The Scripps Research Institute, Department of Cell Biology, La Jolla, California 92037
Movement of cargo through the secretory pathway
of eukaryotic cells occurs as a discontinuous
process involving the activity of 50-80-nm vesicles (28). Vesicles forming on the endoplasmic reticulum
(ER) by the COPII coat machinery (35) were first detected as coated elevations on transitional elements directly facing the Golgi apparatus. Subsequently, intermediates harboring ER-derived cargo were found in regions
distant from the Golgi stack (16, 33). These structures contained a different coat complex, COPI or coatomer, recognized to be involved in vesicle-mediated recycling (20). It
is now apparent that ER export and recycling are tightly coupled. We review recent evidence regarding the organization and role of ER to Golgi intermediates in these
events.
Pleomorphic Elements Function in ER to
Golgi Transport
Stereological analysis of serial thin sections revealed the
general morphological organization and distribution of ER
export/recycling sites (4). Export from the ER was found
to be associated with one or more COPII-coated bud-bearing ER cisternae that face towards a central cavity
(Fig. 1). The central cavity is filled with a collection of
closely opposed vesicles and convoluted tubules containing COPI coats (4). We have termed these central compact clusters of pleomorphic elements as vesicular-tubular clusters (VTCs)1 for their morphological appearance (3), but
they have also been referred to as ERGIC (ER-Golgi
intermediate compartment) (16). The juxtaposition of ER-derived buds and a central VTC composes a morphological unit of organization termed an export complex (4)
(Fig. 1). ER-derived buds cannot be detected outside export complexes, suggesting a local specialization of the cytoplasm that is likely to be enriched in transport components. While VTC-containing export complexes are scattered throughout the cytoplasm, VTCs found in the
perinuclear region of the cell are believed to form a more
extensive array of tubulo-cisternal elements referred to as
the cis-Golgi network (CGN) (24).
Biochemical Composition of VTCs
COPII coats direct the sorting and concentration of cargo
during export from the ER (1, 3, 5). The dissociation of a
COPII vesicular carrier from ER budding elements demarcates the first boundary between the ER and downstream organelles of the secretory pathway. The formation
of this boundary is consistent with morphological observations that VTCs lack continuity with the ER (4, 32, 40). Although there have been reports of apparent connections
between VTCs and the ER, they appear only when selected viral glycoproteins have been overexpressed (18) or
in cells infected with viruses that mature in the early secretory pathway (21). The structures generated under these
conditions are likely to be tubular elaborations of ER or
ER exit sites in response to the presence of viral proteins.
In general, a large body of biochemical and morphological
evidence is consistent with the conclusion that VTCs are
the first distinct compartment downstream from the ER.
Although VTCs define a unique compartment, their composition remains to be firmly established. During export
from the ER, ribophorin and components of the protein
translocation machinery such as Sec61, as well as resident
ER proteins including the folding chaperones calnexin and
BiP, are efficiently excluded from COPII vesicles (5, 31).
Therefore, they are unlikely to be functional components
of VTCs. An important contribution to the identification of ER to Golgi intermediates came with the discovery of
the closely related (90% identity) VTC marker proteins p53
and p58 (16). p53/p58 are type 1 transmembrane proteins
that continuously recycle between the ER and VTCs but
are concentrated in VTCs at steady state. Other proteins
enriched in VTCs are the small GTPases Rab1 (29) and
Rab2 (7), p24 family members that are potentially involved in cargo selection during export from the ER (10,
36), and other membrane components involved in the targeting/fusion of COP II vesicles (11). To date, there are no
markers that can be defined as resident proteins, emphasizing that VTCs are highly dynamic structures.
VTCs Undergo Maturation in a
COPI-dependent Fashion
After release from the ER, COPII vesicles rapidly lose
their coats (2) and become associated with VTCs containing COPI coats. COPI is involved in the formation of vesicles that promote the retrograde transport of proteins containing terminal KKXX motifs (13, 22) and Phe residues
(12, 37). The mechanism by which VTCs initiate the recruitment of COPI coats has been investigated. Using an
assay that reconstitutes budding from ER microsomes in
vitro (31), it was found that uncoated COPII vesicles can
bind COPI components before their fusion to VTCs. This
coupled exchange between COPII and COPI coats was proposed to serve as a tagging mechanism to mark components for rapid retrieval to the ER from VTCs (31). More
recent studies have demonstrated that p53/p58, possibly in
conjunction with p24 family members, is a component of
the recruitment machinery (39). Therefore, segregation of
retrograde- and anterograde-transported proteins is an important activity of the tubular elements comprising VTCs.
Although COPI vesicles are principally associated with
VTCs and early Golgi compartments, they have recently
also been proposed to participate in ER export. They can
form on yeast ER membranes in vitro (6), and COPI components are closely associated with unusual ER cisternae
in mammalian cells in vivo (27). However, the COPI vesicles formed in vitro from yeast ER membranes lack cargo
molecules typically found in COPII vesicles, and the
COPI-enriched region adjacent to the ER elements in
mammalian cells does not contain buds. Therefore, it is
presently more likely that COPII performs an exclusive
role in sorting and concentration of cargo during ER export, while COPI functions in retrograde retrieval from
VTCs and Golgi compartments.
VTCs Can Be Mobilized to the Golgi Region
Along Microtubules
Morphological evidence has now provided clear support
for a role for microtubules in ER to Golgi transport (34).
When cells are incubated at reduced temperature (15°C),
they accumulate VTCs at peripheral sites. After transfer
to 37°C, these VTCs redistribute to the central Golgi region in a microtubule-dependent fashion. Live cell imaging
using a green-fluorescence protein-cargo chimera (Presley, J.F., N.B. Cole, and J. Lippincott-Schwartz. 1996. Mol. Biol. Cell. 7:74a) revealed that VTCs migrated towards the
Golgi in a saltatory fashion at ~1 µm/s. At the cis face of
the Golgi, they appeared to fuse to form the CGN. Combined with the fact that ER buds are localized to the region surrounding VTCs at steady state (4), it is apparent
that the entire export complex serves as a mobile collecting/recycling device promoting acquisition and delivery of
cargo to the Golgi apparatus.
Formation and Consumption of VTCs
At least two opposing models can be envisioned for the
formation and consumption of VTCs. One model (Fig. 2 A)
suggests that the tubular elements of VTC/CGN are
unique compartments that contain a core of nonrecycling,
resident proteins distinct from those found in either the
ER or the Golgi. In this model, ER-derived COPII vesicles undergo heterotypic fusion with these tubular elements. As described above, retrograde recycling would be
mediated by COPI vesicles. Anterograde transport from
these tubular elements to subsequent Golgi compartments
would most likely require a second round of budding to retain compartment identity. However, COPII function is
complete after export from the ER (2, 5, 31), and genetic
experiments in yeast (13, 22) argue against the involvement of known COPI components in anterograde transport. Therefore, vesicle budding will require either currently unrecognized components of the COPI machinery
that distinguish between anterograde or retrograde transport, or a new coat machinery.
In the second model (Fig. 2 B), tubular elements of
VTCs could form de novo from the homotypic fusion of
ER-derived COPII vesicles. Homotypic fusion between
like compartments is a common feature of both the exocytic and endocytic pathways. In this model, the tubular elements of VTCs would then move en bloc to the central Golgi region, where they could undergo further homotypic
fusion with elements derived from other peripheral sites to
form the CGN (3). During transit to the central Golgi region, COPI vesicles would direct recycling components
back to the ER, thereby maintaining a steady-state balance between input and output of membrane. Although this model is attractive, key evidence that COPII vesicles
or VTCs undergo homotypic fusion is missing.
Movement of Cargo through the Golgi Stack
The method by which VTCs are formed and consumed may
provide critical insight into the ensuing mechanism(s) involved in the movement of anterograde-transported cargo
through compartments of the Golgi stack. Transport will
necessarily involve vesicular carriers since morphological
and biochemical evidence favors the view that individual
compartments are not in continuity with one another (17, 24).
One model for Golgi function is an extension of the
model shown in Fig. 2 A. Here, compartments are discontinuous and each is compositionally distinct. Whereas recycling of transport components could be readily accommodated by the COPI machinery, anterograde transport
would require the function of separate vesicular carriers between each compartment. If their formation also involves COPI, as suggested by Rothman and colleagues
(30), a collection of additional novel proteins will be necessary not only to allow COPI coats to discriminate between anterograde and recycling cargo as indicated above,
but also to direct vectorial movement between sequential
Golgi compartments by distinct carriers.
A second possibility is a direct outgrowth of the homotypic assembly model of VTCs (Fig. 2 B). In this view, VTCs,
once formed, do not release anterograde-transported
cargo into carrier vesicles. Rather, the Golgi stack is composed of a series of progressively maturing, discontinuous
compartments initially formed by the fusion of COPII vesicular carriers and peripheral VTCs. A process of "directed maturation" of each compartment would occur
through the activity of the COPI recycling machinery. In
this case, it would only be necessary to propose that the
COPI machinery mediates retrieval of Golgi processing enzymes in response to its relative affinity for these proteins
reflecting their observed steady-state cis to trans distribution across the stack. Processing enzymes in the cis-most
compartments would have a higher probability of being
recruited to a COPI vesicle than those in the trans-most compartments. Selective recruitment of these enzymes
would necessarily be coupled to specificity determinants
directing vesicle targeting and fusion. In this model, a compartment derived from the fusion of VTCs (lacking processing enzymes) would first become selectively enriched
with cis processing enzymes (Fig. 2 B). As the composition of the maturing compartment changes, so does the competition for the COPI machinery by processing enzymes
within the compartment for recycling, thereby ensuring "self-directed" maturation to the medial- and trans-Golgi states
(Fig. 2 B). The apparent polarized organization of the
stack would be a direct consequence of VTCs continuously contributing to the formation of new, cis-most elements (Fig. 2 B). The TGN, where both COPI-mediated
recycling and clathrin-mediated anterograde transport
would be occurring, may be maintained through additional
input of membrane from the endocytic pathway. The concept of directed maturation was proposed as early as 1957 by Grasse (14) and later refined by Morre and co-workers
(25) as cisternal progression. New knowledge of the importance of COPI in retrograde traffic provides a potential
mechanistic basis for this model.
Evidence for the Directed Maturation Model
Considerable evidence supports directed maturation. First,
it is consistent with a major role for COPI in retrograde
transport. Second, it accounts for the striking dependence
of both retrograde and anterograde transport of cargo on
the COPI machinery because processing of anterograde-directed cargo will not occur when COPI-mediated retrieval
is blocked (2, 13, 31). Third, after mitotic disassembly of the
Golgi, COPI vesicles appear to be markedly enriched in
Golgi processing enzymes relative to anterograde cargo (38). Thus, COPI vesicles may sort and concentrate these
enzymes, consistent with their high diffusional mobility in
the membrane (9). Indeed, the lack of precise compartmental localization of various processing enzymes is compatible with the predicted variable affinity of COPI for different Golgi enzymes. Fourth, retrograde transport is
required to maintain the localization of Golgi processing
enzymes (15, 19) and for Golgi enzymes to maintain the
structure of the stack (26). Fifth, all molecules migrate
through the Golgi stack at a uniform rate, as opposed to
variable rates of exit from the ER because of differences in sorting and concentration via COPII vesicles (3). Moreover, assembled structures found in early Golgi compartments
that are too large to enter carrier vesicles, such as ApoE
containing lipoprotein particles, procollagen, casein submicelles, and scale plates in algae and virus particles, are
able to undergo normal processing by Golgi enzymes during their delivery to the surface. Sixth, numerous observations have established that either under- or overexpression of a wide variety of Golgi "marker" proteins leads to dramatic changes in normal Golgi structure, reflecting a dynamic basis for organization of the stack. Finally, directed
maturation could account for the observation that the regeneration of Golgi stacks occurs at export complexes after treatment with brefeldin A (a reagent that collapses
Golgi compartments to the ER) (8) and during recovery
from mitosis (23).
The Future
We have focused on recent results that now clearly establish the importance of VTCs as key intermediates in the
secretory pathway. New approaches that provide insight
into the mechanism(s) of protein retrieval and anterograde flow should help us understand the molecular basis
for the function of the VTCs in transport of cargo from the
ER to and through the Golgi apparatus.
Fig. 1.
Morphological organization of ER export. A diagram
summarizing the three tiers of organization of ER export complexes. An individual ER cisterna contains a collection of closely
opposed buds that define a local transitional region (light zone
with stippled COPII coated buds [Tier I]). A collection of Tier I
budding sites (cylindrical region outlined by dashed lines forming
Tier II) encompass a central cavity containing a collection of tubular elements (VTCs) that have COPI coats (dense coated
buds). Tier III defines the entire export complex consisting of a
local concentration of ER budding sites and the central VTC. See
cover for a morphological rendering of a typical export complex.
Figure taken from reference 4.
[View Larger Version of this Image (70K GIF file)]
Fig. 2.
Potential models for transport of cargo through the
Golgi stack. (A) The first model illustrates that movement of
cargo between the ER, VTC/CGN, and individual cisternae of
the Golgi stack requires distinct vesicular carriers at each step.
Recycling between stacks is mediated by the COPI machinery.
(B) The second model envisions that COPII vesicles fuse to form
VTC/CGN, which subsequently assemble to form the first stack.
Cargo does not exit the CGN; rather, COPI recycling vesicles
harboring Golgi processing enzymes direct its maturation to
transform it into the trans-most cisternae. New VTCs continuously replenish the cis face.
[View Larger Version of this Image (31K GIF file)]
Received for publication 12 March 1997 and in revised form 24 April 1997.
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