REVIEWS

Exiting the Golgi complex

Maria Antonietta De Matteis and Alberto Luini
Abstract | The composition and identity of cell organelles are dictated by the flux of lipids and
proteins that they receive and lose through cytosolic exchange and membrane trafficking.
The trans-Golgi network (TGN) is a major sorting centre for cell lipids and proteins at the
crossroads of the endocytic and exocytic pathways; it has a complex dynamic structure
composed of a network of tubular membranes that generate pleiomorphic carriers targeted
to different destinations. Live-cell imaging combined with three-dimensional tomography
has recently provided the temporal and topographical framework that allows the assembly of
the numerous molecular machineries so far implicated in sorting and trafficking at the TGN.

Early/sorting endosomes
Tubular, vesicular structures
that receive direct input from
the plasma membrane and
that are precursors of the
mature endosomes; they have
a key role in sorting material
for recycling or degradation.
Late endosomes
Endosomes that receive cargo
from early/sorting endosomes
and deliver them to lysosomes,
where the cargo molecules are
degraded. They contain many
vesicles in their lumen, and are
also known as multivesicular
bodies.
Recycling endosomes
Endosomal compartments  
that are mainly composed of
narrow-diameter tubules.   This implies that the selection of any given cargo into
A large fraction of recycling
membrane components pass
through the recycling
endosomes, which might also
be an intermediate station in
trans-Golgi network to plasma
membrane trafficking.
Department of
Cell Biology and Oncology,
Consorzio Mario Negri Sud,
66030 Santa Maria Imbaro
(CH), Italy.
Correspondence to: M.A.D.M.
e-mail:
dematteis@negrisud.it
doi:10.1038/nrm2378
The network of tubular reticular membranes that
emanates from the trans-Golgi cisternae, known as the
trans-Golgi network (TGN)1, has classically been viewed
as the main sorting node of the protein and lipid bio-
synthetic pathways. After synthesis in the endo­plasmic
reticulum (ER) and crossing the Golgi complex, differ-
ent cargo molecules are sorted at the TGN into distinct
pleio­morphic carriers that are targeted to different final
destinations (FIG. 1). Although cargo sorting can occur
in earlier secretory compartments and also continue
beyond the TGN (BOX 1), it reaches a distinctively high
level of complexity and sophistication at the TGN, where
the sorting machinery controls multiple divergent path-
ways directed to spatially segregated acceptor compart-
ments: the apical plasma membrane (PM), the basolateral
PM, early/sorting endosomes or late endosomes, recycling
endosomes, the Golgi stack, secretory granules and other
specialized compartments in specialized cells (FIG. 1).
its carriers involves a distinct set of docking and fusion
factors (such as SNAREs2) that are specific for the correct
acceptor compartment, and that there is the need to
associate specific carriers with the motor and cytoskeletal
tracks that will lead to this acceptor compartment.
In addition to the sorting and delivering of secretory
cargo, the TGN functions as an acceptor compartment for
endosomal traffic, and hence as the interface between the
secretory and the endosomal systems3. The endosomal
membrane input not only contributes to the overall size
of the TGN (see below), but it also has an important role
in the shape and function of the TGN through the ‘quali-
ties’ of the membranes it brings in, with regard to their
protein and lipid contents and their geometry. From the
endosomes the TGN receives those proteins belonging to
its own sorting and transport machineries that are recy-
cling back from the PM and from the endosomes after
they have completed their functions. The input from the
PM–endosomal membranes has a high cholesterol and
sphingolipid content, which contributes to the enrich-
ment of the TGN with these lipids. Moreover, the input
of the tubular endosomal membranes into the TGN is
a way to ‘inject’ membrane curvature into the system,
which might have an important role in the biogenesis of
carriers4 (see below).
Finally, a further important role of the TGN is to
serve as a biosynthetic centre. In the TGN membrane,
many proteins receive their final post-translational
modifications and major lipid classes undergo transfer,
insertion and then completion of their synthesis. These
lipids are subsequently exported to the PM and to the
endosomal system, where they have a major influence
on the composition of these membranes (BOX 2).
Underlying these complex tasks, there is the equally
complex and dynamic structure of the TGN itself, a com-
posite tubular membrane network that emerges from the
stacked flat Golgi cisternae (FIGS 1,2). This fascinating
structure has attracted the interest of cell biologists for
decades. The ‘morphological’ era in the 1970s focused
on morphological and ultrastructural studies of the
TGN5, whereas the next two decades were devoted to
the identification of the sorting signals and the molecular
requirements of the different TGN exit pathways6. In the
past decade, important technological advances (based
on the use of green fluorescent protein (GFP), correlative
light electron microscopy (CLEM), high-voltage electron
microscope (HVEM) tomography and small interfering
RNAs (siRNAs)) have changed our view of several aspects
of the morpho-functional and molecular organization of
the TGN7,8.
Here, we discuss the structure, dynamics and function
of the TGN in mammalian cells, along with the under­
lying molecular mechanisms. This Review will focus on

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© 2008 Nature Publishing Group
REVIEWS

Apical plasma membrane for a long time (which was mainly based on stereoscopic
observations by Rambourg and colleagues9), the TGN
Early in most cell types is a tubular network that emanates
endosome
Late from the last trans-Golgi cisterna, which appears to ‘peel
endosome
3b 1 off ’ from the rest of the Golgi stack (FIGS 1, 2), suggest-
ing that it is being ‘consumed’ in the secretion process9.
3a
Over the past few years, however, this model has been
modified by observations based on electron microscopy
5 II (EM) tomography and cryofixation8,10,11, which have
Recycling

Basolateral plasma membrane

Lysosome endosome Secretory added at least two new important and functionally
IV 4
3 granule significant features.
6 First, these studies have indicated that the TGN can
2
derive not only from the last trans-Golgi cisterna, but
III
also from the two adjacent trans-Golgi cisternae, from
trans-Golgi which tubules project into the space overlying the Golgi
network stacks (FIG. 1). Importantly, only the last cisterna and the
Endoplasmic tubules originating from it show clathrin-coated buds,
reticulum whereas the earlier cisternae and their related tubules
Endoplasmic have different coat profiles5. This differentiation has
Apical cargo reticulum important implications for the core sorting functions of
Basolateral cargo the TGN, because it indicates that only the last cisterna
I 7
is involved in transport to endosomes, which relies on
Endosomal cargo
clathrin-coated carriers (see below), whereas the others
Secretory granule are presumably involved in trafficking towards other
cargo
destinations.
Clathrin
Ribosomes Golgi complex The other remarkable feature of the TGN that was
revealed by tomography is its close relationship with
Figure 1 | The TGN: sorting at the crossroads of the endocytic and exocytic pathways. the ER (FIG. 1). In the original description of the trans-
The trans-Golgi network (TGN) is a tubular network that originates
Nature Reviewsfrom the lastCell
| Molecular trans-
Biology Golgi compartment, Alex Novikoff reported the pres-
Golgi cisternae. It sorts newly synthesized proteins that arrive from earlier Golgi ence of ER‑like cisternae with endosomal cytochemical
compartments (I) towards different destinations (1–5). It also receives input from the
features that were “intimately related to the Golgi sac-
endocytic pathway (II–IV) and sends back components to the earlier Golgi compartments
(7). The exit routes from the TGN include those towards the apical plasma membrane (1),
cule”. He considered these structures as specialized ER
the basolateral plasma membrane (2), recycling endosomes (3), early endosomes (4), membranes and termed them the GERL (Golgi–ER–
late endosomes (5) and specialized compartments such as secretory granules (6) in lysosome), suggesting that they were responsible for
secretory cells. These are the main destinations, and for each of them more than one type the production of lysosomes12. The GERL concept was
of carrier might be involved; for example, basolateral transmembrane and soluble later abandoned1, but the close spatial relationships
trafficking proteins can use different carriers101,156. Apical transmembrane proteins and between the ER and the TGN have been confirmed in
soluble, ciliary and GPI-linked proteins appear to use different pathways73,74,101,102,108. many different cells by recent three-dimensional (3D)
Secretory proteins can also use a transendosomal pathway through recycling endosomes studies8,10,11. These relationships are now categorized
to reach the basolateral (3, 3a)47,48,127–130 or apical surfaces (3, 3b)130,157,158. Golgi-resident among the membrane contact sites and are attracting
proteins (for example, glycosylating enzymes) recycle back to the Golgi stack (7), as
intense interest13.
secretion consumes the last trans-Golgi cisternae. Two important morphological features
have emerged from the three-dimensional tomographic studies. First, the TGN is
Although their composition, dynamics and function
composed of tubules emanating from the last two trans-Golgi cisternae (which are smaller remain unclear, it has been proposed that membrane
and appear to be detaching from the rest of the stack), although only the tubules deriving contact sites function in the transfer of lipids between
from the last cisterna are clathrin coated. Second, the endoplasmic reticulum makes close the ER and the TGN11. This idea stems both from the
contact with the last two trans-Golgi cisternae (black boxes), through which lipid structural homology between these membrane contact
exchange can occur between these two organelles. sites and those of the ER–mitochondria, where the
exchange of lipids between these latter two apposed
organelles is known to take place14, and from the exist-
Golgi stack
the sequential stages of the biogenesis of transport car- ence of lipid-transfer proteins in the TGN, such as the
A stack of ordered, flat, riers at the TGN. In particular, we identify and discuss ceramide-transfer protein CERT and the oxysterol-
membranous cisternae with a the following steps in carrier formation: the sorting and binding protein OSBP1, which contain ER‑targeting
recognizable cis–trans polarity. segregation of cargo into TGN tubular export domains; and Golgi-targeting motifs15 and can therefore shuttle
the extrusion of these domains along microtubules; and between these two organelles. However, whether the
Secretory granules
Organelles present in secretory tubule fission for the generation of carriers that are then ER–TGN contact sites indeed represent areas where
cells (exocrine and endocrine free to move to the cell periphery (FIG. 3). these lipid-transfer proteins operate remains to be veri-
cells) that derive from the trans- fied. A further question that needs to be addressed is
Golgi network. They function as Structure, dynamics and biogenesis whether these junctions only serve for lipid exchange
a reservoir for enzymes and
hormones, and they release
TGN structure. Although the structure of the TGN has or whether they have other roles, which might involve
their contents in response to been studied for many years1, it remains a source of the control of calcium homeostasis in the TGN area,
extracellular stimuli. important insights. According to a view that prevailed again in analogy with ER–mitochondria contact sites16.

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© 2008 Nature Publishing Group
 REVIEWS

amounts of exocytic and endocytic cargo this organelle

Box 1 | Pre- and post-TGN sorting along the biosynthetic pathway
is carrying. Thus, when the secretory cargo input to the
Apical plasma membrane Golgi complex is reduced, a dramatic reduction in TGN
tubules results, and when trafficking resumes and cargo
Post trans-Golgi network sorting

reaches the trans-Golgi, the TGN rapidly grows back to a

Basolateral plasma membrane

normal (or larger) size17. A marked increase in the size of
the TGN has also been reported both in cells exposed to
4 secretory stimuli that impose a cargo load on the Golgi
complex18 and in cells experiencing an increased input
Recycling 5 from the endocytic pathway19.
endosome In addition to changes in cargo input, the size of the
TGN is also affected by its cargo–membrane output. This
can be seen at moderately low temperatures (20 °C), at
trans-Golgi network which cargo continues to arrive at the TGN but cannot
exit. Under these conditions, in cells expressing high
cargo levels (for example, high levels of the secretory
vesicular stomatitis virus glycoprotein ( VSVG)), the
volume and surface area of the TGN increase greatly20,
Pre trans-Golgi network sorting

whereas in the absence of overexpressed cargo, the same

3 Golgi complex
2 ERGIC
1 Endoplasmic
reticulum
Apical cargo ARF1
Basolateral cargo COPI
Endosomal cargo COPII
Secretory granule cargo Clathrin
Step 1 (see figure). Proteins that are destined for export from the endoplasmic reticulum
(ER) are incorporated into transport carriers throughNature Reviews of
the activation | Molecular
the smallCell Biology
GTPase
SAR1, which triggers the assembly of coatomer protein complex-II (COPII), which
recognizes specific motifs on cargo molecules120. The COPII structure is flexible, which
might allow for the formation of differently sized carriers121,122. In addition, different
isoforms of COPII subunits have distinguishable cargo-recognition motifs and
different reciprocal affinities, which contribute to the widening of the repertoire of
carriers leaving the ER122,123. Indeed, it is now clear that different cargoes can be
segregated into distinct carriers when exiting the ER124. Step 2. The ER–Golgi
intermediate compartment (ERGIC) is a key sorting station for retrograde retrieval of
ER‑resident proteins125. This sorting is operated by coatomer protein complex-I (COPI),
which is activated by the small GTPase ARF and which recognizes specific motifs on the
cytosolic tails of ER transmembrane proteins or recognizes the KDEL receptor, which
binds soluble ER proteins that possess the KDEL motif125. Step 3. At the Golgi complex,
secretory cargo is sorted from Golgi-resident enzymes; in addition, some cargo can be
segregated from others126. Step 4. Trans-Golgi network (TGN)-derived carriers can also
intersect with the endocytic pathway at the level of the recycling endosomes,
delineating a transendosomal pathway26,46–48,127–130. Step 5. Sorting can occur through
transcytosis: this is the rule in hepatocytes, where apical proteins are initially
transported to the basolateral plasma membrane and then retrieved to the apical
plasma membrane131, which has also been seen in other epithelial cells for glycosyl
phosphatidylinositol (GPI)-anchored proteins132.
Biogenesis and dynamics: dependence on cargo flux.
One poorly appreciated yet fundamental characteristic
of the TGN is that its tubular components can vary
enormously in size and structure, depending on the
20 °C temperature block results in only an enlargement
of the last cisternae, producing bulging domains at the
rims of the last three cisternae21. The extent of mem-
brane output from the TGN can vary substantially from
cell to cell, and this can have an impact on the size, and
even the presence, of a detectable TGN compartment.
Consequently, the TGN is practically absent from pro-
fessional secretory cells that produce very large secretory
granules9, probably because here the TGN membranes
are used to make the granules themselves.
Altogether, such a tight dependence of TGN size and
ER-resident proteins structure on the extent of the cargo–membrane fluxes
Golgi-resident proteins that traverse it indicates that the TGN is not an inde-
Ribosomes pendent and stable compartment, but rather an ‘induc-
KDEL receptor
ible’ one that is formed and moulded by incoming and
SAR1
outgoing cargo.
The TGN as an assembly of cargo export domains.
In discussing the significance of TGN plasticity, it is
important to consider the morphology and dynamics of
the post-Golgi carriers that emerge continuously from
this organelle. CLEM studies have shown that baso­
lateral VSVG-containing carriers are large pleiomorphic
membrane units that detach from tubular ‘precursors’
that elongate out of the TGN mass by virtue of micro-
tubule-based motors22. Looking at their ultrastructure,
these precursors appear as tubular segments that are
interconnected and have branching and fenestrated
membranes, which are very similar in structure to the
neighbouring TGN7 (FIG. 2d). Moreover, they are seen
to be continuous with the parent membranes in the
trans-Golgi23. This also holds true for endolysosome-
directed carriers, which consist of clusters of clathrin-
coated buds that are connected by tubular regions and
are, again, similar to the parent TGN membranes24,25
(FIG. 2e); and it holds true for carriers that contain other
basolateral or apical cargo26–28, which have a behaviour
and morphology that appear similar to those described
for VSVG carriers. Therefore, these characteristics
of post-Golgi carriers indicate that they are portions of
the TGN membrane into which cargo has been sorted
(see below).

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SNAREs
(Soluble N‑ethyl-maleimide-
sensitive fusion protein (NSF)-
attachment-protein (SNAP)
receptors). A family of
membrane-tethered, coiled-
coil proteins that regulate
fusion reactions and targeted
specificity in the vacuolar
system.
Sorting signals
Sequence motifs or structural
determinants that interact with
specific recognition proteins
and determine the trafficking
or localization of cellular
proteins.
Correlative light-electron
microscopy
(CLEM). A technique by which
the ultrastructure and
dynamics of an individual
object can be observed.
Clathrin
The main component of a
vesicle coating that is involved
in membrane transport in both
the endocytic and biosynthetic
pathways.
Membrane contact sites
Sites of close apposition of
membranes (≤10 nm), either
belonging to the same
organelle (that is, between ER
elements or Golgi cisternae) or
to different organelles (usually
between the ER and another
organelle, such as
mitochondria, the plasma
membrane, vacuoles or the
Golgi complex).
Coatomer protein
complex-II
(COPII). A protein complex
consisting of two heterodimers,
SEC23–SEC24 and SEC13–
SEC31, that are recruited to ER
exit sites by the small GTPase
SAR1. COPII promotes the
formation of Golgi-directed
carriers.
Coatomer protein complex-I
(COPI). A protein complex that
is recruited to membranes by
ARF (ADP-ribosylation factor)
GTPases and that mediates
intra-Golgi and Golgi-to-ER
retrograde transport.
Transcytosis
A process by which materials
are transported across a
polarized cell from one
membrane domain (for
example, the basolateral
plasma membrane) to another
(for example, the apical plasma
membrane).
276 | April 2008 | volume 9 www.nature.com/reviews/molcellbio
Box 2 | The TGN, an important biosynthetic centre for proteins and lipids
The trans-cisternae of the trans-Golgi network (TGN)
provide the location for the final modifications to GlcCer GSLs Cer SM Cholesterol
glycoproteins: mainly sialylation and fucosylation of
Plasma membrane
N‑linked and O‑linked glycans and sulphation of
glycosaminoglycans. In addition, a number of proteins are Early
endosome
processed in the TGN from their pro-forms to their mature
forms .
133
The TGN occupies a key position in the synthesis
of sphingolipids. The common precursor of mammalian
sphingolipids is ceramide, which is synthesized in the
Recycling
endoplasmic reticulum (ER). Ceramide is converted into endosome
sphingomyelin (SM) by SM synthase‑1, which is located in
the trans-Golgi compartments , or it is glycosylated to
134
glucosylceramide (GlcCer) on the cytosolic face of the cis-
Golgi by GlcCer synthase (GCS). GlcCer is then converted
into complex glycosphingolipids (GSLs) in the lumen of the Late
endosome
later Golgi compartments . The view that ceramide and
135
GlcCer use vesicular trafficking to move through the Golgi CERT
stacks, analogous to newly synthesized proteins, has PH
Lysosome
recently been challenged by the demonstration that they
PH
OSBP1
can both be delivered from their sites of synthesis to the
late Golgi by the cytosolic lipid-transfer proteins CERT 136 trans-Golgi
network
and FAPP267, respectively. CERT and FAPP2 are targeted to
PH
the Golgi complex via their pleckstrin homology (PH) FAPP2
domain, as is OSBP1, which is believed to act as a sterol
sensor. CERT and OSBP1 also possess an ER‑targeting
motif53 (black circle).
At the same time as uncovering the existence of non-
vesicular lipid input pathways into the TGN, these
observations have provided new hints for our
understanding of how a non-homogeneous lipid Golgi complex FAPP2 PH Endoplasmic
distribution is created and maintained along the different reticulum
segments of the endomembrane system. Indeed,
Nature Reviews | Molecular
sphingolipids and cholesterol are only found in low amounts in the ER membranes, whereas they are enriched Cell Biology
at the
plasma membrane and in endosomes (thick black outline in figure), with the Golgi complex having intermediate levels65.
Interestingly, an increase in the concentration of SM and cholesterol is also seen in passing from the cis- to the trans-
Golgi137–139. The mechanisms underlying this heterogenous lipid distribution are not completely understood65. They might
include the exclusion of sphingolipids and cholesterol from retrograde membrane carriers140. An additional possibility is a
tight coupling between the synthesis or arrival of sphingolipids and cholesterol at the TGN and their export out of the
Golgi complex. This coupling could be achieved through the lipid-transfer proteins CERT and FAPP2, which
simultaneously promote the synthesis of SM and GSLs and the formation of transport carriers at the TGN67.
From these observations and from the dependence The sorting signals. The best characterized and under-
of the TGN on the flow of cargo, as discussed above, a stood among these principles is based on the recognition
model of the TGN emerges by which this organelle con- of specific sorting motifs in the cytosolic tail of trans­
sists essentially of a dynamic assembly of tubular export membrane cargo and cargo receptors by cytosolic adaptors
domains that form on arrival of cargo and that are then and coat proteins (see below). This principle is shared by
consumed as export carriers. In this context, the ques- other sorting processes along the secretory and endocytic
tions that arise are: how does the arrival of cargo activate pathways (BOX 1). At the TGN, sorting motifs have been
TGN dynamics? How is the segregation of cargo within identified in the endosomal-directed proteins and in some
the distinct tubular domains of the TGN achieved? How basolateral-directed proteins, such as those that are Tyr
are the different tubular domains established? How are based (with NPXY or YXXØ consensus motifs) and those
they converted into export carriers? Below, we will dis- that are based on two Leu residues (with [DE]XXXL[LI]
cuss the underlying mechanisms separately, although or DXXLL consensus motifs)29,30,32.
they might in fact operate concomitantly and in an Post-translational modifications of cargo can also
integrated fashion. affect its sorting. O‑glycosylation and N‑glycosylation act
as lumenal signals for apical targeting33,34, although these
Cargo sorting and segregation seem to be ‘recessive’ in relation to the basolateral cytosolic
Excellent recent reviews have focused on cargo sorting sorting motifs. Ubiquitylation can act as an endosome-
at the TGN, and we refer the reader to these for detailed targeting signal in cooperation with the GGA proteins
descriptions29–32. Here, we will discuss the general principles (Golgi-localized, γ-ear-containing, ADP ribosylation
that guide cargo sorting at the TGN (FIG. 3). factor (ARF)-binding proteins) that bind ubiquitin via
© 2008 Nature Publishing Group

VSVG
(Vesicular stomatitis virus
glycoprotein). A viral
glycoprotein that in its
temperature-sensitive form
(ts045) is used as a
synchronizable secretory
reporter protein, which exploits
its property of being retained
in the ER at 40 °C, and is
transported along the exocytic
pathway upon removal of the
40 °C temperature block.
Adaptors
Proteins that recruit clathrin to
membranes and concentrate
specific transmembrane
proteins in clathrin-coated
areas of the membrane.
GGA protein
(Golgi-localized, γ-ear-
containing, ADP ribosylation
factor (ARF)-binding protein).  
A monomeric clathrin adaptor
that mediates trans-Golgi
network-to-endosome
transport of mannose‑6-
phosphate receptors and other
transmembrane proteins. They
are also involved in targeting
transmembrane proteins to the
multivesicular-body pathway.
Membrane microdomain
A localized membrane region
that differs from surrounding
regions in their lipid
composition and order. There
are probably many types of
lipid microdomains that coexist
within the same membrane
bilayer. Lipid rafts are one type
of membrane microdomain.
nature reviews | molecular cell biology volume 9 | April 2008 | 277
REVIEWS
a b
c d e
1 2 3 4
Figure 2 | Ultrastructure of the TGN and post-Golgi carriers. a | Three-dimensional tomographic reconstruction of the
Golgi complex, showing the cis-Golgi network (white) and the stack with the terminalNature Reviews | Molecular
three trans-cisternae Cellred
in pink, Biology
and
cyan. The TGN (violet) appears as a tubular network that emerges from the lateral part of the last trans-cisterna (cyan).
Bar, 200 nm. b | Initial extrusion of a potential carrier precursor that contains the TGN marker TGN46–HRP (arrow) from the
TGN of a Golgi stack, as visualized by immuno-electron microscopy peroxidase technique. Bar, 300 nm. c | Formation of
a TGN-derived carrier that contains vesicular stomatitis virus glycoprotein (VSVG)–green fluorescent protein (GFP) during
its elongation out of the Golgi mass (arrowheads), as visualized by video microscopy, showing the extrusion and elongation
of a tubular carrier precursor (panels 1–3) and the detachment of a roundish carrier (larger arrowhead; panel 4) from the
tip of the tubular precursor (smaller arrowhead; panel 4). Bar, 6 µm. d | Ultrastructure of basolateral plasma-membrane‑
directed carriers: the carriers appear as tubular segments with branched and fenestrated (arrows) membranes, respectively;
immuno-gold labelling of VSVG. Bar, 380 nm. e | Ultrastructure of late-endosome-directed carriers: the carriers are grape-
shaped because they are made of clusters of clathrin-coated buds (arrows); immuno-gold labelling of the mannose-6-
phosphate receptor. Bar, 380 nm. Part d is modified, with permission, from REF. 23  2003 American Society for Cell Biology.
Part e is modified, with permission, from Ref. 25  2006 Blackwell Publishing.
their GAT (GGA and Tom1) domain35,36. Phosphorylation from that of the earlier secretory compartments in terms
of membrane proteins can also influence their trafficking of sphingolipid and cholesterol content. These lipids
at the TGN, either by modulating the activity of a sorting have a tendency to coalesce and to form liquid-ordered
motif or by generating new sorting motifs29,37. membrane domains for which some proteins have a
The formation of protein complexes or aggregates particular affinity, such as glycosyl phosphatidylinositol
might be another way to segregate cargo proteins, with (GPI)-anchored proteins. This is the basis for the ‘lipid raft
clustering-induced sorting having a pivotal role in secre- hypothesis’, which in its initial formulation41 posited that
tory granule biogenesis38. Oligomerization can either be the lipid rafts and associated proteins act per se as apical
due to the intrinsic properties of the cargo protein, as sorting platforms. This original model has been recently
in the case of granins and other secretory granule pro- updated and integrated into the clustering model42, and
teins38, or be induced by the clustering of cargo receptors, it now envisages that the clustering of lipid rafts, achieved
as proposed for the sorting of some glycoproteins to the by different means33,43, is the main mechanism for seg-
apical PM39,40. regating the apical raft-associated proteins from the
The affinity of selected proteins for membrane micro­ basolateral proteins.
domains enriched in sphingolipids and cholesterol has
also been implicated as one of the mechanisms responsi- Cargo segregation to tubular TGN subdomains. The pres-
ble for apical targeting of cargo proteins. Indeed, owing ence of sorting signals in cargo molecules provides the
to its lipid biosynthetic activity and its intense exchange basis for the physical segregation of cargo into different
with the endocytic pathway, and owing to the activities TGN domains. This occurs through the involvement of
of TGN-localized lipid-transfer proteins, the TGN pro- machineries that function by deciphering sorting motifs
vides a lipid environment that is substantially different or by creating a lipid and protein microenvironment
© 2008 Nature Publishing Group
 REVIEWS

a Apical plasma membrane for which specific cargo have a selective and distinctly
higher affinity. Key components of these machiner-
Sorting, segregation and tubulation

ies include small GTPases of the ARF and Rab family

Basolateral plasma membrane

Late (see BOX 3), adaptors, GRIP-golgins and lipids, such as
endosome phospho­inositides, sphingolipids and cholesterol.
Recycling Secretory
endosome granule Once activated by specific nucleotide exchange fac-
tors, ARF1 promotes the recruitment of cytosolic protein
complexes that can recognize the aforementioned cargo
sorting motifs and that can also bind clathrin, thus func-
trans-Golgi tioning as an adaptor between cargo proteins and the
network clathrin coat. These coat adaptors include the AP1A and
AP3 complexes and the GGAs, which mediate endosomal
sorting44. Clathrin has recently been shown to be involved
in transport between the TGN and the PM, although the
b Apical plasma membrane
clathrin adaptor involved remains unknown45. In addi-
Late endosome Recycling tion, even though the AP1B and AP4 complexes mediate
endosome sorting of basolateral cargo to the PM, AP1B operates

Basolateral plasma membrane

only in the recycling endosomes along the transendosomal
route that many newly synthesized proteins take to reach
Tubule extrusion

Secretory
granule the basolateral PM46-48 (see also BOX 1) and AP4 does not
KIF13A KIFC3 appear to require clathrin44 (FIG 3).
Apical Activation of ARF can also trigger the generation of
?
cargo
distinct TGN microdomains through action on the local
Basolateral
KIF5B cargo lipid composition. In particular, active ARF stimulates
Endosomal the production of phosphatidylinositol 4‑phosphate
cargo (PtdIns4P), as it can recruit and activate PtdIns 4‑kinase
Secretory (PI4K)IIIβ49. As with phospho­inositides in general,
granule cargo PtdIns4P acts as a gathering point for the recruitment
c Apical plasma membrane AP1A of a specific set of cytosolic proteins that contribute to
Late endosome
Recycling AP1B the local specialization of this membrane. Indeed, this
endosome ARF–PtdIns4P pairing can be considered as a marker
Basolateral plasma membrane

AP3
for TGN domains, as is the Rab5–PtdIns3P pairing for
Secretory AP4
granule early endosomes50. In combination with ARF, PtdIns4P
KIF13A GGAs increases the affinity of AP1 and the GGAs for the TGN
KIFC3
membranes51,52, and thus contributes to the organization
Fission

BARS
KIF5B
PKD Dynamin
Dynamin BARS
Figure 3 | The formation stages of post-Golgi carriers.Nature a | Sorting,
Reviews segregation
| Molecular and
Cell Biology
tubulation. At the trans-Golgi network (TGN), cargo directed to different final
destinations are sorted into distinct carriers. Specific sorting signals in endosomal cargo
are recognized by coat adaptors such as AP1A and the GGAs (Golgi-localized, γ-ear-
containing, ADP ribosylation factor (ARF)-binding proteins). Among the apical cargo, the
glycosyl phosphatidylinositol (GPI)-anchored proteins are sorted owing to their affinity
for cholesterol- and glycolipid-enriched domains and to their propensity to cluster.
Basolateral cargo can follow a direct or transendosomal pathway through recycling
endosomes, where they are sorted using an endosome-like sorting motif recognized by
AP1B (Ref. 47). AP3 and AP4 have also been implicated in basolateral transport44. Other
proteins involved in the early stages of formation of basolateral carriers (possibly at the
tubulation stage) include FAPP2 and the GRIP-golgins58,66. b | Tubule extrusion. When a
TGN export domain is mature, it interacts with a suitable microtubule-based motor and
is extruded out of the Golgi area. The motors identified to date include: KIF5B, which is
involved in the transport of the apical cargo p75 (Ref. 72) and in the Rab6-dependent
trafficking of vesicular stomatitis virus glycoprotein (VSVG)75; KIFC3, which is involved in
apical transport of haemagglutinin78; and KIF13A, which is involved in the transport of
endosomal cargo77. c | Fission. During the elongation process, fission occurs at the
thinnest sections of the tubular precursor. The distinct fission machineries described to
date are the dynamin-based machinery (endosome-directed and apically directed
carriers in polarized cells) and the protein kinase D (PKD)- and brefeldin A-ribosylated
substrate (BARS)-based machineries (basolaterally-directed carriers).
Clathrin
FAPP2 of clathrin microdomains.
PtdIns4P also promotes the recruitment of the lipid-
GRIP-golgins
transfer proteins OSBP1, CERT and FAPP2 (Ref 53),
GPI-protein thereby inducing further changes in the local lipid
PKD clustering composition of these ARF–PtdIns4P-enriched mem-
N-O-glycans
brane domains. CERT and FAPP2 transfer ceramide and
glucosylceramide (the precursors of sphingo­myelin
and glycosphingolipids, respectively) to the TGN,
whereas OSBP1 potentially senses and controls the levels
of cholesterol in the TGN membranes (BOX 2). As noted
above, sphingolipids and cholesterol have a tendency to
coalesce to form a liquid-ordered membrane bilayer for
which some proteins, such as the GPI-anchored proteins,
have particularly high affinities. This could therefore
eventually lead to the segregation of GPI-anchored cargo
proteins from other cargo.
Finally, the GRIP-golgins 54 are believed to have
a role in establishing and maintaining distinct TGN
subdomains. This concept arises from the observation
that the simultaneous expression of two different GRIP-
golgins results in the expansion of distinct regions of the
TGN54. However, the mechanisms of action of the GRIP-
golgins in this process remain to be defined. Some of
them have been implicated in retrograde trafficking from
endosomes, so they might be required for the retrieval of
essential components of the TGN55,56. Alternatively, they

278 | April 2008 | volume 9 www.nature.com/reviews/molcellbio

© 2008 Nature Publishing Group

Box 3 | Small GTPases at the TGN
The ARF family
The small GTPases of the ADP ribosylation factor (ARF) family are master regulators
of the structure and function of the Golgi complex141 and they are involved in multiple
trans-Golgi network (TGN) exit pathways. Many ARF regulators and effectors are
present at the TGN, including the brefeldin A-sensitive exchange factors BIG1 and
BIG2, GBF1 (Ref. 141), the GTPase-activating proteins ARAP1, which is enriched at the
TGN (T. Daniele and M. A. D. M., unpublished observations) and, possibly, AMAP2
(Ref. 142). The ARF effectors at the TGN include the adaptors AP1–4, the GGAs (Golgi-
localized, γ-ear-containing, ARF-binding proteins) and Mint3 (Refs 29,44,143); the
lipid-metabolizing enzymes PI4KIIIβ and PLD; the lipid-transfer proteins FAPP2 and,
possibly, OSBP1 and CERT53; and the arfaptins (Ref. 62; A. Di Campli and M. A. D. M.,
unpublished observations). ARF-like protein 1 (ARL1) is localized at the TGN, where its
activation requires the ARF-related protein ARPR1 (Ref. 141). The effectors of ARL1 at
the TGN include the arfaptins and the GRIP-golgins p97 and p230.
The Rab family
Rab6 is present on TGN-derived carriers and regulates exocytosis by enhancing the
processive kinesin-dependent motion of secretory vesicles75. Rab8 controls AP1B-
dependent trafficking of basolateral carriers102,104,144, and recent data from the Rab8
knockout mouse also indicate its involvement in apical trafficking in intestinal cells145.
The regulation of carrier motility is the candidate sphere of action for Rab8,
considering that it associates with optineurin, which binds myosin VI and FIP2 and
which can associate with actin through hungtintin118,146,147.
Rab10 is localized to the Golgi complex148 and when it is overexpressed it promotes
the inhibition of TGN-to-plasma membrane transport and the mis-sorting of basolateral
cargo to the apical membrane149. Rab11a is localized at the TGN, on recycling
endosomes and in specialized membrane compartments in secretory cells150,151. It has
also been implicated in basolateral trafficking26,152.
Rab14 and Rab22B (or Rab31) have been localized to the TGN. Interfering with Rab22b
impairs TGN-to-endosome trafficking153, disrupts the distribution of TGN46 and inhibits
the transport of vesicular stomatitis virus glycoprotein (VSVG) to the cell surface.
The Rho family
The state of activation of CDC42 at the Golgi complex is under the control of Fgd1
(Ref. 154) and ARHGAP21, an ARF1-regulated CDC42 GTPase-activating protein
(GAP)155. CDC42 is selectively involved in basolateral trafficking.
might be directly involved in the formation of enzyme
domains57 or of tubular protrusions that contain specific
cargoes58,59.
Mechanisms of TGN tubulation. Following cargo segre-
gation, cargo-containing tubular domains are generated.
Interestingly, this is not unique to the TGN. The creation
of tubular domains is an effective mechanism to sort
transmembrane proteins into separate containers that is
also used in endosomal sorting60. The conversion of cis-
ternal membranes into highly bent, tubular, reticular TGN
structures can in principle be achieved by several protein-
based or lipid-based mechanisms. Unfortunately, although
GPI anchor the general principles of membrane bending are relatively
The function of this post-
translational modification is to
well understood4, a focused analysis of the specific tubula-
attach proteins to the tion mechanisms that operate at the TGN has not yet been
exoplasmic leaflet of carried out. There are, however, several pieces of evidence
membranes and possibly to that should provide direction for future studies.
specific domains therein.  
Possible protein-based mechanisms include curvature-
The anchor is made of one
mole­cule of phosphatidyl­ inducing proteins, such as the BAR family members. Among
inositol, to which a these, the γ-BAR protein is known to be associated with
carbohydrate chain is linked the TGN61, as is arfaptin-2 (Ref. 62) and possibly endo-
through the C6 hydroxyl of the philin B63. Moreover, the aforementioned GRIP-golgins
inositol, and it is linked to the
protein through an
have also been proposed to be involved in membrane
ethanolamine phosphate tubulation54, although their mechanisms of action have
moiety. yet to be established.
nature reviews | molecular cell biology volume 9 | April 2008 | 279
© 2008 Nature Publishing Group
REVIEWS
Among the lipid-based mechanisms, the lipid-
metabolizing enzyme phospholipase A2 (PLA2) has been
shown to be required for tubulation of TGN membranes
in vitro64. By removing an acyl chain from phospholipids,
PLA2 can generate wedge-shaped lysolipids in the external
leaflet of membranes, thereby inducing spontaneous posi-
tive curvature4. Other possible players are transmembrane
or inter-organelle lipid-transfer proteins (for example,
DRS2, CERT, FAPP2 and OSBP1 (Ref. 65); see BOX 2).
FAPP2 has been visualized on tubular carrier precursors66
and has been proposed to transfer glucosylceramide from
the Golgi into the TGN67. Here, tubulation could result
from the selective transfer of lipids into the cytosolic
TGN membrane leaflet, creating transmembrane area
asymmetry and hence curvature4. The other transfer
proteins CERT and OSBP1 might have important roles
too, through their insertion of cholesterol and ceramide,
respectively, into the TGN13, which can facilitate mem-
brane curvature4,68,69. The lipid flippases are also potential
curvature-inducing enzymes, again through their gener­
ation of transmembrane asymmetry4. Although these
flippases have not specifically been examined in the
context of TGN tubulation in mammalian cells, the flip-
pase DRS2 has been shown to be involved in the exit of
cargo from the Golgi complex in yeast70.
Another class of events that might facilitate TGN
tubulation is a direct consequence of the trafficking and
sorting processes. One such event is the input of tubular
endosomal membranes into the TGN. The curvature of
these membranes might be transferred to the acceptor
organelle, the TGN, and induce it to tubulate. Another
event might be the segregation of Golgi enzymes from the
TGN. Because the flat morphology of the Golgi cisternae
has been reported to be partly due to the stabilizing prop-
erties of large Golgi-enzyme polymers71, the exclusion of
Golgi enzymes from a given TGN domain could favour
its cisterna-to-tubule transformation. Finally, TGN
tubulation might be favoured by the loss (as seen by the
‘peeling off ’ of the tubulated trans-Golgi­cisternae) of
the mechanical stabilization that is provided in the stack
by the presence of an adjacent cisterna.
In summary, a multiplicity of mechanisms are available
at the TGN that might be involved in membrane bend-
ing. Dedicated studies are now needed to elucidate the
relevance of each of these mechanisms in the formation
of TGN tubules.
The release of post-Golgi carriers
When a TGN export domain is ready for export, it inter-
acts with a suitable motor and is extruded from the Golgi
area (FIG. 3b). This is followed by the fission of these tubular
precursors into centrifugally moving carriers (FIG. 3c).
Mechanisms of extrusion of carrier precursors. The
elongation of carrier precursors from the TGN and
the translocation of carriers through the cytosol occur
along microtubules (towards either their plus or minus
ends; see Ref. 72 for a recent discussion), by virtue of
microtubule-based motors (usually kinesins), in nearly
all animal species72 (FIG. 3b). In mammalian cells, motors
interact with transport carriers in a remarkably, but not
REVIEWS
GRIP-golgins
A family of coiled-coil proteins
that includes golgin-245,
golgin-97, CCG88 and
CCG185, all of which have a
C‑terminal, 50-amino-acid
domain that is known as the
GRIP domain, which is
responsible for their targeting
to the trans-Golgi network.
Transendosomal route
A route that connects the
trans-Golgi network and the
plasma membrane, and passes
through recycling endosomes.
BAR family
A family of proteins that
contains the BAR (Bin,
amphiphysin and Rvs) domain,
a banana-shaped domain that
binds to highly curved
membranes.
Lipid flippase
A protein that facilitates the
translocation of a phospholipid
or glycosphingolipid from one
leaflet of a membrane bilayer
to the other.
Mannose‑6-phosphate
receptor
(M6PR). Receptor that is
located at the TGN and, at low
levels, at the plasma
membrane. It is responsible for
targeting several soluble
lysosomal hydrolases from the
Golgi complex, through
endosomes, to the lysosomes.
280 | April 2008 | volume 9 www.nature.com/reviews/molcellbio
completely, selective fashion. A well-studied example is
kinesin heavy chain-5B (KIF5B), which mediates apical
trafficking of p75 in confluent Madin–Darby canine
kidney (MDCK) cells72. This role of KIF5B is not shared
by other apically directed motors, such as the kinesin-
like proteins KIF3A, KIF3B and KIF17 (Ref. 72), which
are instead involved in the transport of ciliary apical
proteins73,74. Moreover, KIF5B itself is not active in the
transport of other apical proteins, such as prominin and
GPI–GFP, or of basolateral markers, such as E‑cadherin
and the LDL receptor72. However, these motors can also
show a degree of promiscuity, as KIF5B has also been
implicated in Rab6-dependent trafficking of VSVG in a
non-polarized cell line75.
Multiple mechanisms appear to participate in the
recognition between motors and carriers. The simplest
is direct binding between the motor and the cytosolic
domain of a cargo protein, as is seen for dynein light chain
and the cytoplasmic tail of rhodopsin76. Alternatively,
adaptor proteins serve as bridges between motors
and their cargo. For example, KIF13A transports the
mannose‑6-phosphate receptor (M6PR) through its inter-
action with the AP1 complex77. In addition, both motor
and cargo can associate with the same specific lipid micro­
domain, as is the case for KIFC3 and the apically targeted
annexin-XIIIb, for example78.
A further element of selectivity in carrier extru-
sion and translocation mechanisms can arise from the
microtubule tracks themselves72. A recent interesting
example is the formation of microtubules at the Golgi
complex by virtue of the CLASP (cytoplasmic linker
protein (CLIP)-associating protein) microtubule-binding
proteins. The CLASPs are recruited to the TGN by the
GRIP-golgin GCC185 and initiate the polymerization of
microtubules that are preferentially orientated towards
the leading edge of the cell in fibroblasts, hence selectively
supporting post-Golgi transport to that area of the cell79.
Thus, microtubules and motors contribute significantly
to the efficiency and selectivity of transport out of the
Golgi complex. It is therefore surprising to find that
cargo export from the Golgi complex appeared to occur
normally in cells with depolymerized microtubules17,80.
However, this is due to the ability of the Golgi complex
to reorganize itself into peripherally scattered mini-stacks
that do not rely on long-range, microtubule-mediated
transcytosolic transport for their carriers to reach their
destinations17,80, although they do lose the apical polarity
of trafficking81.
The molecular machinery of carrier fission. Fission takes
place anywhere along the length of tubular carrier pre-
cursors at sites corresponding to their thin tubular com-
ponents23 (FIGS 2c,3c), rather than in regions containing
tubular networks or vacuoles. Obviously, the precise point
of fission will define the size and morphology of a carrier:
if it occurs close to the tip of a precursor, the carrier will
be small, whereas large and structurally complex carriers
will form through cleavage at the base of a carrier precur-
sor, which explains, in a simple way, the pleiomorphic and
variable aspects of these carriers (FIG. 2c, d). In a similar
way, endosome-directed carriers can detach from the
© 2008 Nature Publishing Group
TGN as simple vesicles (albeit rarely) or as entire pieces
of TGN membranes containing 2–3 clathrin-coated buds,
resulting in a grape-like morphology25 (FIG. 2e).
From a mechanistic viewpoint, it is of note that the
fission event almost always coincides with the elongation
of these precursors out of the Golgi mass. This suggests
that the pulling force exerted by the motors facilitates
fission, presumably by generating tension within the pre-
cursor membrane23,27. In agreement with this concept,
membrane tension has recently been proposed to have
an important role in fission in vitro82; and a ‘pull-and-
cut’ model for TGN tubule fission has been proposed83.
However, even though mechanically generated tension
might be required, it does not appear to be sufficient
for fission. Indeed, a number of molecules have been
identified that have key roles in fission at the TGN,
although it remains unclear whether they participate in
the actual fission event or have regulatory or preparatory
roles instead.
An important fission-related machine involves the
sequential activation and recruitment of a series of sig-
nalling proteins with roles in the fission of basolateral
carriers (transporting VSVG, n‑CAM and SEC6), but
not of p75-containing apical carriers. This sequence
includes specific isoforms of the βγ subunits of the heter-
otrimeric G proteins84, phospholipase C‑β (presumably
to produce diacylglycerol and free cytosolic Ca2+) and,
finally, protein kinase C‑η (PKCη) and protein kinase
D (PKD), which both require diacylglycerol85. When
PKD is inhibited, TGN-derived tubules extend out of
the Golgi mass but do not detach, retracting elastically
back into the TGN86; conversely, the expression of con-
stitutively active PKD results in the vesiculation of the
TGN and of most of the Golgi complex.
Another player in fission at the TGN is dynamin, a
protein that is well known to have a key role in fission at
several other trafficking stages87,88. Dynamin is present
in the Golgi complex89 and is required for the export of
the apical protein p75 from the Golgi in polarized cells.
Its inhibition induces the formation of anom­alously
long p75-containing tubules that do not detach from
the Golgi mass and do not deliver their cargo to the
PM27. Dynamin is also involved in the export of pro­
collagen from the TGN, of VSVG from fibroblasts90 and
of the M6PR towards the late endosomes91. It has been
proposed that this Golgi-localized dynamin is part of
a complex with actin and cortactin that is recruited to
the Golgi complex by activated ARF91. Another dynamin
interactor that interfaces with this actin polymerization
machinery is syndapin-2, a tubule-inducing protein that
is required for VSVG export92.
The sequential recruitment and activation of ARF,
PI4KIIIβ and the lipid-transfer protein FAPP2 discussed
above might also be important in fission. FAPP2 pro-
motes the trafficking of VSVG in non-polarized cells66
and of GPI-anchored proteins and haemagglutinin in
polarized cells93. This partly occurs at the tubule-fission
stage, because a truncated form of FAPP2 (the FAPP2
pleckstrin-homology (PH) domain) induces the forma-
tion of extremely long, cargo-containing TGN tubules,
from which functioning carriers are never liberated66.

Actin comets
Short actin filaments that
function to propel intracellular
organelles or pathogens.
nature reviews | molecular cell biology volume 9 | April 2008 | 281
The two roles of FAPP2, one in lipid transfer and the other
in cargo export, appear to be linked, although the nature
of this link is not clear67.
Finally, C-terminal-binding protein-1 (CTBP1)/
brefeldin A-ribosylated substrate (BARS) is a bifunc-
tional protein that has been implicated in carrier for-
mation at several trafficking steps94 and is required for
the fission of VSVG-containing basolateral carriers in
epithelial cells90. The inhibition of CTBP1/BARS induces
the formation of TGN tubules that do not undergo fis-
sion and are reabsorbed into the TGN90. Although the
mechanism of action and the precise role (either essen-
tial or preparatory) of CTBP1/BARS in fission are also
not clear, some of its fission-relevant interacting proteins
have now been identified94,95.
Interestingly, several of the fission-inducing pathways,
including actin–cortactin–dynamin, ARF–PI4KIIIβ–
FAPP2, CTBP1/BARS and βγ–PLCβ–PKCη–PKD,
im­pinge on the formation of basolateral, VSVG-containing
carriers. It is therefore possible that they represent ‘frag-
ments’ of a single, larger and more complex pathway
that is involved in the control of basolateral trafficking.
Indeed, some potential relationships between these path-
way fragments are beginning to emerge: PKD can phos-
phorylate PI4KIIIβ and thus modulate its activity96 and
consequently the activity of FAPP2 too; and PI4KIIIβ
interacts with CTBP1/BARS through a linker protein
(C. Valente, A. L. and D. Corda, unpublished observa-
tions), which could be used to recruit CTBP1/BARS to
an area where carrier formation is taking place. However,
caution is necessary in discussing these potential links.
VSVG is a viral protein that might have evolved to hijack
more than one export route and can use both a direct and
a transendosomal pathway to the PM (FIG. 1 and BOX 1).
It is therefore unclear whether all of the molecules that
have been shown to be involved in VSVG export to the
PM operate in the same or in parallel (or sequential)
pathways, and hence whether it is legitimate to assemble
the available information as proposed.
Actin in exit from the Golgi
Although the above molecules have been shown to be
engaged in defined stages of TGN carrier formation, it
cannot be ruled out that they might also function at other
steps in the life cycle of a carrier. In addition to these,
other molecules have been shown to have roles in carrier
formation at the TGN, although their effects have not
been attributed to a specific formation stage. Several of
these molecules are actin related. Actin-based machiner-
ies were shown long ago to have multiple roles in endo­
cytosis97,98, and a similar concept might well emerge for
the actions of actin in the structure and function of the
Golgi complex. Following the demonstration that actin,
spectrin and ankyrin are involved in trafficking and can
be recruited onto Golgi membranes in an ARF-dependent
way99, several reports have indicated that actin-related
proteins can function in Golgi trafficking (reviewed
in Ref. 100).
The small GTPase CDC42A is a regulator of actin
polymerization that has additional important func-
tions in signalling and binds to the Golgi complex in an
© 2008 Nature Publishing Group
REVIEWS
ARF-dependent fashion 100. In the Golgi complex,
CDC42 regulates the dynamics of a local pool of short
actin filaments101–103 and through this effect might con-
trol the polarization of trafficking markers104, in addition
to the efficiency of transport to the basolateral and apical
membranes in epithelial cells101,102,105. LIMK1 is possibly
linked to CDC42, as a kinase that resides on the Golgi
complex in neurons and that regulates the polymeriza-
tion of a local pool of actin through interaction with
cofilin. This machinery has differential effects on the
trafficking of neuronal cargo proteins106. In epithelial
cells, the trafficking of the M6PR from the Golgi com-
plex to late endosomes relies on HIP1R, a cortactin inter­
actor that interfaces with both the clathrin coat and the
actin polymerization machinery107. In further support
of a role for actin dynamics at the Golgi complex, agents
that stabilize or depolymerize actin inhibit basolateral
and apical transport out of the Golgi complex (although,
interestingly, not of GPI-linked cargoes)108. In addition
to these, several other actin-related proteins have been
implicated in exit from the TGN, including coronin-7
(Ref. 109) , CDK5–p35 kinase 110, the actin organizer
SPIR-1 (Ref. 111), the actin–tubulin crosslinking pro-
tein MACF1 (Ref. 59) and PIP5K, which induces actin
comets112,113. Some of the above mechanisms have been
suggested to function by controlling a Golgi pool of actin
filaments. These might operate through the deforma-
tion of membranes or the recruitment of further players
during the carrier formation process, or as tracks for
actin-based myosin motors. For example, it has been
suggested that non-muscle myosin II mediates export
of the basolateral cargo VSVG from the TGN114-116; how-
ever, the involvement of myosin II has been subsequently
questioned (Ref. 117). The minus-end myosin VI motor
participates with optineurin and RAB8 in a complex that
mediates TGN export of some (VSVG, LDLR9) but not
all (Fc receptor) cargoes118.
Conclusions and perspectives
What emerges from every overview of TGN research is
the complexity of this organelle, which is arguably the
most complex organelle among the eukaryotic traffick-
ing stations. With its multiple functions as a bio­synthetic
and sorting centre that can control a large number of
outgoing pathways, the TGN must be endowed with
an unusual number of diverse molecular machineries
that function in coordination. In the past few decades,
we have gained some degree of understanding of the
morpho-dynamics of this organelle, and also the aware-
ness that our knowledge of the underlying molecular
machineries is still incomplete and fragmentary. We
have discovered a number of pieces of a large mosaic,
but a coherent picture still escapes us. The challeng-
ing task is now to continue uncovering the multitude
of pieces that remain hidden and to reveal the overall
pattern of the mosaic. To this end, we feel that it will be
important to generate a comprehensive list of the path-
ways that enter and exit the TGN and also to assign each
newly discovered machinery to the correct pathway, in
addition to one of the four stages into which special-
ized export carrier genesis can be divided: cargo sorting
REVIEWS

and segregation, membrane bending and tubulation,
extrusion by motors and, finally, membrane fission.
Another major challenge will be to understand how
the sorting and trafficking processes at the TGN are
regulated. Three layers of regulation will have to be
explored. The first concerns the local regulatory events
that trigger the formation of a carrier. An interesting
hypothesis is that these events involve classical signal-
ling cascades that can be triggered by the secretory cargo
and that are transduced through (as yet undefined)
cargo receptors, coupled to hetero­trimeric G proteins83.
This and other potential regulatory modalities also need
to be experimentally tested. A second layer is at the
‘organelle’ level, and concerns the coordination of TGN
trafficking with the previous and successive transport
steps. The third layer of regulation is at the global cel-
lular level, which allows the integration of TGN-related
processes within global cell functions and responses.
This is likely to involve signalling mechanisms that can
communicate with other cell districts and that have
been reported to be present on the membranes of the
Golgi complex119.

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Acknowledgements
We are extremely grateful to R. Polishchuk for providing the
images shown in FIG. 2, we thank the members of the
Department of Cell Biology and Oncology (DCBO) for useful
discussions, C. P. Berrie for editorial assistance and E. Fontana
for artwork. The authors acknowledge the support of Telethon
and Italian Association for Cancer Research (AIRC). We would
also like to apologize to those whose primary research could
not be cited owing to space constraints.
DATABASES
InterPro: http://www.ebi.ac.uk/interpro/
GAT | PH
UniProtKB: http://beta.uniprot.org/
annexin-XIIIb | AP1B | AP3 | AP4 | ARF | arfaptin-2 | CDC42 |
CERT | coronin-7 | DRS2| endophilin B | FAPP2 | GCC185 |
HIP1R | KIF17 | KIF3A | KIF3B | KIF5B | KIFC3 | LIMK1 | MACF1 |
n‑CAM | optineurin | OSBP1 | PKCη | PKD | PLA2 | Rab5 | Rab6 |
SAR1 | SEC6 | SPIR-1 |
FURTHER INFORMATION
Maria Antonietta De Matteis’ homepage: http://www.
negrisud.it/en/dcbo/DeMatteis
All links are active in the online pdf