distribution of cis- and trans-Golgi SNAREs (Volchuk et al.
2004) could in principle account for the distribution of the fusogenic SNARE
complexes, the authors argue that i-SNAREs would enhance this phenomena –
essentially fine-tuning the specificity of membrane fusion events in the Golgi.
While this is a particularly attractive notion, the concept of i-SNAREs functioning in the Golgi still awaits in vivo validation.
Localization of SNAREs to the Golgi
In general SNAREs are predominantly localized to the vesicles and compartments on which they function and are absent from those on which they do
not. How are SNAREs localized to the Golgi? Accumulating evidence suggests
that both the transmembrane domain of SNAREs as well as signals in their
cytoplasmic domains accounts for their steady-state distributions. A requirement of the transmembrane domain in the localization of Golgi enzymes is
well established in mammalian cells. Such studies have led to the proposals
that (1) the length of the transmembrane is an important factor in Golgi
localization and that sorting/or localization is the result of Golgi membrane
bilayer thickness (Bretcher and Munro 1993) or (2) that the TMDs of Golgi
residents oligomerize and are prevented from exiting the Golgi (Nilsson et al.
1993). The transmembrane domains of the yeast SNAREs Sed5p and Sft1p
contribute to their Golgi localization (Banfield et al. 1994; Rayner and Pelham
1997) and TMD length has been shown to be important for the Golgi
localization of Syn5 in mammalian cells (Watson and Pessin 2001). However
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in yeast, TMD length alone is not sufficient to ensure exclusive Golgi localization of SNARE protein chimeras (Rayner and Pelham 1997).
The steady-state distribution of SNAREs proteins in the Golgi appears be
dynamic – relying on the active retrieval of SNAREs from distal cisternae and
their retrieval to earlier sub-compartments or to the ER, from where they
return to Golgi. In yeast, Sed5p (Wooding and Pelham 1998), Sec22p (Ballensiefen et al. 1998) and Bos1p (Ossipov et al. 1999) have been shown to cycle
between the Golgi and the ER. Although yeast Bet1p does not undergo such
cycling (Ossipov et al. 1999), in mammalian cells Bet1 and Sec22b have been
shown to continually cycle between the Golgi and the ER (Hay et al. 1998). The
recycling of Bet1 does not appear to require interactions with its cognate
SNAREs (Joglekar et al. 2003). In the case of yeast Sec22p and Bos1p, the
retrieval of these SNAREs from the Golgi requires a functional COPI coat
(Ballensiefen et al. 1998; Ossipov et al. 1999). An analysis of the lateral
distribution and vesicle incorporation of SNAREs in the mammalian Golgi
using electron microscopy is also consistent with a dynamic localization
mechanism (Cosson et al. 2005).
The Golgi SNARE Ykt6 does not contain a TMD, but rather is dually lipid
modified at its C-terminus (Fukasawa et al. 2004). The farnesylated form of
Ykt6 resides in the cytoplasm whereas farnesylated, palmitoylated Ykt6 is
found predominantly on Golgi membranes in non-neuronal mammalian cells
(Fukasawa et al. 2004; Hasegawa et al. 2004). How Ykt6 is targeted to Golgi
membranes remains to be determined.
SNAREs and COPI interactions
Several SNAREs have been shown cycle within or from the Golgi in a COPIdependent manner, data that implies an interaction between these SNAREs
and the coat protein complex. The COPI coat is comprised of the heptameric
complex termed coatomer, together with the GTPase Arf1. Arf1 cycles on and
off Golgi membranes as a function of its nucleotide-bound state. GTP-bound
Arf localizes to membranes whereas GDP-bound Arf is found in the cytoplasm. The nucleotide status on Arf1 is controlled through the action of its
exchange factor (Arf GEF) and its activating protein (Arf GAP). In vitro studies
using yeast Golgi SNAREs revealed that the Arf1p GAPs, Glo3p and Gsc1p, act
catalytically on Golgi SNAREs promoting a conformational change that
facilitates stoichiometric recruitment of Arf1p to SNAREs (Rein et al. 2002).
In agreement with these findings, studies in mammalian cells have identified
a motif on Arf that is required for the recruitment of Arf to Golgi membranes
by the Qb-SNARE membrin (Honda et al. 2005). Schindler and Spang (2007)
have shown that Gcs1p accelerates the formation of SNARE complexes in vitro
and suggested that Arf GAPs may function as folding chaperones for SNAREs.
Such mechanisms may function to couple SNARE recruitment to vesicle
formation in cells, thus ensuring that each vesicle carries sufficient SNAREs
capable of forming cognate SNARE complexes at its target compartment.
SNAREs
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The role of SNAREs in the morphological and functional
organization of the Golgi
Presumably recycling Golgi SNAREs is important for ensuring efficient vesiclemediated transport, as this would allow these proteins to be employed in
successive rounds of trafficking. Mathematical modeling using a minimal
system, in which the variables were restricted to cytoplasmic coat protein
complexes and SNAREs, was sufficient to generate stable non-identical
compartments (Heinrich and Rapoport 2005). A requirement of Heinrich and
Rapoport’s (2005) model was that each vesicle generating coat complex
preferentially bound and packaged a characteristic set of SNAREs. The lateral
distribution of Golgi SNAREs observed by Cosson et al. (2005) may similarly
reflect differential affinity of Golgi SNAREs for the COPI coat in vivo. Thus, the
affinity of vesicle coats, or their cargo sorting affiliated partners, may function
to promote and maintain the compositional integrity of Golgi cisternia through
their intrinsic ability to bind different SNAREs with varying affinities.
Regulators of Golgi SNARE function
The activity of SNAREs is regulated at various stages of their action including the assembly post-translational of the t-SNARE and the assembly of the
trans-SNARE complex (Fig. 5). A variety of proteins have been identified that
modulate the activity of SNAREs. In addition, post-translational modifications
such prenylation, palmitoylation and phosphorylation also influence the
activity and or localization of SNAREs.
NSF/Sec18p and a-SNAP/Sec17p
NSF and a-SNAP represent two co-operating core regulators of SNARE protein
activity. These proteins are responsible for the disassembly of cis-SNARE
complexes (Fig. 5), an activity that frees-up SNAREs to be used in successive
rounds of vesicle fusion. Three molecules of a-SNAP link the cis-SNARE
complex with a hexamer of the ATPase NSF/Sec18p and together this complex
is referred to as the 20 S complex (Hohl et al. 1998; Wimmer et al. 2001; Furst
et al. 2003; Brunger and DeLaBarre 2003). NSF contains two ATPase domains
termed D1 and D2. The D2 ATPase domain mediates the formation of the NSF
hexamer whereas the D1 ATPase domain effects the dissociation of the cisSNARE complex. The association of NSF with a-SNAP into the 20 S complex
stimulates the ATPase activity of NSF (Marz et al. 2003).
The Sec1/Munc-18 like (SM) proteins
Sec1/Munc-18 (SM) proteins bind directly to SNAREs and act downstream of
vesicle tethering events. Sly1p, the yeast SM protein which binds to Sed5p,
was identified because a mutant of this protein (sly1-20p) could suppress the
loss of the essential Rab/Ypt GTPase Ypt1p (Dascher et al. 1991). The association of Sly1p with Sed5p has been shown to enhance Sed5p-containing transSNARE complexes (Kosodo et al. 2002), and to be important for the specificity
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of Golgi SNARE complex assembly (Peng and Gallwitz 2002) whereas the
assocation of Sly1 with Syntaxin 5 has been demonstrated to be important for
ER–Golgi transport (Williams et al. 2004). However, the interaction between
Sed5p and Sly1p is dispensible for transport (Peng and Gallwitz 2004). Unlike
the exocytic and neuronal syntaxins and their requisite SM protein interactions, the interaction between Sly1p and Sed5p is mediated by a short
stretch of amino acids at the N-terminus of the protein which does not involve
either the Habc or SNARE-motif domains (Yamaguchi et al. 2002; Bracher and
Weissenhorn 2002; Dulubova et al. 2003) and thus this association does not
promote a folded-back conformation for Sed5p. A similar mode of binding is
also evident between the SM protein Vps45/Vps45p and the Qa-SNARE
Syntaxin 16/Tlg2p (Dulubova et al. 2002).
It is now apparent that Sly1p is capable of binding to non-syntaxin SNAREs
as well as to SNARE complexes and that this SNARE binding property of Sly1p
is important in the specificity of cognate SNARE complex formation (Peng and
Gallwitz 2002, 2004; Li et al. 2005).
The Golgins
The Golgins are a class of large coiled-coil containing Golgi localized proteins
with roles in tethering vesicles to the Golgi. Some golgins contain a single Cterminal transmembrane domain whereas other members of the family are
peripherally associated with Golgi membranes. The peripheral membrane
protein p115 has been shown to bind directly to SNAREs involved in ERGolgi intermediate compartment (ERGIC) as well as ERGIC-Golgi transport
(Allan et al. 2000; Shorter et al. 2002). A SNARE-related coiled-coil region of
p115 interacts with many Golgi SNAREs and such interactions likely promote
the formation of trans-SNARE complexes (Shorter et al. 2002). The functional
consequences of such interactions may be to ensure a direct connection
between the tethering machinery and SNAREs as well as to facilitate the
recruitment of p115 to membrane sites where unassembled SNAREs are
located (Brandon et al. 2006; Bentley et al. 2006). Mutational analysis of
p115 suggests that the SNARE-modulating activity of the protein is more
important than its tethering activity in maintaining the structure and function of the Golgi (Puthenveedu and Lindstedt 2004). Uso1p is the yeast
homologue of p115 (Sapperstein et al. 1995, 1996; Cao et al. 1998).
Other Golgins have also been shown to bind to Golgi SNAREs, including
GM130 which interacts directly with Syntaxin 5 (Diao et al. 2007) and GCC185,
which binds directly to Syntaxin 16 (Ganley et al. 2008). The emerging picture
of the role of Golgins is one in which these proteins sequester Rab GTPases
and Qa-SNAREs/syntaxins, and in so doing keep these two key proteins in
close proximity to the tether.
Conserved oligomeric Golgi (COG)
The COG complex is a member of the oligomeric vesicle tethering factor
family which comprise a structurally diverse group of peripheral membrane
SNAREs
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57
protein complexes involved in vesicle-organellar tethering events prior to
SNARE complex assembly (Oka and Krieger 2005; Stzul and Lupashin 2006).
The COG complex is involved in retrograde trafficking of Golgi-resident
proteins and extensive genetic interactions have been documented between
the COG complex and SNAREs. In addition the yeast and mammalian COG
complexes co-immune precipitate with Golgi SNAREs (Suvorova et al. 2002;
Zolov and Lupashin 2005) and the localization and stability of Golgi SNAREs is
altered in cells with defective COG complex components (Oka et al. 2004;
Fotso et al. 2005; Zolov and Luphasin 2005; Shestakova et al. 2007). More
recently, the yeast COG complex has been shown to interact with the SNAREmotif of Sed5p and to preferentially bind to Sed5p-containing SNARE complexes, leading the authors to propose that one function of the COG complex
is to stabilize intra-Golgi SNARE complexes (Shestakova et al. 2007).
GATE-16
GATE-16, a member of the ubiquitin-fold (UF) protein family, is localized to
the Golgi and interacts with NSF as well as GS28 (Sagiv et al. 2000). NSF/a-SNAP
facilitates the interaction of GATE-16 with GS28 in a manner that requires
ATP-binding but not ATP hydrolysis. Interestingly, GATE-16 binding prevents
GS28 from interacting with Syntaxin 5 and in so doing prevents the formation
of a functional t-SNARE (Muller et al. 2002). In addition, the yeast GATE-16
homologue, Aut7p, interacts with Bet1p, a Qc-SNARE involved in ER-to-Golgi
transport and shows genetic interactions with BET1 and the ER-Golgi RSNARE SEC22 (Legesse-Miller et al. 2000).
FIG
FIG (also known as CAL, PIST and GOPC) localizes to the TGN where it interacts
with the Qc-SNARE Syntaxin 6 (Charest et al. 2001). FIG contains two coiledcoil regions and a single PDZ domain and the protein’s interaction with
Syntaxin 6 is mediated via the second coiled-coil region and its C-terminal
flanking region. Although the biological significance of this interaction
remains to be determined, knock-out of the FIG gene in mice results in
selective ablation of acrosome formation during spermatogenesis (Yao et
al. 2002). The acrosome is believed to form from the Golgi apparatus and the
absence of FIG leads to fragmented acrosomal vesicles suggestive of a role for
FIG in the fusion of vesicles into the acrosome. Curiously, FIG also interacts
with Golgin-160 (Hicks and Machamer 2005).
Phosphorylation
Many SNAREs and their regulatory proteins are known to be phosphorylated
by a variety of kinases (Gerst 2003; Snyder et al. 2006). The yeast Golgi
Qa-SNARE Sed5p is a phosphoprotein and Weinberger et al. (2005) have
shown that amino acid substitutions to an evolutionarily conserved protein
kinase A phosphorylation site adjacent to the transmembrane domain of the
protein has dramatic effects on Golgi morphology. While expression of the
58
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D. K. Banfield and W. Hong
pseudophosphorylated form of Sed5p (Ser317Asp) results in the accumulation of ER membranes and vesicles, expression of the non-phosphorylatable
form of the protein (Ser317Ala) results in the accumulation of Golgi membranes reminiscent of the mammalian cell Golgi (Weinberger et al. 2005). The
Ser317Ala mutant also shows an increased affinity for the COPI coat, suggesting that phoshorylation status of Sed5p in cells may play a role in regulating
Golgi morphology.
Palmitoylation
Several SNAREs are known to be palmitoylated. Some of these SNAREs lack
transmembrane domains and are anchored to the membrane by their palmitate moieties, such is the case for SNAP-25, SNAP-23 and Syntaxin 11 (Vogel and
Roche 1999; Veit 2000; Prekeris et al. 2000). In contrast, the Golgi SNARE Ykt6p
is anchored by a combination of prenylation and palmitoylation (Fukasawa
et al. 2004). In addition, it is now apparent that several SNAREs bearing TMDs
are also palmitoylated (Valdez-Taubas and Pelham 2005) including the TGN/
endosomal Qc-SNARE, Tlg1p. The yeast DHCC-CDR family member Swf1p is
required for palmitoylation of Tlg1p and prevention of Tlg1p palmitoylation
results in its ubiquitination and transportation, via the multivesicular body, to
the vacuole for degradation. While palymitoyation of TMD-anchored SNAREs
does not appear to be essential for their function, this modification may play a
role in the membrane partitioning of these SNAREs and or in dissociation of
these modified proteins from other SNAREs, following fusion (Valdez-Taubas
and Pelham 2005). Based on amino acid sequence similarities with Tlg1p, the
mammalian Golgi resident SNAREs Syntaxin 6, Syntaxin 10 and VAMP4 may also
be substrates for palymitoylation (Valdez-Taubas and Pelham 2005).
Unlike the TMD-anchored SNAREs, which are modified via DHHC-CDR
palmitoyltransferases, Ykt6 appears to be capable of mediating its own palymitoylation (Veit 2004) via its longin fold (Dietrich et al. 2004). Ykt6 is found in
two pools in cells–a cytoplasmic pool and a membrane associated pool. Ykt6
lacks a proteinaceous membrane anchor but contains a prenylation consensus
sequence (a so-called CAAX box) at its C-terminus. The cytoplasmic pool of Ykt6
has been shown to farnesylated and the farnesylation of Ykt6 is prerequiste for
the subsequent palymitoylation and membrane association of the protein
(Fukasawa et al. 2004). Fukasawa et al. (2004) propose a cycle of membrane
association of Ykt6 in which the farnesylated fold-back conformation of Ykt6p
(mediated by an interaction between the longin domain and SNARE-motif,
Tochio et al. 2001) is targeted to membranes, whereupon the protein is
palymitoylated. This dual lipid modification may be required for stable membrane association of Ykt6 (Fukasawa et al. 2004).
Golgi SNAREs and apoptosis
During programmed cells death (apoptosis) the Golgi loses its cisternal
organization and is fragmented into clusters of tubulovesicular elements
SNAREs
*
59
(Lane et al. 2001) and the early secretory pathway is blocked (Lane et al.
2002). While this phenotype is associated with the proteolytic cleavage of
members of the Golgin family (Mancini et al. 2000; Chiu et al. 2002; Lane
et al. 2002) Lowe et al. (2004) have shown that Syntaxin 5 is cleaved by
caspase during apoptosis. Caspase-3 cleaves Syntaxin 5 at Asp 263, separating the SNARE-motif from the N-terminal Habc domain. Syntaxin 5
participates in several SNARE complexes in the Golgi, including partnerships with Bet1 (Qc-), membrin/GS27 (Qb-) and Sec22b (R-); Bet1 (Qc-),
GS28 (Qb-) and Ykt6 (R-) as well as with GS15 (Qc-), GS28 (Qb-) and Ykt6
(R-) (Hay et al. 1998; Zhang et al. 2001; Xu et al. 2002). Thus, cleavage of
Syntaxin 5 by caspase-3 is likely to affect several trafficking steps to and
within the Golgi.
Future perspectives
While much has been learned about the role of SNAREs in the Golgi many
important questions remain to be addressed. These include identification of
the sorting signals/motifs on SNAREs as well as the macromolecular details
governing interactions between SNAREs and the COPI vesicle generating
machinery. Establishing, whether like SNAREs and the ER vesicle coat COPII
(Morsomme et al. 2003), Golgi SNAREs influence the incorporation of particular cargo proteins into COPI-coated vesicles. Finally, a more detailed understanding of the mechanisms governing the steady-state localization of
SNAREs to the Golgi will make important contributions to our understanding
of how Golgi SNARE trafficking impacts the morphological and functional
organization of this fascinating organelle.
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