Download Golgins and GTPases, giving identity and structure to the Golgi

Biochimica et Biophysica Acta 1744 (2005) 383 – 395
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Review
Golgins and GTPases, giving identity and structure to the Golgi apparatus
Benjamin Short, Alexander Haas, Francis A. Barr*
Intracellular Protein Transport, Independent Junior Research Group, Max-Planck-Institute of Biochemistry, Martinsried, 82152, Germany
Received 3 November 2004; received in revised form 9 February 2005; accepted 9 February 2005
Available online 16 March 2005
Abstract
In this review we will focus on the recent advances in how coiled-coil proteins of the golgin family give identity and structure to the Golgi
apparatus in animal cells. A number of recent studies reveal a common theme for the targeting of golgins containing the ARL-binding GRIP
domain, and the related ARF-binding GRAB domain. Similarly, other golgins such as the vesicle tethering factor p115 and Bicaudal-D are
targeted by the Rab GTPases, Rab1 and Rab6, respectively. Together golgins and their regulatory GTPases form a complex network,
commonly known as the Golgi matrix, which organizes Golgi membranes and regulates membrane trafficking.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Golgin; Rab; ARF-like; ADP-ribosylation factor; GTPase; Golgi matrix
1. The Golgi apparatus, together apart
The Golgi apparatus is a series of flattened, cisternal
membrane structures forming the heart of the secretory
pathway. Strikingly, in most animal and plant cells these
cisternae, unlike the endoplasmic reticulum, do not fuse
into one continuous reticular structure, but are closely
juxtaposed in a stack-like organization. This raises the
obvious question of how cisternae are held closed together
in stacks, but do not fuse, while at the same time vesicles
that are tethered to these stacks can fuse with the Golgi
cisternae. In animal cells, these stacks are then arranged
end to end to form the dGolgi ribbonT [1]. The Golgi stack
is a polarized structure with a cis-face exchanging proteins
and lipids with the endoplasmic reticulum (ER), and a
trans-face communicating with the plasma membrane and
compartments of the endocytic pathway. Complex networks are found at each of these faces, referred to as the
cis-Golgi network (CGN) and the trans-Golgi network
(TGN) [2,3], respectively. As secretory material passes
* Corresponding author. Present address: Laboratory of Mammalian Cell
Biology and Development, The Rockefeller University, 1230 York Avenue
Box #300, New York, NY 10021, USA. Tel.: +49 89 8578 3135; fax: +49
89 8578 3102.
E-mail address: [email protected] (F.A. Barr).
0167-4889/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2005.02.001
through the Golgi in a cis to trans fashion, it becomes
post-translationally modified in a sequential order before
being sorted at the TGN for delivery to its final destination
within the cell. Despite this highly organized structure the
Golgi is a highly dynamic organelle. For example, it is
estimated that, in the exocrine pancreas, more secretory
material enters the Golgi from the ER every 5 min than
there is protein in the Golgi [1]. Nevertheless, the Golgi
manages to maintain its high degree of structural organization and, indeed, must do so in order to ensure that
secretory proteins are correctly modified and sorted. Many
Golgi-resident proteins are, therefore, involved in forming
a matrix that helps maintain the structure of this highly
dynamic organelle.
One of the first clues that a subset of Golgi proteins
played such a role came from the electron microscopy
studies, which identified proteinaceous bridges linking
adjacent cisternae together [4,5]. This was followed by the
demonstration that isolated Golgi membranes retaining their
stacked cisternal structure could be detergent-extracted to
leave a proteinaceous exoskeleton with the characteristic
organization of Golgi cisternae that is capable of binding
Golgi resident glycosyltransferases [6].
More recently, the components of the Golgi matrix have
begun to be characterized at a molecular level. An important
feature of many but not all of these components is that they
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the sera of patients with a variety of autoimmune disorders
[11]. All contain large regions of coiled-coil, a common
protein motif known to form an extended rod-like structure
[12]. Exactly why golgins are autoantigens is still unclear,
but autoantibodies to them may well arise as a result of the
breakdown of the Golgi during apoptosis and necrosis
leading to the generation of multiple antigenic fragments
from the repetitive coiled-coil domains [13].
More recently, several additional examples of coiled-coil
proteins localizing to the Golgi apparatus have been found.
Although no autoantibodies to these proteins have been
found so far, they have also been classified as golgins due to
their extensive coiled-coil structures [14,15]. Examples of
the different types of golgin and a summary of the
mechanisms they used to target to the Golgi are shown in
Fig. 1, and discussed later in this review.
Apart from Golgi localization and the presence of coiledcoil motifs, the only other common feature so far identified
in the golgin family is that many members interact with
small GTPases. This diversity amongst the golgins is
reflected in the wide range of functions that they carry
out. Indeed, the family is so diverse in both structure and
function that different organisms appear to have different
complements of golgins in their genome. For example,
behave differently to Golgi-resident enzymes when cells are
treated for short periods with reagents blocking ER-to-Golgi
transport (for example, with the fungal metabolite brefeldin
A (BFA) or dominant-negative forms of the COPII vesicle
coat component Sar1p). Under these conditions, many Golgi
resident proteins relocalize to the ER [7], yet some Golgilike structures containing components of the Golgi matrix
remain distinct from the ER [8]. Principally, these are
members of the golgin family of Golgi-localized coiled-coil
proteins and the GRASP family of Golgi stacking proteins.
As we shall see, it is a network of interactions between these
proteins, and their regulation by small GTPases of the Rab,
ADP-ribosylation factor (ARF), and ARF-like (ARL)
families that is responsible for the dynamic organization
of the Golgi apparatus. This review will focus on the
molecular characteristics of the golgin family of proteins
and their regulation by small GTPases, other aspects such as
Golgi is biogenesis have been reviewed elsewhere [9,10].
2. Golgins: a diverse family of coiled-coil proteins
The golgins were originally identified as a family of
Golgi-localized autoantigens, using antibodies derived from
c) Rab-recruited golgins
b) Adaptor-associated
p115 (Rab1)
BicaudalD1/D2 (Rab6)
TMF (Rab6)
golgins
a) Transmembrane
golgins
-
d) ARL-recruited
GRIP-domain golgins
Golgin-245 (ARL1)
Imh1p (Arl1p)
Golgin-97 (ARL1)
e) ARF-recruited
GRAB-domain golgins
GMAP-210 (ARF1?)
Rud3p (Arf1p)
GRASP65-GM130
GRASP55-Golgin45
+
GTP
GTP
Giantin
Golgin-84
CASP
Cytoplasm
Lumen
Coiled-coil
ARL-GTPase
ARF-GTPase
Interaction domain Rab-GTPase
Fig. 1. Golgins associate with Golgi membranes in a variety of ways. Golgins are a diverse family of Golgi-localized coiled-coil proteins and interact with
Golgi membranes in a variety of different ways. (a) Some have a transmembrane domain near their C-terminus while others are peripheral membrane proteins.
(b) Peripheral membrane golgins may associate with the membrane by an interaction with an adaptor protein of the GRASP family. The GRASP proteins
themselves target via an N-terminal myristoyl group. Other golgins are recruited to Golgi membranes in a nucleotide-dependent manner by small GTPases of
the (c) Rab, (d) ARL, and (e) ARF families. Rabs target to membranes via C-terminal geranylgeranyl modifications, while ARFs and most but not all ARLs
target by N-terminal myristoylation. The mode of interaction between golgins and Rabs is unknown but ARL1 binds to the well-conserved GRIP domain that is
found in a subset of golgins. A dimeric GRIP domain binds two ARL1 molecules, an interaction for which an absolutely conserved tyrosine residue is essential.
By analogy with the GRIP domain, a dimeric GRAB domain is likely to bind two ARF1 molecules. A conserved bulky hydrophobic residue, typically leucine,
rather than tyrosine is needed for this interaction. See the text for more details and references. Diagram is not to scale.
B. Short et al. / Biochimica et Biophysica Acta 1744 (2005) 383–395
while p115 can be found in all eukaryotes and GRASPs are
found in all eukaryotes except plants, golgin-45 is present
only in vertebrates, and GM130 is present only in mammals.
This doesn’t mean that they are unimportant. Rather, it
probably indicates that the Golgi is structurally adapted in
different species to meet the specific needs of different cell
types. For example, the recent finding that GM130 is not
required for mammalian cell viability at 34.5 8C, but is
essential at higher temperatures could indicate that this
golgin has arisen in response to the requirements of growth
at higher body temperatures. It is interesting to speculate
that organisms which typically live at an ambient temperature of between 4 and 20 8C such as nematodes and yeasts
may therefore get away with a simpler complement of
golgins, and thus not require a GM130 homologue. The
modular structure of golgins, with central coiled-coil regions
and functional domains at the N- and C-termini, is thus ideal
for such wide adaptations. A similar situation exists with the
cognate GTPases regulating particular golgins, Rab1 like
p115 is found in all eukaryotes, while Rab2, like its partner
golgin-45, is also only present in vertebrates.
One of the best-characterized functions of golgins is their
role in membrane tethering events, as exemplified by the cisGolgi golgins, p115, GM130 and giantin (Fig. 2a–c). The
tethering factor p115 was originally identified using the
Rothman intra-Golgi transport assay [16]. It is a peripheral
membrane protein mainly localized to the vesicular-tubular
clusters of ER to Golgi transport intermediates (VTCs) and
the cis-Golgi [16,17] and was shown by rotary shadowing to
be a homodimer with an N-terminal globular head domain
and a C-terminal coiled-coil domain with a length of 45 nm
[18]. A short acidic patch is located at the extreme Cterminus of the protein. The budding yeast homologue of
p115, Uso1p, is similar in structure but is much larger in size
(approximately 150 nm) [19].
Studies on Uso1p provided insight into its function as a
tethering factor. Along with the small GTPase Ypt1p (yeast
Rab1), it is required for the docking of ER-derived COPIIcoated vesicles with Golgi membranes in an in vitro assay
[20,21]. Recent studies on mammalian p115 have suggested
a direct interaction with SNARE proteins that catalyze
membrane fusion. Not only could p115 bind to certain early
Golgi SNARE proteins but an in vitro assay showed that it
could actually stimulate SNARE complex assembly, thereby
linking initial membrane tethering to fusion [22]. Additionally, it has been shown that p115 is important for COPI, as
well as COPII, vesicle docking [23]. To accomplish these
functions, p115 interacts with two additional golgins at the
cis-Golgi: GM130 and giantin.
GM130 was originally identified (as golgin-95) using
antisera from an autoimmune disease patient and, subsequently, by raising antisera against solubilised Golgi matrix
[24,25]. Localized to the cis-Golgi, GM130 is predicted to
contain extensive regions of coiled-coil forming a homodimer, and has a basic domain of 75 amino acids at its Nterminus, which binds to the C-terminal acidic patch of p115
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[26]. GM130 is targeted to cis-Golgi membranes by its tight
binding to GRASP65, an interaction that requires a motif at
the extreme C-terminus of GM130 [27].
Giantin, on the other hand, is an integral membrane
protein of 400 kDa, with a C-terminal transmembrane
domain and a very large cytoplasmic domain containing
extensive regions predicted to form coiled-coil [28]. It is
localized to the edges of the Golgi stack and on COPI
vesicles and also binds p115 via its N-terminus [23].
How do p115, GM130, and giantin act together to
mediate docking events at the cis-Golgi? The presence of
giantin in COPI vesicles and GM130 on Golgi membranes,
and the ability of both proteins to bind p115, suggest a
model in which COPI vesicles are linked to their target
membranes by a giantin–p115–GM130 ternary complex
[23]—although this wouldn’t apply to ER-targeted COPI
vesicles. Such a model is supported by an in vitro assay in
which the docking of COPI vesicles with Golgi membranes
is inhibited if the vesicles are pre-incubated with anti-giantin
antibody but is unaffected if the Golgi membranes are preincubated with anti-giantin. Anti-GM130 antibodies, on the
other hand, only inhibit the assay if they are pre-incubated
with Golgi membranes but have no effect when preincubated with COPI vesicles. The same assay shows a
dependence on p115 since addition of increasing amounts of
the protein increases the rate of vesicle docking [23].
The idea of a giantin–p115–GM130 ternary complex is
contradicted however by evidence that GM130 and giantin
compete for the same binding site on p115 [29]. Also, studies
on trafficking between the ER and the Golgi suggest that
the three proteins act at kinetically distinct stages since antip115 antibodies inhibit transport at the VTC stage while antiGM130 and anti-giantin inhibition occurs at the Golgi—with
giantin inhibition occurring later than GM130 inhibition
[30].
Since p115, GM130, and giantin are implicated in such a
variety of different processes (including both retrograde and
anterograde transport steps as well as cisternal stacking), it
is possible that they act in discrete ways during different
events. The effects of antibody inhibition may vary depending on precisely which process is being measured in a
particular assay. In any case, the interactions of these
golgins, both with each other and with other proteins, are
clearly important for vesicle tethering events at the cisGolgi. Additionally, these three golgins have also been
implicated in the stacking of Golgi cisternae when the Golgi
reforms following mitosis, a case of membrane tethering
without subsequent fusion [31]. The complex formed by
p115, GM130, and giantin may therefore be the proteinaceous material seen linking Golgi cisternae as well as the
dstringsT seen between tethered vesicles and their target
membranes in electron micrographs [5,32].
Recently, the question of whether GM130 is essential for
maintaining Golgi structure has been investigated in a
conditional lethal mutant cell line, ldlG, which lacks
detectable levels of GM130 protein [33]. The fact that, at
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a)
d)
COPII
Vesicle
Recycling Vesicle
p150
Rab1
glued
Dynein
COPII
Vesicle
b)
GTP
GTP
Bicaudal D1/2
p115
Arp2/3
-
cis-Golgi cisternae
Microtubule
+
GRASP65
GM130
+
d
-
Golgi apparatus
p115
c
COPI
b
VTCs
Rab1
a
Vesicle/VTC
COPII
cis-Golgi cisternae
c)
Endoplasmic
reticulum
GRASP65
+
p115
-
Giantin
Rab1
COPI Vesicle
GM130
Fig. 2. Membrane tethering systems at the Golgi. The role of the p115 the tethering system in ER to Golgi, and intra-Golgi traffic. (a) COPII vesicles formed at
ER exit sites are clustered together by the action of the golgin tethering factor p115. Rab1 recruits p115 on to these vesicles by interactions with the globular
head and neck regions of p115. These tethered vesicles then fuse in an NSF and SNARE dependent mechanism to form VTCs. (b) VTCs then dock with the
cis-Golgi, this event is thought to be facilitated by an interaction between the Rab1–p115 complex on the VTCs and GM130–GRASP65 on the cis-Golgi. An
acidic C-terminal patch on p115 directly binds to a complementary basic patch at the N-terminus of GM130. (c) p115 and GM130 may also play additional
roles in docking COPI vesicles to the cis-Golgi. This event is mediated via an interaction of the transmembrane golgin giantin on COPI vesicles with GM130 at
the cis-Golgi. p115 can catalyse this event, although the mechanism is unclear. (d) Recruitment of the dynein–dynactin complex to vesicles by Rab6. Activated
Rab6 recruits the dynein motor to Golgi membranes and transports vesicles via multiple interactions with the dynactin complex and the golgin Bicaudal-D.
Bicaudal-D acts as an attachment factor for the dynactin complex, and binds Rab6 via its C-terminal domain. In addition Rab6 interacts with the dynactin
subunit p150glued. We speculate that dynactin at microtubule tips can capture Rab6 bearing Golgi vesicles, and that dynein then transports these membranes
towards the microtubule minus end. See the text for more details and references. Diagram is not to scale.
higher temperatures, these cells have a disrupted Golgi,
show defects in secretion, and eventually die demonstrates
that, indeed, GM130 is essential for normal Golgi structure
and function. On the other hand, ldlG cells do manage to
maintain a normal Golgi at the lower temperature of 34.5
8C, indicating that other proteins are able to compensate for
the absence of GM130 under these conditions [33].
While GM130 and its interaction partners may control
the formation of the Golgi stack, another golgin, golgin-84,
appears to regulate Golgi structure at a different level—
namely the lateral organization of stacks into the Golgi
ribbon [34,35]. Primarily localized to CGN, golgin-84 is an
integral membrane protein that interacts with the GTPase
Rab1 [34,35]. The transmembrane region of golgin-84 is
similar to that of giantin and another golgin, CASP [36].
Both the overexpression and depletion of the protein
resulted in the fragmentation of the Golgi ribbon [34]. On
the other hand, the addition of the cytoplasmic Rab1-
B. Short et al. / Biochimica et Biophysica Acta 1744 (2005) 383–395
binding domain of golgin-84 to an in vitro Golgi reassembly assay promoted the lateral growth of the Golgi
cisternae [35]. Depletion also resulted in an increase in the
amount of ER membrane relative to the Golgi membrane,
and a two-fold reduction in transport from the ER to the cell
surface [34]. This latter result implies that p115/GM130/
giantin are not the only golgins regulating ER-to-Golgi
transport. Diao et al. [34] proposed that while p115,
GM130, and giantin have a direct role in ER-to-Golgi
transport, golgin-84 regulates formation of the Golgi ribbon.
The depletion of golgin-84 might then inhibit protein
transport if trafficking occurs less efficiently through
individual Golgi stacks than through a fully formed ribbon.
Other golgins perform different roles that contribute to
efficient transport at the Golgi. The two related golgins
Bicaudal-D1 and Bicaudal-D2 have recently been shown to
localize to the trans-Golgi through their interaction with
Rab6 [37,38]. In addition, the Bicaudal-D proteins interact
with dynactin, an adaptor for the microtubule-based, minusend directed motor protein dynein [39]. Given that the Golgi
apparatus is positioned in a dynein-dependent manner in the
pericentriolar region of animal cells [40,41], and that Rab6
controls microtubule-dependent retrograde transport from
endosomes to the trans-Golgi and from the Golgi to ER
[42–44], Rab6 and Bicaudal-D probably combine to tether
transport vesicles directed toward the Golgi to the cytoskeleton and collect them in the pericentriolar region. Thus,
rather than tethering membranes together, Bicaudal-D is a
golgin that tethers vesicles and possibly some Golgi
membranes to the microtubule cytoskeleton.
3. GRASP proteins stack the Golgi together
A second important component of the Golgi matrix is a
family of proteins identified using a functional assay for the
post-mitotic reassembly of Golgi stacks. The inhibition of
this assay by the alkylation agent NEM allowed the
identification of two GRASPs (for Golgi Reassembly
Stacking Proteins), with molecular weights of 55 and 65
kDa [45,46]. GRASP65 is localized to the cis-Golgi while
GRASP55 is predominantly at the medial Golgi. Both
proteins are peripheral membrane proteins on the cytosolic
face of Golgi cisternae, and are associated with the
membrane by N-terminal myristoylation (although how
the proteins are targeted specifically to Golgi membranes
remains unclear). Both proteins are phosphorylated during
mitosis, which may be significant for the mitotic disassembly of the Golgi apparatus. Their role in the reassembly of
the Golgi following mitosis was made clear by experiments
in which blocking antibodies specific for either of the
GRASPs, or non-myristoylated (i.e. non-membrane targeting) versions of the proteins could inhibit cisternal stacking
in an in vitro assay [45,46]. More recently, the microinjection of anti-GRASP65 antibody into semi-permeabilised cells was shown to impair the breakdown of the Golgi
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apparatus induced by mitotic cytosol and, indeed, microinjection into intact cells prevented mitotic entry, suggesting
a dGolgi checkpointT in which fragmentation of the Golgi is
required for the cells to enter mitosis [47]. This effect is
mediated through the polo-like kinase (Plk1; see [48] for a
review of Plk1 function), which docks to the C-terminal
domain of GRASP65 once it has been phosphorylated by
Cdk1–cyclin B [49]. At present it is unknown what the
downstream targets of Plk1 are in this context.
One important way in which the GRASP proteins
regulate Golgi structure is likely to be their interactions
with members of the golgin family. GRASP65 is an adaptor
for GM130, and this interaction is necessary for both
proteins to localize to Golgi membranes [27]. Similarly,
GRASP55 is a specific binding partner of the medial-Golgi
localized golgin-45. Disruption of this complex by the
depletion of golgin-45 results in the dispersal of the Golgi
apparatus and inhibition of protein transport [50]. In
addition, GRASP65 has been shown to interact with
members of the p24 family of cargo receptors involved in
recycling between the ER and Golgi [51] and GRASP55
binds to Transforming Growth Factor-a (TGF-a ), an
interaction important for TGF-a’s expression at the cell
surface [52]. Thus, GRASP proteins and their binding
partners are important for both Golgi structure and function,
providing a link between the Golgi matrix and certain
integral membrane cargo proteins.
4. Dynamics of the Golgi matrix
As discussed above, the Golgi matrix must be a highly
dynamic structure in order to facilitate the constant
remodelling of the Golgi apparatus as it fulfils its function.
Components of the matrix interact with Golgi membranes in
a variety of ways. Some golgins are integral membrane
proteins but many are peripherally associated, potentially
allowing their rapid recycling from one part of the Golgi
membrane to another via the cytosol. The variety of ways in
which golgins associate with Golgi membranes is illustrated
in Fig. 1.
The GM130–GRASP65 complex associates with high
affinity to Golgi membranes through the myristoyl group of
GRASP65 [27,45]. However, photobleaching experiments
using green fluorescent protein (GFP)-tagged proteins
suggests that the complex undergoes rapid recycling
between the Golgi membranes and the cytosol [7]. It is
unclear what the receptor for the GM130–GRASP65
complex is on Golgi membranes and how the association
and disassociation with this receptor is regulated. In
principal, the myristoyl group of GRASP65 could associate
with any membrane and yet newly-synthesized GRASP65
associates directly with the cis-Golgi and not with any other
membranes such as the ER [53]. One possibility is that
GRASP65 recognizes Golgi membranes by binding to the
cytoplasmic domains of transmembrane proteins such as the
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B. Short et al. / Biochimica et Biophysica Acta 1744 (2005) 383–395
p24 family [51]. GRASP65 is able to distinguish between
the different oligomeric states of the p24 proteins and might
therefore be able to determine its specific localization to
Golgi membranes in vivo by only binding the Golgilocalized pool of p24 proteins, which are thought to exist in
a higher oligomeric state than the ER-localized pool [51,54].
The localization of other peripheral membrane golgins to
Golgi membranes is dependent upon their interactions with
small GTPases such as the Rab proteins (see below). These
GTPases themselves cycle between cytosolic and membrane-bound states in a nucleotide-dependent manner and
are subject to regulation by accessory factors such as
specific GTPase Activating Proteins (GAPs) and Guanine
nucleotide Exchange Factors (GEFs). Membrane-associated, GTP-bound (active) Rabs are then able to recruit
golgins to the membrane.
Integral membrane golgins, on the other hand, may cycle
between the Golgi and the ER. There are three type-II
transmembrane golgins in animal cells, namely giantin,
golgin-84, and CASP (CCAAT-displacement proteins alternatively spliced product) [28,36,55,56]. A fourth golgin,
golgin-67, has been proposed to be a transmembrane protein
[56], but there are good reasons for thinking that this is not
the case since the Golgi targeting sequence does not fulfil
the criteria expected for a true transmembrane domain [15].
The transmembrane domains of giantin, golgin-84, and
CASP show a high degree of sequence similarity. In
particular, key tyrosine and histidine residues are completely
conserved. When the conserved tyrosine residue is mutated
to leucine, CASP no longer localizes to the Golgi and
accumulates in the ER instead [36]. Whether the mutant
protein fails to exit the ER or is not retained in the Golgi is
unknown. If the latter case is true, it could mean that wild
type CASP cycles between the ER and Golgi. This is
supported by the observation that CASP redistributes to the
ER upon BFA treatment [36].
Golgin-84 is also likely to cycle between these compartments since BFA treatment also causes its redistribution to
the ER [34]. Strictly speaking, this means that golgin-84 and
CASP are not part of the Golgi matrix according to the
definition that matrix components remain separate from the
ER upon BFA or dominant negative Sar1p treatment.
Nevertheless, these two golgins are clearly important
structural components of the Golgi and have several other
features in common with bona fide matrix proteins.
Thus, it seems that all structural components of the Golgi
recycle to either the ER or the cytoplasm. This leads to the
question of the extent to which the Golgi apparatus can be
thought of as an independent organelle with its own identity
[9]. An analysis of the effects of BFA and dominant
negative Sar1p suggests that the Golgi depends, at least in
part, on the ER for its existence [7]. Indeed, confocal video
microscopy studies of the yeast Pichia pastoris have
demonstrated, at least in this organism, that the Golgi forms
de novo at sites intimately linked with the transitional ER,
where COPII coated vesicles form to deliver material to the
Golgi [57]. Golgins may play a key role in maintaining this
link, since the depletion of Drosophila melanogaster p115
results in the disorganization of both the Golgi stack and
transitional ER sites [58].
However, since some matrix components like GM130
and GRASP65 cannot be induced to relocalize to the ER by
treatment with BFA and dominant negative Sar1p [8], the
Golgi does have a separate identity from the ER as defined
by these proteins. There are some caveats to this, as under
conditions where ER exit is blocked by the drug H89 in the
presence of BFA even Golgi matrix proteins appear to be
redistributed to the ER [59]. Recent evidence suggests that it
may only be under a restricted set of experimental
conditions that components of the Golgi redistribute to the
ER [60]. Pecot and Malhotra used a protein-trapping
approach to demonstrate that, contrary to some theories of
Golgi dynamics, ER-resident proteins and Golgi-resident
enzymes do indeed colocalize upon BFA treatment but do
not come into contact with one another during mitosis,
suggesting that the Golgi remains a distinct organelle
throughout the mammalian cell cycle [60].
Nevertheless, considerable changes do occur to Golgi
structure during mitosis [1]. We will now briefly consider
how these changes are regulated by kinases and how
morphologically similar changes during apoptosis are
regulated by caspases.
5. Changes in Golgi structure during mitosis and
apoptosis
At the onset of mitosis in animal cells, the Golgi
apparatus fragments into numerous tubulo-vesicular structures known as mitotic Golgi fragments (MGFs), which
contain various structural components of the Golgi such as
GM130 and GRASP65. During cytokinesis, the MGFs
become partitioned between the two daughter cells and then
coalesce to reform a functional, perinuclear Golgi with a
typical ribbon-like appearance [61]. These large-scale
changes in Golgi structure are accompanied by changes in
the phosphorylation states of many resident Golgi proteins
through the activities of many mitotically regulated kinases
and phosphatases. A full review of these events is beyond
the scope of this article; for a more detailed review see
Preisinger and Barr (2001) [62]. Nevertheless, a few
examples will serve to illustrate the principles by which
Golgi structure is regulated during mitosis.
The paradigm for the regulation of the Golgi structure by
phosphorylation is the interaction between p115 and
GM130, which has been reported to contribute to COPIand COPII-coated vesicle docking and cisternal stacking
[20,21,23,31]. This interaction is disrupted at the onset of
mitosis by the phosphorylation of GM130 on serine25 by
Cdk1 [63]. This is thought to be of significance for the
mitotic breakdown of the Golgi apparatus due to the
continued budding of transport vesicles while p115–
B. Short et al. / Biochimica et Biophysica Acta 1744 (2005) 383–395
GM130 mediated vesicle tethering is inhibited. However,
this phosphorylation event alone is unlikely to cause a
complete disruption of the Golgi and, accordingly, many
other mitotic kinases and Golgi substrates have been
identified.
In addition to phosphorylating GM130, Cdk1–cyclin B
also phosphorylates Rab1, golgin-84, and GRASP65
[34,49,64,65]. Indeed, GRASP65 is the major phosphoprotein at the Golgi in mitosis and is also phosphorylated by
polo-like kinase (Plk1) [65]. The mitotic phosphorylation of
GRASP65 appears to alter its ability to self-oligomerise, and
thus may contribute to the unstacking of Golgi cisternae at
the onset of mitosis [66,67]. GRASP55, on the other hand,
has been shown to be an in vitro substrate for the mitogen
activated protein (MAP) kinase ERK2, which possibly acts
downstream of MEK1 [68]. This fits with reports that MEK1
(and its upstream activator Raf1) is required for the fragmentation of the Golgi during prophase [69–71]. The relative
importance of some of these phosphorylations is disputed
however [63,72] and a full picture of the events leading to the
mitotic breakdown of the Golgi is still to emerge.
The Golgi also fragments during apoptosis although the
proteolytic cleavage of Golgi structural proteins, rather than
phosphorylation, is the key regulatory mechanism. Again,
the reader is referred to a more detailed review for a fuller
discussion of this topic [73].
The reason for the apoptotic breakdown of the Golgi is
unclear but it may be necessary in order to prevent
unregulated protein transport to the cell surface during cell
death. Indeed, the cleavage of giantin and the SNARE
protein syntaxin5 by caspase-3 is accompanied by a block in
ER to Golgi transport [74].
Golgin-160 is cleaved by caspase-2 and GRASP65 is a
target of caspase-3 during apoptosis [75,76]. The expression
of cleavage-resistant forms of these proteins delays apoptotic Golgi fragmentation, demonstrating the importance of
these proteins for Golgi structure. Another golgin, p115, is a
target for caspases 3 and 8 and, again, Golgi fragmentation
was slowed by the expression of cleavage resistant p115
[77]. Interestingly, the C-terminal cleavage product of p115
appears to translocate into the nucleus and have a proapoptotic effect, indicating a signalling function for the
Golgi during apoptosis [77].
Thus, large-scale alterations in Golgi morphology during
mitosis and apoptosis are regulated by the actions,
respectively, of kinases and caspases on Golgi structural
components. Under normal, interphase conditions however,
small GTPases are more important for the dynamic
regulation of Golgi structure and function.
6. Regulation by small GTPases
The Rab family of small, Ras-related GTPases consists
of over 60 members in the human genome [78]. They have
been shown to be involved in almost all stages of vesicle
389
transport, from vesicle budding and motility to tethering and
fusion [79]. They accomplish all of these through the
nucleotide-dependent recruitment of a variety of effector
proteins.
Different members of the Rab family are able to target to
specific membrane compartments within the cell, and
several Rab proteins localize to specific sub-domains of
the Golgi. Precisely how this targeting is achieved is unclear
but, once a Rab protein is localized and activated, it is able
to recruit its effectors either from the cytosol or from within
the two-dimensional plane of the membrane. These effectors
can then be organized and a series of protein–protein and
protein–lipid interactions set up to establish a specific
membrane sub-domain. At the Golgi, these effectors include
numerous members of the golgin family [14].
Thus, through their ability to cycle between the
cytoplasm and membranes, Rab proteins play a key role
in specifying the identities of particular membrane domains
[80,81]. The constant flux of protein and lipid through the
Golgi in both anterograde and retrograde directions would
lead to a rapid mixing of all the sub-domains of the Golgi if
Rab proteins were unable to dynamically regulate the
golgins and allow the assembly and disassembly of functional membrane domains. The participation of Rab proteins
at all stages of vesicle transport, from cargo selection to
docking and fusion, suggests that vesicles too may be
looked on as Rab-organized membrane sub-domains, which,
during their lifetime, are able to physically separate from
one membrane and join with another.
Some golgins are specifically recruited to the Golgi by
their interaction with their cognate Rab protein. For
example, Rab6 recruits both Bicaudal-D (and its associated
dynactin subunits) as well as the golgin TMF to Golgi
membranes [37,38,82]. Similarly, p115 is recruited to
membranes by Rab1 [83], although its interaction with
GM130 probably also plays a role in its membrane
association at the cis-Golgi [26]. GM130 itself relies on
its interaction with GRASP65 for its localization to Golgi
membranes [27]. Nevertheless, GM130 interacts with three
different Golgi-localized Rab proteins, Rabs 1, 2, and 33b
[50,84–86]. Likewise, the GRASP55-binding golgin, golgin-45, is a Rab2 effector [50], while the integral membrane
golgin, golgin-84, interacts with Rab1 [34,35]. The function
of the interaction between Rabs and golgins already
associated with Golgi membranes may be to organize the
golgins within the plane of the membrane or Rab binding
may induce conformational changes in the golgin resulting
in alterations in the golgins activity. Rab binding might
similarly alter the affinity of a tethering factor for its binding
partner. On the other hand, larger Rab-induced conformational changes in a golgin might directly assist vesicle
docking and fusion by dcollapsingT a tethered vesicle onto
its target membrane. Breaks in the coiled-coil sequence of a
golgin might act as hinges to facilitate such a process.
Rab proteins are not the only small GTPases known to
regulate components of the Golgi matrix. The ARL family
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B. Short et al. / Biochimica et Biophysica Acta 1744 (2005) 383–395
of ARF-related GTPases, of which there are ten members in
humans, seems to have diverse functions in vivo. However,
at present only two members of the family, ARL1 and
ARL3/ARFRP1, are known to be important for Golgi
structure and function. Other ARLs may function elsewhere
in the cell in processes with little to do with membrane
traffic, since ARL2 is found to complex with both a
phosphodiesterase and the tubulin-folding cofactor D
[87,88].
Mammalian ARL1 was shown to be important for Golgi
structure since the overexpression of a constitutively active
mutant (Arl1Q71L) resulted in large, unstacked Golgi
cisternae while a dominant negative mutant (Arl1T31N)
caused the Golgi to disappear [89]. ARL1 was then shown
to bind the GRIP domain of golgin-245 [90]. The GRIP
domain is an ~45 amino acid motif found in at least four
mammalian golgins (p230/golgin-245, golgin-97, GCC88,
and GCC185) and one yeast golgin (Imh1p), and targets
these proteins to the trans-Golgi/TGN. The mutation of an
invariant tyrosine residue in the GRIP domain abolishes
Golgi localization [91–93], and, in the case of golgin-245,
abrogates the interaction with ARL1 [90]. Studies in
budding yeast demonstrated that Arl1p was required for
the Golgi localization of Imh1p and that the GRIP domain
of Imh1p mediated this interaction [94,95]. In addition,
Arl1p was also required for the localization of exogenously
expressed mammalian GRIP domains to the yeast Golgi
[95]. The latter result suggests that ARL1 recruits several
GRIP domain-containing golgins to the trans-Golgi and that
this mechanism is conserved throughout evolution.
The crystal structure of ARL1 bound to the GRIP domain
of golgin-245 has recently been solved, providing an
intriguing insight into the mechanism by which this
complex is targeted to the Golgi [96,97]. The GRIP domain
of golgin-245 forms a homodimer, with each monomer
separately binding an ARL1-GTP molecule. The critical
tyrosine residue of the GRIP domain protrudes into a
bselectivity pocketQ in ARL1 consisting of the switch
regions of the protein. When ARL1 is bound to GDP, the
switch regions are in a different conformation and the
bselectivity pocketQ is closed [96,97]. The ARL1–GRIP
complex is thought to associate with membranes by
orientating itself such that the N-terminal myristoyl groups
of ARL1 and tryptophan residues near the C-termini of the
GRIP domains can all insert into the lipid bilayer. The
simultaneous binding of two ARL1 molecules by the GRIP
domain dimer is likely to stabilize the association of the
golgin with the membrane [96,97].
But how does ARL1 specifically target to the Golgi?
Yeast Arl3p (ARFRP1 in humans) is required for the
association of Arl1p and Imh1p with the Golgi although it
doesn’t interact directly with either of these proteins [94,95].
Arl3p is also not myristoylated at its N-terminus. Rather, the
membrane association of Arl3p is dependent upon it being
acetylated at its N-terminus by the NatC complex [98,99].
The Golgi integral membrane protein Sys1p (Sys1 in
humans) is also required for Arl3p localization, possibly
acting as a receptor for acetylated Arl3p. Thus, Sys1p
localizes Arl3p to the Golgi, resulting in the recruitment of
Arl1p and Imh1p [98,99]. How Sys1p is correctly localized,
and how Arl3p mediates the recruitment of Arl1p are
outstanding questions. Nevertheless, this GTPase cascade
demonstrates the exquisite regulation involved in localizing
golgins such as the GRIP proteins and maintaining correct
Golgi structure and function. It is also reminiscent of the
GTPase cascade leading to the recruitment of the RabGTPase Sec4p on to Golgi to plasma membrane targeted
vesicles in budding yeast [100]. In this case, two GTPases
Ypt31p and Ypt32p, recruit Sec2p, the exchange factor for
Sec4p, to the vesicles and this promotes GDP–GTP
exchange on Sec4p and thus its membrane association
[100].
Whether other GTPases interact with golgins in a similar
way to the ARL–GRIP domain system remains to be seen.
An indication that this may be a common theme comes from
the recent finding that the golgin GMAP-210 and its yeast
homologue Rud3p contain a GRIP-related ARF-binding
(GRAB) domain [101]. Like the GRIP domain the GRAB
domain is positioned toward the C-terminus, and binds to a
small GTPase of the ARF/ARL family, in the case of Rud3p
this is Arf1p [101]. However, this is insufficient for Golgitargeting and Erv14p, a small recycling transmembrane
protein of the ER-Golgi cargo receptor family is also needed
for Golgi localization of Rud3p.
7. The role of the cytoskeleton in maintaining Golgi
structure
So far, we have only considered the role of Golgi matrix
proteins and their regulatory molecules in organizing the
Golgi apparatus. But additional factors are also required to
maintain normal Golgi structure, including microtubulebased motor proteins and other microtubule binding
proteins.
The mammalian Golgi apparatus depends on an intact
microtubule network to maintain its normal morphology. If
microtubules are disrupted by, for example, treatment with
nocodazole, the Golgi fragments into bmini-stacksQ which,
although scattered throughout the cytosol, are still capable
of mediating membrane traffic [102]. Repolymerisation of
microtubules allows the mini-stacks to move to the microtubule organizing centre (MTOC) of the cell where they
coalesce to reform the Golgi ribbon [103,104].
Maintenance of an intact Golgi at the MTOC appears to
require a balance of plus- and minus-end directed motor
protein activity. The minus-end directed motor protein
cytoplasmic dynein 1 localizes to the Golgi and the
inhibition of its function by the overexpression of the
dynactin subunit p50 results in a dispersal of the Golgi
similar to that seen upon nocodazole treatment [105,106]. A
second isoform of cytoplasmic dynein also localizes to the
B. Short et al. / Biochimica et Biophysica Acta 1744 (2005) 383–395
Golgi and plays a role in maintaining its organization [107].
Dynein requires the multi-subunit protein dynactin to target
it to specific cargo and stimulate its motor activity [108]. As
discussed above, Rab6 and the golgin Bicaudal-D are
important for the specific recruitment of dynactin and
dynein to Golgi membranes [37,38], and a model for this
process is presented in Fig. 1d.
Plus-end directed motors of the kinesin family are also
found at the Golgi. Conventional kinesin is Golgi localized
and its inhibition by either anti-sense application or
antibody microinjection causes the Golgi to collapse into a
compact structure at the MTOC [109–111]. Thus, a
continuous btug of warQ between the plus- and minus-end
directed microtubule-based motors determines the position
and contributes to the organization of the Golgi apparatus
[112]. In addition to Bicaudal-D, other proteins are likely to
be involved in linking Golgi membranes to microtubules
and motor proteins. Candidates include Hook3 at the cisGolgi [113], and CLIP-59 at the trans-Golgi [114]. Another
golgin that has been suggested to function in linking the
Golgi to the cytoskeleton is GMAP-210 [115–117]. GMAP210 may act as a recruitment factor for gamma-tubulin, and
thus promote the nucleation of microtubules that help to
organize the Golgi ribbon around the centrosome [116].
However, these findings are somewhat controversial since
the gamma-tubulin binding and centrosome targeting
portion of GMAP-210 identified by Rios and coworkers
[116] maps to the same region as the Golgi-targeting ARF1binding GRAB domain discussed previously [101]. This
matter is discussed in more detail elsewhere [118].
The actin cytoskeleton is also important for maintaining
Golgi structure in mammalian cells. Treatment of cells with
cytochalasin D to disrupt actin filaments caused the Golgi to
adopt a more clustered morphology instead of the usual
extended ribbon [119]. An extensive actin network is
thought to be associated with the Golgi in mammalian cells
[120] and, accordingly, several myosin motors localize to
the organelle, where they are thought to contribute to the
formation and transport of Golgi vesicles [112,121]. In
addition, Golgi-specific isoforms of spectrin and its binding
partner ankyrin have been identified in animal cells [122–
125], although in the case of spectrin the molecular nature of
the isoform is undefined. This spectrin–ankyrin network
may contribute to Golgi integrity as well as being involved
in the sorting and selection of cargo transiting from the ER
to the Golgi [123]. Interestingly, small GTPases may also
regulate the recruitment of the spectrin-ankyrin network.
There is some evidence that active ARF stimulates the
production of phosphatidylinositol-4,5-bisphosphate
(PtdIns(4,5)P2) by recruiting phosphatidylinositol kinases
to the Golgi. Increased levels of PtdIns(4,5)P2 result in the
binding of spectrin to Golgi membranes through its
pleckstrin homology (PH) domain [126,127]. This mechanism is unlikely to be a universal determinant of Golgi
structure since spectrins are not present in all eukaryotes.
There is also some controversy regarding the role of
391
phosphatidylinositol-4-phosphate (PtdIns(4,5)P2) at the
Golgi, since PtdIns(4)P could be a precursor of
PtdIns(4,5)P2 or alternatively have direct functions in its
own right. While the expression of dominant negative forms
of phosphatidylinositol-4-kinaseR dramatically alters Golgi
organization [127], this does not indicate per se that
perturbation of PtdIns(4,5)P2 levels is the mechanism. A
family of Golgi specific PH domain proteins has been
shown to bind to PtdIns(4)P, and studies in yeast suggest
that Golgi PtdIns(4)P does not function as a precursor of
PtdIns(4,5)P2 [128–130]. Furthermore, PH domains that
interact with PtdIns(4,5)P2 are typically found together with
the bulk of this lipid at the plasma membrane [131,132].
Finally, the Golgi has also recently been observed to
associate with the vimentin intermediate filament cytoskeleton [133]. Thus, numerous cytoskeletal networks and their
associated motor proteins cooperate to dynamically regulate
the structure and function of the Golgi apparatus. Of
particular interest is the function of the Golgi in polarizing
cells when, typically, the Golgi reorients to face the leading
edge of the cell and contributes to the establishment and
maintenance of cell polarity (reviewed in [134]). Interactions between the cytoskeletal networks and components of
the Golgi matrix are likely to be of key importance in this
and other processes.
8. Conclusions and future perspectives
Numerous different proteins are involved in organizing
the Golgi apparatus into its typical, stacked, ribbon-like
structure. The main player is the Golgi matrix, a complex
network of proteins such as the GRASPs, the expanding
family of coiled-coil golgins, and their regulatory GTPases
of the ARF, ARL, and Rab families. The dictionary
definition of the word dmatrixT is b. . .a surrounding
substance within which something else originates, develops,
or is containedQ and this fits well with the role of the Golgi
matrix.
Many golgins act as membrane tethers although many of
the coiled-coil proteins labelled as such may, in fact, be
much more active in their function than previously thought.
Golgin-84, for instance, has been proposed to have a
specific role in the lateral organization of Golgi stacks
[34], while p115 may actively promote SNARE complex
assembly for membrane fusion [22]. One can imagine other
possible functions for coiled-coil dtethering proteinsT such as
negative regulation of membrane fusion by keeping
membranes apart and providing transient binding sites for
vesicles in order to limit their diffusion. The stacking of
Golgi cisternae, for example, would represent a case in
which membranes are tethered together but do not undergo
fusion. Additional components of the matrix surely await
discovery, and the recent demonstration that the cis-Golgi
golgin GM130 activates two kinases involved in cell
migration and polarity hints at hitherto unexpected roles
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B. Short et al. / Biochimica et Biophysica Acta 1744 (2005) 383–395
for these proteins [135]. What is clear is that no single
golgin alone can be said to be responsible for maintaining
Golgi structure. Rather, it is the complex network of
interactions between these proteins that is important.
As the Golgi apparatus is a highly dynamic organelle, the
regulation of the matrix is essential for it to fulfil its function.
Small GTPases of the Rab and ARL families appear to carry
out this regulatory role, and an appreciation of how these
GTPases themselves are controlled will be a key goal of
future research. The identification of specific GEFs and
GAPs will help our understanding of how GTPases and their
effector proteins are organized in time and space to actively
maintain both Golgi structure and function. A number of
important studies have begun to define the structural basis of
how a small set of these factors act [136–138], but much more
work is needed to identify the full set of regulators acting on
the nearly 60 human Rab GTPases. Larger changes in Golgi
morphology during mitosis and apoptosis are regulated by
protein kinases and caspases respectively. Again, only a few
proteins have been examined in any detail, so for the most
part the exact molecular details of the changes seen in mitosis
and apoptosis remain to be elucidated.
Finally, various cytoskeletal networks also play a role in
organizing the Golgi. Microtubules and associated motor
proteins appear to be of particular importance. An understanding of how the cytoskeleton interacts with components
of the Golgi matrix will undoubtedly help us in the future to
form a complete picture of how the Golgi apparatus is held
together.
Acknowledgements
We thank the reviewers for making a number of
important suggestions and the members of the Barr group
for reading the manuscript. The Max-Planck Society
supports research in the laboratory of FAB.
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