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Cargo Capture and Bulk Flow
in the Early Secretory Pathway
Charles Barlowe1 and Ari Helenius2
1
Biochemistry Department, Geisel School of Medicines, Dartmouth College, Hanover,
New Hampshire 03755; email: [email protected]
2
Institute of Biochemistry, ETH Zurich, Zurich CH-8093, Switzerland
Annu. Rev. Cell Dev. Biol. 2016. 32:10.1–10.26
Keywords
The Annual Review of Cell and Developmental
Biology is online at cellbio.annualreviews.org
ER, Golgi, coat proteins, trafficking, protein secretion
This article’s doi:
10.1146/annurev-cellbio-111315-125016
Abstract
c 2016 by Annual Reviews.
Copyright All rights reserved
Transport of newly synthesized proteins from the endoplasmic reticulum
(ER) to the Golgi complex is highly selective. As a general rule, such transport is limited to soluble and membrane-associated secretory proteins that
have reached properly folded and assembled conformations. To secure the
efficiency, fidelity, and control of this crucial transport step, cells use a combination of mechanisms. The mechanisms are based on selective retention
of proteins in the ER to prevent uptake into transport vesicles, on selective capture of proteins in COPII carrier vesicles, on inclusion of proteins
in these vesicles by default as part of fluid and membrane bulk flow, and
on selective retrieval of proteins from post-ER compartments by retrograde
vesicle transport.
10.1
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Contents
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INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2
SECRETION IS SELECTIVE AND CAREFULLY CONTROLLED . . . . . . . . . . . . . 10.4
ER ARCHITECTURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5
The ER Lumen Is Dynamic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6
Is There a Retention Matrix? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7
ERES AND THE ROLE OF COPII COATS AND VESICLES . . . . . . . . . . . . . . . . . . . . 10.8
CARGO CAPTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9
SOLUBLE CARGO RECEPTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9
ERGIC-53: A Cargo Receptor for Soluble Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . .10.10
p24 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.10
Erv29 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.10
TRANSMEMBRANE CARGO RECEPTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.11
Cornichon Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.11
SREBP and SCAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.11
iRhoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.12
Erv26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.12
ERGIC AS THE NEXT SORTING STATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.12
RETROGRADE SORTING SIGNALS AND RECEPTORS . . . . . . . . . . . . . . . . . . . . . .10.13
BULK FLOW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.14
The Rate of Bulk Flow Is High . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.14
Nonnative Proteins Are Exported . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.16
There Are Few Receptors, Adaptors, and Signal Motifs . . . . . . . . . . . . . . . . . . . . . . . . . . .10.16
Depletion of Receptors and Removal of Signals Do Not Prevent Transport. . . . . . . .10.16
Some Cargo Do Not Concentrate in ERES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.16
WHY CARGO CAPTURE? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.16
Fine-Tuning of Protein Deployment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.17
Acceleration of Secretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.17
Bulky Cargo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.17
Assistance in Folding and Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.17
Maturation Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.17
Inhibition of Premature Ligand Association or Enzyme Activity . . . . . . . . . . . . . . . . . . .10.18
Release from Hydrophobic Mismatch. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.18
Masking of Retrieval Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.18
Creating Membrane Microdomains in ERES that Promote Bulk Flow . . . . . . . . . . . . .10.18
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.18
INTRODUCTION
The endoplasmic reticulum (ER) is responsible for the synthesis, modification, and maturation
of a broad spectrum of proteins that are transported to the extracellular space, the plasma
membrane, and the organelles of the endocytic and secretory pathways. In spite of great diversity
in the amount and nature of the secretory products, the operational principles for selective
transport are shared among eukaryotic cells. The process depends on continuous forward-directed
(anterograde) and backward-directed (retrograde) vesicle transport.
The selectivity in transport depends on five major interdependent strategies. Cargo capture
involves receptor-mediated export of proteins from the ER to the Golgi complex in COPII vesicles.
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CARGO CAPTURE, BULK FLOW, RETENTION, RETRIEVAL, AND ERAD
Cargo capture is a general term for mechanisms in which transport depends on the recognition of selected cargo
by receptors, adaptors, and components of the vesicle coat. In the ER, cargo capture involves loading of secretory
cargo into COPII transport vesicles at ER exit sites (ERES). Such loading requires the presence of export signals
and motifs in fully folded and oligomerized client proteins. The receptors and adaptors usually dissociate from their
ligands when the COPII coat is lost or when the vesicle fuses with the donor compartment. The receptors usually
return to the ER by retrieval pathways.
Export by bulk flow is not dependent on receptors or export signals. Inclusion of soluble or membrane-associated
proteins into COPII vesicles is by default. The cargo molecules are simply included as components of the bulk
membrane and fluid. For selectivity, bulk flow is dependent in part on retention.
Retention is a term used for mechanisms that prevent molecules in the ER from exiting via COPII vesicles. By
limiting the molecules that can enter the vesicles, retention is compatible with both bulk flow and cargo capture
and is involved in establishing selectivity in vesicle transport.
Retrieval is required for the recycling of membrane and some fluid from acceptor compartments [ER-Golgi
intermediate compartment (ERGIC) and cis-Golgi]. Its role is to replace membrane and volume lost from the ER
by anterograde vesicle traffic. Retrieval is also needed to return factors lost from the ER. Because some of the
secretory cargo may also recycle back to the ER, retrieval can influence the rate of cargo secretion.
ER-associated degradation (ERAD) provides a mechanism for the elimination of proteins that fail to achieve
proper folding. These proteins are retrotranslocated from the ER lumen or membrane to the cytosol, where they
are degraded. Given the low folding efficiency of many secretory proteins, the ERAD pathway is essential for
maintaining ER homeostasis.
Bulk flow occurs when proteins and lipids are included in COPII vesicles by default as part of
the fluid and membrane. Retention prevents proteins from entering the transport vesicles, and
retrieval drives retrograde transport from the ER-Golgi intermediate compartment (ERGIC) and
early Golgi compartments back to the ER. Finally, ER-associated degradation (ERAD) eliminates
proteins that fail to fulfill the conformational requirements that allow export to the Golgi complex.
These mechanisms are described in more detail in Figure 1 and the sidebar Cargo Capture, Bulk
Flow, Retention, Retrieval, and ERAD.
The principles that bring about selectivity during cargo capture and bulk flow are distinct but
complementary. During cargo capture, selective loading of mature cargo into transport vesicles is
promoted by receptors, adaptors, and coat proteins that recognize specific signal motifs in folded
and assembled secretory proteins. The motifs may not be available until folding and assembly are
completed. In contrast, during bulk flow, the incompletely folded proteins are recognized and
prevented from entering the vesicles. In this case, the resident molecular chaperones and folding
enzymes determine which proteins are incompletely folded and assembled, and hold them back.
The membrane and fluid continuously lost from the ER during anterograde vesicle transport
must be replaced. At least for membrane components and a selection of soluble proteins, such
replacement occurs by retrograde vesicle traffic from the ERGIC and cis-Golgi (Figure 1). To
prevent a futile transport cycle back to the ER, mechanisms may be in place to limit reflux of
secretory cargo while allowing recycling of components of the membrane container and important
ER factors.
In this review, our main goal is to address some of the general issues regarding the logistics of
membrane traffic in the early secretory pathway. To what extent does transport of protein cargo
from the ER depend on cargo capture and bulk flow? Why do some secretory proteins use one
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cis-Golgi
COPI
Rab6
COPI
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COPII
ERES
ERAD
Endoplasmic
reticulum
Figure 1
Interdependent pathways for selective protein transport between endoplasmic reticulum (ER) and Golgi
compartments. Folding and modification of nascent secretory proteins in chaperone-rich regions of the ER
produce transport-competent cargo. Proteins that fail to reach a fully folded conformational state are
targeted for ER-associated degradation (ERAD) by retrotranslocation from the ER and proteasomedependent proteolysis. Folded cargo migrate to ER exit sites (ERES) and are transported from the ER in
COPII-derived intermediates. For retrograde transport from ERGIC (ER-Golgi intermediate
compartment) and the cis-Golgi, COPI vesicles are formed to retrieve transport machinery, membrane, and
escaped ER-resident proteins. A Rab6-dependent pathway also returns proteins and membrane from Golgi
compartments to the ER through tubular membrane elements. In addition to balanced anterograde and
retrograde trafficking pathways, a robust ER quality control system is critical for secretory cargo to advance.
or the other mechanism? What role does retention play, and how does it work? How important
is retrieval in securing selectivity? These topics have been under intensive discussion for many
years. The literature spans a period of almost 50 years. Where do we stand today? For some
influential reviews from different periods in this ongoing debate, see Bonifacino & Glick (2004),
Borgese (2016), Geva & Schuldiner (2014), Hauri et al. (2000), Herrmann et al. (1999), Hurtley
& Helenius (1989), Hutt & Balch (2013), Lee et al. (2004), Lippincott-Schwartz et al. (2000),
Mayor & Riezman (2004), Palade (1975), Pelham (1994), Pfeffer & Rothman (1987), Warren &
Mellman (1999), and Zanetti et al. (2012).
SECRETION IS SELECTIVE AND CAREFULLY CONTROLLED
The secretory products that we refer to as cargo constitute an extremely heterogeneous group
of molecules with a multitude of special properties, requirements, and conformational subtleties.
Each cell type has its own panel of secretory products. More than a fifth of mammalian proteins
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defined by the genome originate in the ER (Kanapin et al. 2003). During co- and posttranslational
folding in the ER lumen and membrane, a majority of proteins acquire N-linked glycans and
disulfide bonds. Many assemble into homo- or hetero-oligomers. Some represent complexes with
large dimensions such as procollagens, prochylomicrons, and viral particles.
The environment in the ER is optimized for the folding and maturation of proteins for export
(Braakman & Bulleid 2011). Conditions are oxidizing, ATP is available as an energy source, and
the ionic environment is pH neutral with a high concentration of calcium. The lumen is filled with
molecular chaperones, thiol oxidoreductases, prolyl isomerases, and N-linked glycan–modifying
enzymes. The total concentration of protein has been estimated to equal or exceed 100 mg/mL,
which means that the space is extremely crowded (Booth & Koch 1989). When resident proteins
escape to the Golgi complex, which happens at a low rate, they are retrieved by retrograde vesicle
transport (Letourneur et al. 1994, Semenza et al. 1990).
The main role of ER luminal residents is to support the folding and maturation process by interacting with nascent, incompletely folded, misfolded, and unassembled proteins. This is part of an
intricate, carefully balanced system termed proteostasis that involves hundreds of cellular factors
(Hutt & Balch 2013). For individual proteins, the final outcome depends on the thermodynamic
stability of folded and incompletely folded forms and on the kinetics by which various conformational states are reached, maintained, and lost within the ER proteostasis environment (Kowalski
et al. 1998, Wiseman et al. 2007). In this unique milieu, proteins are continuously challenged and
“massaged” by a relentless folding machinery. The selective export of mature secretory proteins
is referred to as ER quality control (QC) (Hurtley & Helenius 1989). QC poses a formidable
challenge because the secretory cargo in the ER is present as a heterogeneous mixture of diverse
proteins in different states of conformation, modification, oligomerization, and aggregation.
ER ARCHITECTURE
The ER is a compartment with a single continuous membrane and luminal space that can occupy
up to 30% of cell volume. It possesses several subdomains with distinct morphology, composition,
and function (Lynes & Simmen 2011, Voeltz et al. 2002). These subdomains include the nuclear
envelope, ribosome-rich sheets, tubular domains, cortical ER sheets, and ER exit sites (ERES). The
size, morphology, and composition vary with cell type and cell function, with conditions tightly
adjusted by the unfolded protein response (UPR) to meet variable growth and stress demands
(Walter & Ron 2011).
The soluble, luminal pool of resident proteins appears to be rather uniformly distributed
throughout the volume of the ER. Most of these proteins have C-terminal H/KDEL sequences
that provide a signal for retrieval from the Golgi complex (Pelham 1991). Many possess, in addition,
negatively charged sequences that serve as low-affinity, high-capacity calcium-binding sites (Booth
& Koch 1989, Sonnichsen et al. 1994).
In contrast to soluble proteins, resident membrane proteins, with some exceptions such as
the chaperone calnexin and BAP31, are restricted to subdomains of the ER membrane (Shibata
et al. 2010, Voeltz et al. 2002). Some of these resident membrane proteins have cytosolic signal
motifs, such as C-terminal KKXX/RRXX, for COPI-dependent retrieval should they leak to postER compartments. In addition to integral membrane proteins, the ER membrane has numerous
peripheral proteins permanently or transiently attached to the cytosolic surface.
ERES represent specialized subdomains enriched in COPII budding machinery and some
secretory cargo. These are the zones where secretory cargo departs in anterograde-directed transport carriers (Bannykh et al. 1996, Orci et al. 1991) (Figure 2). In mammalian cells, ERES
occur as relatively stable structures in approximately 50 to a few hundred locations distributed in
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Sec13-Sec31
complex
Sec23-Sec24
complex
Collagen
trimers
Sar1
GTPase
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Sec16
Tango1-cTAGE5
Folding
Retention
Endoplasmic
reticulum lumen
Figure 2
COPII-dependent protein transport at ER exit sites (ERES). Nascent soluble and integral membrane secretory proteins (brown) are
folded in the ER lumen and are released from ER retention machinery. Cargo capture is catalyzed by the Sec23-Sec24 adaptor protein,
which assembles into ternary cargo complexes with activated Sar1 GTPase. The Sec24 subunit has distinct cargo-binding sites for
recognition of different ER export signals displayed by certain membrane cargo. Next, polymerization of extended Sec13-Sec31
proteins around inner-layer cargo complexes produces a flexible cage that deforms the ER membrane and produces anterograde
transport vesicles. Coat assembly is controlled by the Sar1 GTPase cycle; Sar1 is activated to the GTP-bound state by ERES-localized
Sec12 and is returned to the GDP-bound form by Sec23. Certain soluble, GPI-anchored, and transmembrane cargo are bound by
receptors for efficient linkage to COPII coat subunits. Soluble and membrane-bound proteins not retained by the ER retention
machinery can also exit in COPII vesicles by bulk flow as part of the fluid or the membrane without association with COPII coat
subunits. Large cargo molecules depend on specialized factors for coordination of coat assembly with cargo incorporation. Fully
assembled collagen trimers, which depend on Tango1-cTAGE5, provide an example. COPII subunits and assembly factors such as
Sec12 and Sec16 are highly enriched at ERES.
ribosome-free elements of the so-called transitional ER (Budnik & Stephens 2009, Farhan et al.
2008).
Mobile ER-resident proteins such as calreticulin, BiP, PDI, ERp57, and calnexin are not
detected in the ERES (Balch et al. 1994, Mezzacasa & Helenius 2002). Certain transmembrane
proteins with a single, short transmembrane domain (TMD) are also virtually excluded, although
freely diffusible elsewhere in the ER membrane (Borgese 2016). Given the distribution of a mutant
glycoprotein of vesicular stomatitis virus (VSV-G) and misfolded carboxypeptidase Y (CPY∗ ),
incompletely folded cargo proteins are not only excluded but actively removed from ERES (Hsu
et al. 2012, Mezzacasa & Helenius 2002). These observations indicate that there are mechanisms
that limit access of ER-resident components and incompletely folded proteins to ERES.
The ER Lumen Is Dynamic
Observations using imaging [fluorescence recovery after photobleaching (FRAP), fluorescence
loss in photobleaching (FLIP), and fluorescence correlation spectroscopy (FCS)] indicate that,
in spite of the high protein concentration and the fixed subdomains in the ER, the environment
in both the ER lumen and membrane bilayer is dynamic. Many of the components are mobile.
The diffusion coefficients reported for a small folded reporter protein (GFP-KDEL) is in the
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5–30 μm2 /s range (Dayel et al. 1999, Sbalzarini et al. 2005, Snapp et al. 2006). Diffusion is 3–6fold slower than in the cytosol and 9–18-fold slower than in water. Small transmembrane protein
reporters are also highly mobile (Nehls et al. 2000, Ronchi et al. 2008).
Resident chaperones, like BiP, calreticulin, and calnexin, are mobile but diffuse more slowly,
consistent with a role in engaging client proteins and interacting with other chaperones (Lai et al.
2010, Malchus & Weiss 2010, Ostrovsky et al. 2009, Rolls et al. 2002, Snapp et al. 2006). Large
ER-resident proteins such as the translocon complex are virtually immobile (Nikonov et al. 2002).
When massive misfolding is induced, BiP’s motility is dramatically reduced, and conversely, when
the load of newly synthesized protein is reduced, diffusion of chaperones increases.
For newly synthesized secretory cargo, mobility is variable and depends on the cargo’s folding
status. Some secretory proteins, such as gp120-GFP and tyrosinase-YFP, are mobile when folded
but are effectively immobilized when incompletely folded (Costantini et al. 2015, Kamada et al.
2004). FRAP analysis shows that other cargo, such as the temperature-sensitive folding mutant
(tsO45) of VSV-G and the cystic fibrosis transmembrane conductance regulator (CFTR), are
mobile both when folded and when incompletely folded (Haggie et al. 2002, Nehls et al. 2000).
However, FCS data indicate that the misfolded form of VSV-G at a nonpermissive temperature
undergoes strongly obstructed, anomalous diffusion, consistent with biochemical data that show
association with ER-resident proteins (Malchus & Weiss 2010). Thus, the mobility of severely
misfolded cargo proteins is limited, but some less severely misfolded proteins are readily diffusible.
Is There a Retention Matrix?
The mobility of proteins in the ER is often taken as an argument against retention and in favor of
cargo capture models. Indeed, if cargo capture were the sole mechanism for selective transport,
proteins that lacked the appropriate export signals would simply be left behind. However, there are
observations that speak for active retention. The overexpression of some misfolded proteins leads,
for example, to their export, indicating that they can be exported in their misfolded form when a
retention system is saturated (Hammond & Helenius 1994, Spear & Ng 2003). A similar message
is provided by studies showing that misfolded, mutant proteins retained in the ER are secreted
when binding to ER chaperones is inhibited (Marcus & Perlmutter 2000, Zhang et al. 1997).
Similarly, when the concentration of calcium is lowered, incompletely folded and misassembled
proteins as well as ER-resident proteins escape (Booth & Koch 1989, Pena et al. 2010, Sonnichsen
et al. 1994). It is evident that many incompletely folded and resident proteins are actually transport
competent. That they exit inefficiently under normal conditions suggests that they are held back.
It is well documented that ER-resident proteins have multiple interactions with each other
and with client proteins ( Jansen et al. 2012, Shimizu & Hendershot 2007). For example, BiP,
the Hsp70 family member in the ER, interacts with itself and at least 12 other resident partners.
Between the molecular chaperones, such interactions occur whether client proteins are present
or not. Extensive cross-linking of luminal proteins with each other after addition of chemical
cross-linkers has been interpreted as evidence for interacting networks (Meunier et al. 2002, Tatu
& Helenius 1997). Whereas client proteins are chemically cross-linked to large luminal protein
complexes when they are incompletely folded, they are no longer cross-linked after reaching the
folded state. It has been proposed that the ER contains distinct phases: an immobile phase formed
by resident proteins and incompletely folded proteins and a mobile phase containing proteins in
transit (Pfeffer & Rothman 1987).
It has also been suggested that a luminal network may not only be stabilized by calcium ions but
be rendered dynamic by oscillations in the free calcium concentration, which in the lumen ranges
from 0.1 to 0.8 millimolar (Booth & Koch 1989, Meldolesi & Pozzan 1998, Sambrook 1990). All
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major luminal chaperones and folding factors (PDI, BiP, GRP94, ERp72, and calreticulin) are
high-capacity calcium binders (Nigam et al. 1994). Calreticulin and calsequestrin have sequences
rich in negative residues at their C termini that can bind 20–50 calcium ions. When calcium
concentration in the ER is reduced, interactions between luminal proteins are reduced, some
of the chaperones become nonfunctional, and importantly, both misfolded client proteins and
resident proteins are secreted (Booth & Koch 1989, Meldolesi & Pozzan 1998, Pena et al. 2010,
Sonnichsen et al. 1994).
The question is not whether retention exists. The important inquiries are, rather, how does
retention work, and how is it compatible with the extensive mobility of proteins? One possible explanation is that, rather than producing a rigid matrix, the protein- and calcium-rich environment
promotes the formation of transient, dynamic liquid phases. Various types of liquid phases and
hydrogels have recently been described in the cytosol, nucleus, and nuclear pores, where protein
concentration is high (Hulsmann et al. 2012, Hyman & Simons 2012, Li et al. 2012). Components that partition into these phases remain mobile, but long-distance movement is limited by
phase boundaries. Under conditions resembling the ER, calcium-stabilized, stimulus-responsive
hydrogels can be generated using proteins with C-terminal calcium-binding domains similar to
those in calreticulin and calsequestrin (Dooley et al. 2014). Such liquid phases undergo sol-gel
transitions and are highly dynamic. This property may explain why chaperones and incompletely
folded clients are mobile but still essentially excluded from the ERES.
For membrane proteins, TMD length appears to influence access to ERES. Certain folded
proteins with a TMD shorter than 17 amino acids are excluded from the ERES, whereas proteins
with a 22-amino-acid TMD are not (Dukhovny et al. 2009, Ronchi et al. 2008). TMD length can
also determine whether membrane proteins are exported (Borgese 2016). The lipid composition
and biophysical properties of the membrane in ERES may differ from the rest of the ER, and ERES
may be less accepting of short TMDs. Moreover, some integral membrane cargo with TMDs of
more than 24 amino acids depend on transmembrane adaptor proteins for efficient partition into
ERES, as discussed below.
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ERES AND THE ROLE OF COPII COATS AND VESICLES
Key proteins for assembly of COPII coats at ERES include the integral membrane protein Sec12
and the peripheral protein Sec16 (Figure 2). These factors localize to ERES and regulate binding
of the Sar1 GTPase as well as binding of the two major coat subunits Sec23-Sec24 and Sec13Sec31 (Connerly et al. 2005, Saito et al. 2014, Zanetti et al. 2012). Whereas coat subunits bind to
these subdomains dynamically (with a half-time t1/2 of ∼3 s), ERES punctae can be visually tracked
as stable domains for minutes by live-cell fluorescence imaging (Forster et al. 2006, Shindiapina
& Barlowe 2010). Over time, ERES can form de novo, fuse, and divide. In addition to changes
in size, the total number of ERES per cell varies from one to several hundred, depending on cell
type and status (Bevis et al. 2002; Farhan et al. 2008, 2010; Stephens 2003; Warren 2013; Yelinek
et al. 2009).
The dynamic structure of ERES is consistent with their function as a major sorting hub in the
secretory pathway. The COPII coat performs a critical role not only in carrier vesicle formation
but also in selective loading of cargo (Figure 2). Here Sec23-Sec24 adaptor complexes catalyze
cargo capture through direct binding of export signals to the Sec24 subunit (Miller et al. 2003,
Mossessova et al. 2003). Extended Sec13-Sec31 proteins then polymerize around these inner-layer
Sec23-Sec24 complexes to produce cage-like structures that deform the ERES membrane and bud
anterograde transport vesicles (Fath et al. 2007, Matsuoka et al. 2001, Stagg et al. 2006). Sar1 GTP
recruits Sec23-Sec24 to ERES membranes and stabilizes interactions with cargo. However, cargo
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binding to Sec23-Sec24 is dynamic, and the Sar1 GTPase cycle can be harnessed to concentrate
cargo into forming vesicles (Tabata et al. 2009). ERES localization of the Sar1 guanine nucleotide
exchange factor (Sec12) and GTPase-activating protein (Sec23) likely drives flux through this
GTPase cycle to enrich cargo complexes at vesicle formation sites (Miller & Barlowe 2010).
To accommodate the diversity of cargo that must transit through the ERES, cells express
multiple paralogs of the Sec24 adaptor protein, each with up to four nonoverlapping cargo recognition sites. Moreover, there are multiple paralogs of the Sar1, Sec23, and Sec31 subunits as well
as specific covalent modifications that permit flexible assembly of the COPII coat to package
cargo of various shapes and sizes, including large, rod-like procollagen molecules (∼300 nm) and
prechylomicrons (Zanetti et al. 2012).
CARGO CAPTURE
A growing number of transmembrane secretory proteins have been found to display defined
cytosolic sorting signals, such as the diacidic DXE motif, that bind directly to Sec24 adaptor
subunits and increase efficiency of packaging into ER-derived transport carriers (Miller et al.
2003, Mossessova et al. 2003). However, cargo diversity and requirements for regulated ER export
appear to have dictated a need for adaptors and accessory factors that operate in concert with the
core COPII machinery. Some 13 different transmembrane cargo receptors and adaptors have been
identified in yeast, and approximately 24 have been identified in mammalian cells.
For some soluble secretory cargo and many membrane proteins, efficient sorting into COPII
carriers depends on transmembrane receptors that physically link cargo with coat subunits. In
these cases, receptors accompany their cargo into anterograde carriers until cargos are released
in post-ER compartment and then the empty receptors are retrieved. Other accessory factors
facilitate packaging of proteins into COPII carriers but do not travel with their clients. Illustrative
examples of each type of cargo receptor and adaptor are considered below. However, for many
membrane and most soluble cargo, ER export signals and machinery for recognition have not
been defined. Without a more comprehensive inventory of factors and underlying mechanisms,
it is challenging to predict which cargo depends on capture for ER export.
The clients for cargo capture are the mature versions of secretory proteins. In some cases,
it is clear why the receptors may not interact with incompletely folded proteins also present
in the ER. Some of the receptors are lectins specific for extensively trimmed, high-mannose
N-linked glycans in proteins that have passed the QC steps imposed by the calnexin-calreticulin
cycle (Appenzeller et al. 1999). GPI-anchored proteins provide another example wherein binding
occurs with p24 protein receptors once an ethanolamine-phosphate group has been enzymatically
removed from the glycan group by a QC enzyme (Manzano-Lopez et al. 2015). In this context,
it is important to mention that cargo capture is fully compatible with retention mechanisms that
prevent incompletely folded proteins from accessing ERES regions. If a cargo receptor is localized
in the ERES, an export signal may be present but not available for receptor binding until the client
protein is fully folded.
SOLUBLE CARGO RECEPTORS
Quantitative immunoelectron microscopy showing concentration of certain soluble secretory proteins between ER and Golgi compartments suggested that cargo was selectively enclosed within
membrane-bound compartments (Bendayan et al. 1980). In vitro assays that reconstituted packaging of cargo into COPII vesicles documented concentration of cargo into carrier vesicles and
physical linkage of soluble cargo to COPII subunits (Kuehn et al. 1998, Salama et al. 1993). These
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findings suggested that uptake of soluble cargo is receptor mediated. Studies on ERGIC-53 and
the p24 proteins provided the first candidates for transmembrane cargo receptors that mediate
ER export of soluble cargo.
ERGIC-53: A Cargo Receptor for Soluble Glycoproteins
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ERGIC-53 (LMAN1) is a single-pass transmembrane protein that rapidly cycles as a homohexamer between ER and Golgi compartments and has both COPI and COPII sorting signals in its
cytoplasmic tail (Kappeler et al. 1997). The luminal domain consists of an L-type lectin domain
that binds to high-mannose oligosaccharides in a calcium-dependent manner (Itin et al. 1996).
Hauri and colleagues showed that ERGIC-53 acts as a cargo receptor for procathepsin and α1 antitrypsin after they have passed QC in the calnexin-calreticulin cycle (Appenzeller et al. 1999,
Nyfeler et al. 2008).
Study of human blood clotting disorders also converged on ERGIC-53, as the combined
deficiency of coagulation factor V and factor VIII mapped to the genetic locus that encodes it
(Nichols et al. 1998). Loss-of-function mutations cause a 70–90% decrease in the serum levels of
coagulation factors (Zhang 2009). A second gene termed multiple coagulation factor deficiency
gene 2 (MCFD2), which produces a similar phenotype, encodes a small, peripheral calcium-binding
protein that associates with the ERGIC-53 lectin domain. MCFD2 is thought to specify ER export
of certain glycoprotein cargo (Nyfeler et al. 2006).
The recognition mechanism is clearly carbohydrate based, although additional protein elements within folded cargo can be important for efficient binding and ER export (AppenzellerHerzog et al. 2005). Interestingly, in cells that lack functional ERGIC-53, clients ultimately reach
their proper destination. The slower transport may represent bulk flow or may depend on loweraffinity binding to other broad-specificity cargo receptors. Notably, ERGIC-53 belongs to a family
of calcium-dependent L-type lectins in animal cells; this family includes ERGL, VIP36, and VIPL
(Nufer et al. 2003). Defined cargo for these potential receptors has not been described.
p24 Proteins
Proteomic analyses identified the p24 proteins as major constituents of COPI- and COPII-coated
vesicles (Schimmoller et al. 1995, Stamnes et al. 1995). They share membrane topology and
dynamic ER-Golgi cycling with ERGIC-53 and are abundant in the early secretory pathway. The
large p24 family has been divided into four subfamilies termed p24α, p24β1, p24γ, and p24δ
(Dominguez et al. 1998). Vertebrates express 10 distinct p24 proteins. These proteins often occur
in heteromeric complexes, and in some cases the stability of specific subunits is interdependent
(Strating et al. 2009).
Yeast strains that lack p24β (Emp24) or p24δ (Erv25) display a delay in the transport of the
soluble cargo protein invertase and the GPI-anchored membrane protein Gas1 (Schimmoller
et al. 1995). Mammalian cells also have p24 family members that bind directly to specific GPIanchored cargo and promote their export (Bonnon et al. 2010, Takida et al. 2008). Recent work
indicates that the p24 receptor complex binds the fully remodeled GPI-glycan moiety as a lectin
(Manzano-Lopez et al. 2015).
Erv29 Proteins
Biochemical approaches show that gpαf is linked to COPII subunits through the transmembrane
protein Erv29 (Belden & Barlowe 2001). Concentrative sorting of gpαf into COPII vesicles is
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increased 20-fold relative to the bulk flow marker GFP-HDEL, and ER membranes lacking Erv29
package both gpαf and GFP-HDEL at comparably low levels (Malkus et al. 2002). In vivo, erv29
yeast cells display a 5–10-fold decrease in transport rates of client proteins, including gpαf (Belden
& Barlowe 2001, Caldwell et al. 2001). Moreover, Erv29 exhibits the properties expected of a
cargo receptor; overexpression of Erv29 cargo results in ER export saturation, which is alleviated
by increasing the expression level of Erv29 (Belden & Barlowe 2001). The proregion of gpαf
contains a hydrophobic I-L-V signal that is necessary and sufficient for Erv29-dependent export
(Otte & Barlowe 2004).
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TRANSMEMBRANE CARGO RECEPTORS
Although certain soluble cargo proteins may rely on transmembrane receptors for ER export,
transmembrane secretory cargo should be able to present ER export signals directly to the COPII
budding machinery. However, there are a growing number of examples in which integral membrane secretory proteins depend on transmembrane adaptors for linkage to COPII subunits and
for efficient uptake into COPII vesicles. The Cornichon proteins were among the first to be identified; Drosophila cornichon mutants failed to deliver a TGFα family member to the oocyte cell
surface (Roth et al. 1995). Studies of a homologous Cornichon protein in yeast, termed Erv14,
demonstrated that the protein actively cycles between ER and Golgi compartments and binds to
specific integral membrane cargo for transport from the ER (Powers & Barlowe 1998).
Cornichon Proteins
The Cornichon proteins are small hydrophobic proteins with three transmembrane segments
and cytosolic regions that contain potent COPI and COPII transport signals (Pagant et al. 2015,
Powers & Barlowe 2002). To date, several integral membrane cargo, including TGFα, AMPA receptors, and G protein–coupled receptors (GPCRs), are known to depend on Cornichon function
for efficient delivery (Bokel et al. 2006, Sauvageau et al. 2014, Schwenk et al. 2009). Systematic
studies in yeast have identified more than 30 transmembrane cargo proteins that accumulate in
the ER when cells are deleted for Erv14 (Herzig et al. 2012, Pagant et al. 2015). Most of these proteins are polytopic plasma membrane proteins with the longer transmembrane segments that are
common among late endomembrane proteins (Sharpe et al. 2010). Indeed, experimental evidence
indicates that TMD length controls Erv14 dependence for ER export of specific cargo (Herzig
et al. 2012).
Current data support a model in which Erv14 binds to proteins with lengthy TMDs and ushers
them into COPII vesicles through interactions with Sec24. Recent findings also show that some
transmembrane secretory cargo depend on direct interactions with both Erv14 and Sec24 adaptor
subunits for efficient ER export (Pagant et al. 2015). The use of multiple signals in receptorcargo complexes and the oligomeric structure of many of the known COPII cargo receptors (e.g.,
ERGIC-53 and p24 proteins) may provide a means to increase affinity and selectivity of ER export.
SREBP and SCAP
Reliance on cargo receptors provides the cell with opportunities to regulate traffic. Perhaps the
best-characterized example of ER export control is the sterol regulatory element–binding proteins (SREBPs). They constitute a family of membrane-bound transcription factors that are proteolytically processed at the Golgi complex to produce soluble transcription factors that turn
on nuclear genes necessary for increasing cholesterol levels. SREBP2 binds to and depends on
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the multimembrane-spanning SCAP protein for packaging into COPII vesicles (Goldstein et al.
2006). SCAP also contains a sterol-sensing domain, and under high ER cholesterol levels, the
protein adopts a conformation that shields its potent COPII export signal and retains SREBP2
in the ER. When cholesterol levels fall, SCAP undergoes a conformational change that exposes
this COPII-binding motif for efficient transport of SCAP-SREBP2 complexes to the Golgi complex, where resident proteases liberate the cytosolic transcription factor for activation of sterol
biosynthetic genes (Zhang et al. 2013). This is an example of how a transmembrane cargo protein
depends on a membrane adaptor for regulated ER export.
iRhoms
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Other examples in which integral membrane secretory cargo rely on transmembrane COPII
adaptor proteins are the iRhoms (inactive homologs of rhomboid proteases) and yeast Erv26.
Members of the iRhom family of catalytically inactive intramembrane proteases have preserved
active-site architecture to bind specific type I transmembrane client proteins in the ER. In the
case of mammalian cells, the single-pass transmembrane protein ADAM17 fails to exit the ER
in the absence of iRhom2 (Adrain & Freeman 2012). In contrast, the Drosophila melanogaster
iRhom binds to EGF-like ligands in the ER, but instead of incorporating these proteins into
COPII vesicles, the cargo clients are directed to ERAD (Zettl et al. 2011). The iRhoms thus play
a role in regulating cellular expression of certain signaling proteins. How the iRhoms interact
with ERAD components and COPII export machinery remains to be worked out. But the use of
pseudoenzymes as transmembrane adaptors to regulate protein delivery may be a more general
phenomenon (Adrain & Freeman 2012).
Erv26
A tetraspanning transmembrane protein, Erv26 cycles between the ER and Golgi complex for
anterograde transport of several type II transmembrane proteins. The cargo includes vacuolar
alkaline phosphatase and several Golgi-localized glycosyltransferases (Bue & Barlowe 2009, Herzig
et al. 2012). Typically, these cargo proteins have larger luminal domains and short cytosolic tail
sequences. Erv26 appears to bind to the luminal domain of alkaline phosphatase (Dancourt &
Barlowe 2009), which may be distinct from the TMD interactions exhibited by Cornichon proteins
and iRhoms. However, the Erv26 cargo receptor may perform a similar role in regulated transport
of specific cargo. Alternatively, the topology of certain type II membrane cargo may necessitate
adaptors for efficient linkage to COPII coat subunits.
ERGIC AS THE NEXT SORTING STATION
After shedding their coat, COPII vesicles loaded with biosynthetic cargo either fuse with one
another to form the ERGIC or fuse with preexisting ERGIC elements (Figure 1) (Hauri &
Schweizer 1992). Imaging of GFP-tagged VSV-G secretory cargo shows dynamic movement of
pleomorphic vesicular-tubular clusters along microtubules to the cis-Golgi (Presley et al. 1997).
Whether the move to the cis-Golgi involves maturation of ERGIC membranes into early Golgi
cisternae or detachment of cargo-rich domains from a more stable ERGIC compartment remains
an open question (Appenzeller-Herzog & Hauri 2006).
For retrograde membrane traffic back to the ER, two distinct mechanisms are known: COPI
vesicles and Rab6-mediated tubular membrane elements. The COPI vesicles are generated on
ERGIC and Golgi membranes (Scales et al. 1997). Activation of the small GTPase Arf1 results
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in the recruitment of coatomer proteins to the cytosolic surface. The assembled COPI coat, in
turn, binds membrane proteins that have retrograde sorting signals in their cytosolic domains
(Letourneur et al. 1994). The mechanism involving the Rab6 GTPase is tubulation of cis-Golgi
elements without involvement of a visible coat (Heffernan & Simpson 2014). The cargo identified
so far includes Golgi enzymes, some bacterial toxins, and Erp44. Although information about this
pathway is limited, evidence indicates an important role for this pathway in Golgi homeostasis
(Sengupta et al. 2015). The high surface-to-volume ratio provided by the tubular geometry may
help to limit retrieval of soluble proteins by bulk flow.
The volume of retrograde transport must be high to balance the loss of membrane and critical
components from the ER caused by anterograde traffic. Estimates of COPII vesicle traffic in
mammalian tissue culture cells indicate that a volume and membrane area corresponding to those
of the full volume and surface of the ER are exported every 1 or 2 h. Whereas water and solutes
may be replaceable without membrane traffic, retrieval of membrane components must depend
on such traffic. To avoid a futile cycle for secretory cargo, transport has to be selective. That
selective retrieval can result in the enrichment of secretory cargo in the ERGIC has been observed
for amylase and chymotrypsinogen in pancreatic acinar cells (Martinez-Menarguez et al. 1999).
However, it is likely that retrieval of outward-directed cargo occurs to some extent and influences
the rates of anterograde protein transport (Fossati et al. 2014).
RETROGRADE SORTING SIGNALS AND RECEPTORS
Retrieval of many proteins from the ERGIC and cis-Golgi to the ER is based on cargo capture.
Transmembrane proteins that cycle between the ER and the ERGIC/Golgi complex have, in their
C termini, dilysine sorting signals (KKXX or KXKXX) that are recognized by the COPI coat.
Context-specific recognition of dilysine motifs by COPI are also thought to direct transmembrane
cargo to their proper cellular compartment (Papanikou & Glick 2014). Some soluble cargo depend
on transmembrane receptors that link up with COPI coat subunits. The C-terminal H/KDEL
motifs on soluble ER-resident proteins, including BiP and PDI, allow for specific binding to the
transmembrane KDEL receptor (Munro & Pelham 1987, Semenza et al. 1990). Multiple KDEL
receptor isoforms are present in animal cells to efficiently retrieve ER residents that contain
variations of the KDEL motif (Raykhel et al. 2007).
Type II transmembrane proteins such as Sec12 cannot present C-terminal dilysine motifs for
COPI recognition. In this context, genetic screening uncovered the conserved Rer1 protein, a
multispanning transmembrane protein with a COPI sorting signal (Boehm et al. 1997, Sato et al.
2003). Rer1 binds to escaped ER membrane proteins in post-ER compartments through TMD
interactions (Fullekrug et al. 1997, Sato et al. 2003). More recently, a third class of retrograde sorting receptor was identified and characterized through proteomic analysis of ER-derived transport
vesicles (Otte et al. 2001). The Erv41-Erv46 complex is required for ER localization of soluble
ER-resident proteins that lack a C-terminal KDEL motif, including glucosidase I and the prolyl
isomerase FKBP-13 (Shibuya et al. 2015). The signal recognized in these soluble cargo remains
to be determined.
Retrograde traffic serves a backup QC function by retrieving incompletely folded or assembled
secretory proteins that escape the ER either alone or in complex with chaperones (Hammond &
Helenius 1994, Sato et al. 2004, Vavassori et al. 2013, Yamamoto et al. 2001). For heteromeric
membrane proteins such as potassium channels, exposure of arginine-based RXR retrieval signals
in unassembled subunits results in COPI recognition and retrieval to the ER. When the channels
are fully assembled, specific 14-3-3 proteins are recruited to mask the signals and permit forward
transport (Michelsen et al. 2005).
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Molecular chaperones localized in ERGIC and Golgi compartments are in some cases responsible for retrieval of incompletely folded or assembled secretory cargo to the ER. After association
with incompletely assembled IgM subunits, ERp44 (a PDI family member) exposes its C-terminal
KDEL motif and escorts the IgM back to the ER, where the more neutral pH allows for IgM
dissociation (Vavassori et al. 2013). In addition to serving as a retrieval device, ERp44 together
with ERGIC-53 assists the oxidative oligomerization of IgM and possibly other clients such as
adiponectin (Cortini & Sitia 2010, Sannino et al. 2014, Vavassori et al. 2013, Wang et al. 2007).
The distribution of ERp44 across early secretory compartments is thought to impart the progressive QC necessary in professional secretory cells (Anelli et al. 2015). In this manner, the ERGIC
and cis-Golgi can be viewed as partners with the ER in promoting protein maturation in an iterative process that ensures that only legitimate cargo advances whereas incompletely folded proteins
are retrieved.
The possibility that a retention-based process also plays a role in retrograde traffic has recently
emerged. The diacidic DXE motif was found to limit backward transport of VSV-G and other
membrane cargo via the Rab6 pathway (Fossati et al. 2014). This finding implies that a motif that
promotes recruitment into COPII vesicles may later ensure unidirectional progression through the
Golgi complex, perhaps by excluding cargo from recycling vesicles. A C-terminal valine residue,
which is common in type I membrane proteins, has also been proposed to serve as an anterograde
signal by associating with Golgi matrix proteins (GRASP65 and -55) (D’Angelo et al. 2009).
Finally, COPI acting in concert with CDC42 may promote forward transport in the Golgi complex
by targeting cargo to tubular Golgi elements (Park et al. 2015).
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BULK FLOW
The existence of several cargo capture mechanisms at the levels of both anterograde and retrograde
traffic in the early secretory pathway is undisputed. A role for bulk flow is not as well established.
That bulk flow takes place is a given; every transport vesicle has the capacity to carry lipids,
membrane proteins, soluble proteins, and solutes by default. It is also clear that a secretory pathway
based entirely on bulk flow (i.e., free diffusion of cargo in and out of anterograde and retrograde
vesicles) can, in principle, support net movement of cargo from the site of synthesis to downstream
organelles simply by partitioning.
It has not been easy to obtain experimental evidence that bulk flow actually does play a role
in the transport of secretory proteins from the ER. The main reason for this difficulty is that the
bulk flow model implies by definition that no factors are involved beyond the machinery needed
for retention and ongoing vesicle traffic. Thus, most arguments in favor of bulk flow are indirect
or based on negative data. Taken together, the observations and arguments discussed below do,
however, make a strong case for bulk flow combined with retention and retrograde retrieval as a
central player in secretory transport.
The Rate of Bulk Flow Is High
An important question to consider first is whether the rate and efficiency of bulk fluid and membrane flux from the ER are high enough to support the rates observed in protein export. To
measure bulk flow, inert tracer molecules that are formed in the ER, a closed compartment, must
be used. The tracers must be released rapidly, and they cannot be delayed or accelerated en route
by interactions with cell components. When the secretion of such an inert tracer (or a secretory
protein) is followed, for example, by a pulse-chase approach, the time course and efficiency of exit
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can be determined, and estimates can be obtained for the fastest possible transport as well as for
the half-times (t1/2 ) for arrival in the medial Golgi and plasma membrane.
In a highly influential paper, Wieland and colleagues measured fluid flow in CHO and HepG2
cells using a membrane-permeable, iodinated acyl-tripeptide as a soluble bulk flow tracer (Wieland
et al. 1987). The authors observed that the first glycosylated tripeptides were secreted 7–22 min after addition of the tracer. As expected of a bulk flow process, the rate was independent of tripeptide
concentration over several orders of magnitude. Secretory proteins moved through the pathway
more slowly than the tracer did. Although no t1/2 was measured, and although there are concerns
about the assumptions of these experiments, including the possibility that glycotripeptides follow
a nonvesicular route out of cells (see references in Romisch & Schekman 1992 and Thor et al.
2009), the study was important because it introduced bulk flow (combined with retention) as a
realistic mechanism for selective ER export.
A more recent pulse-chase study used a soluble protein as a fluid phase tracer in CHO and
MDCK cells (Thor et al. 2009). The protein was a small, rapidly folding, cytosolic protein of viral
origin devoid of disulfide bonds and glycosylation sites. The first labeled tracer was detected in
the extracellular fluid within 12 min after synthesis, and the t1/2 of secretion was 40 min. This
finding implied that an amount of fluid equivalent to half of the ER volume is transported out
of the ER every 40 min. This rate corresponds to 155 COPII vesicles per second, assuming no
backflow of tracer. Given the number of ERES in CHO cells (approximately 400), this rate of
secretion adds up to one COPII vesicle every 3 s from each ERES. Interestingly, a similar rate has
been estimated in Trypanosoma brucei cells, which have a single ERES (Warren 2013). In polarized
epithelial MDCK cell monolayers, the rate of bulk flow of the viral tracer to the apical and the
basolateral surfaces was the same, but with five times more secretion apically.
Importantly, the t1/2 of export of the viral capsid tracer was higher than for endogenous secretory proteins and, in fact, equal to or faster than has been measured for any secretory protein
in mammalian cells. In mammalian cells, the lowest t1/2 values for arrival of secretory proteins
to the medial Golgi are 10–15 min and for arrival to the plasma membrane are ∼35–40 min
compared with ∼8 min in yeast (Fries & Lindstrom 1986, Geiger et al. 2011, Losev et al. 2006,
Wieland et al. 1987). Significantly, the large differences in transport rates observed for different
proteins were initially used as an argument for receptor-mediated mechanisms of export (Fitting &
Kabat 1982, Lodish et al. 1983). Later studies showed that proteins become export competent
only once properly folded and oligomerized and that the differences in secretion are caused mainly
by differences in folding rates (Hurtley & Helenius 1989).
For membrane components, the bulk flow rate is not so clear. Direct measurements using inert
tracers do not exist to our knowledge. If 155 spherical COPII vesicles with a radius of 35 nm
(Barlowe et al. 1994) leave the ER per second, our calculations show that the t1/2 of bulk export of
membrane protein to the plasma membrane would be approximately 37 min, assuming that the
surface area of the ER in a CHO cell is half that of the twice larger BHK21 cells (Griffiths et al.
1984). The rate of membrane flow is thus likely to be similar to that of bulk flow of fluid and to
be sufficient to support the rate observed for the fastest among membrane proteins (Boncompain
et al. 2012, Copeland et al. 1986, Fossati et al. 2014, Nishimura et al. 1999, Strous & Lodish 1980,
Williams et al. 1985). After release from an artificial, streptavidin-based “hook” by the addition
of biotin, TGFα and VSV-G can be seen in videos to enter ERES within 2–5 min (Boncompain
et al. 2012). In tissue culture cells, the fastest membrane proteins arrive at the medial Golgi with
a t1/2 of 10–15 min and at the plasma membrane after approximately 30 min.
Taken together, the available data, although limited, indicate that bulk flow of fluid is likely to
be fast enough to support the rate of protein transport observed in the secretory pathway. Although
less well documented, the outward bulk flow of membrane probably occurs at comparable rates.
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Nonnative Proteins Are Exported
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Heterologous proteins, proteins of cytosolic and bacterial origin, and foreign secretory proteins are
often efficiently secreted when targeted to the ER. Examples include GFP, horseradish peroxidase,
peroxisomal proteins, and bacterial beta-lactamase (Bard et al. 2006, Brandizzi et al. 2004, EidenPlach et al. 2004, Hirz et al. 2013, Shi et al. 2007, Thor et al. 2009, Wiedmann et al. 1984).
The lack of selectivity in secretion is often exploited to produce secreted biopharmaceuticals and
industrial catalysts (De Meyer & Depicker 2014, Fitzgerald & Glick 2014, Loos et al. 2011).
CHO cells, fungi, and plants are usually employed. The bottleneck for secretion usually involves
folding and apoptosis, not trafficking (Kim et al. 2012). All these findings argue for bulk flow in
these cases because nonnative, heterologous proteins are unlikely to carry export signals, and cells
would not be expected to possess cargo receptors for cytosolic proteins and cargo that they do not
normally produce. Furthermore, it is not necessary to express full-length proteins to have efficient
secretion. Truncated folding-competent domains are often efficiently secreted (El Omari et al.
2014, Gaudin et al. 1999, Singh et al. 1990, Zhang et al. 1997). Because each domain is unlikely
to have an export signal, bulk flow seems likely for such proteins.
There Are Few Receptors, Adaptors, and Signal Motifs
The number of cargo receptors and adaptors identified so far especially for soluble proteins is
small and does not cover more than a small fraction of the cargo. It is possible that additional
receptors/adaptors exist and that some of them have multiple clients. However, that some of the
cargo exits the ER without cargo capture seems more likely.
The number of known export motifs in secretory proteins is also modest. Especially for soluble
proteins, few motifs have been identified. Those motifs that are known do not seem to mediate
association of more than a handful of clients. Although there are probably more sorting signals
on cargo and additional binding sites on the Sec24 isoforms, there is a real possibility that some
proteins exit the ER without export signals.
Depletion of Receptors and Removal of Signals Do Not Prevent Transport
Loss of signal motifs, adaptors, and receptors does not block export but only delays it. For example,
the loss of p24-binding sites on the Lst1p of COPII causes a 40% reduction in the rate of GPIanchor protein export (Castillon et al. 2011). For erv29 deletions, the proalpha factor and CPY
cargo accumulate in the ER, and transport rates in yeast are 5–10-fold reduced. The slowed export
may in such cases depend on bulk flow.
Some Cargo Do Not Concentrate in ERES
Whereas cargo capture ligands are concentrated in ERES, many secretory proteins are not. One
example is provided by the zymogens in pancreatic cells (Martinez-Menarguez et al. 1999).
WHY CARGO CAPTURE?
If bulk flow of fluid and membrane is as fast and efficient as implied above, one must consider
why cells have evolved elaborate systems for cargo capture and other transport-promoting and
transport-accelerating mechanisms. Why not rely on bulk flow for all cargo?
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Fine-Tuning of Protein Deployment
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An important reason in favor of cargo capture is the opportunity it provides to regulate posttranslational protein deployment. It allows for regulation at the level of individual proteins and
protein classes without affecting other cargo. Thus, the expression of a specific receptor protein
in the plasma membrane may, for example, be adjusted dynamically in response to external or
physiological stimuli. Regulation can be coupled to signaling pathways.
In addition to the role of SCAP and iRhom2 discussed, a well-studied example of regulation
is provided by GPCRs, for which many membrane-associated and cytosolic effector molecules
that regulate ER-to-Golgi traffic have been identified (Achour et al. 2008, Dong et al. 2007,
Sauvageau et al. 2014). One such effector is Rab acceptor family 2 (PARF2), a protein that serves
as a gatekeeper in the ER for the GB1 subunit of the GABAB receptor (Doly et al. 2015).
Acceleration of Secretion
Accelerating export from the ER beyond the bulk rate can be advantageous for cargo that must
rapidly reach its destination organelle or be rapidly removed from the ER. Rapid arrival of VSV-G
at the plasma membrane may, for example, be important during acute virus infection, when the
virus competes with the immune system. The rate of secretion of TGFα in Drosophila development
(Roth et al. 1995) and of Axl2, a polarity factor in yeast, is likely to be important in fast-growing
cells (Herzig et al. 2012).
Bulky Cargo
For large cargo such as procollagens and prechylomicrons, systems that recognize the cargo and
promote the formation of larger vesicles are needed (Malhotra et al. 2015). The inclusion of large
membrane protein clusters or membrane proteins with awkward geometries may also require
special arrangements. Large-molecular aggregates with slow diffusion may also depend on special
loading mechanisms.
Assistance in Folding and Assembly
Some factors may combine the functions of a molecular chaperone and escort by stabilizing poorly
folding proteins and accompanying them to the Golgi complex. The receptor-associated protein
(RAP) assists as a soluble molecular chaperone involved in the proper oxidative folding of a family
of receptors (Bu 2001). In addition, it prevents premature interaction with ligands expressed in the
same compartment and accompanies the receptors to the Golgi complex. Shr3, Gsf2, Pho86, and
Chs7 in yeast are chaperones that associate with amino acid and hexose permeases during their
folding and prevent aggregation as well as ERAD (Kota & Ljungdahl 2005, Kota et al. 2007).
7B2 is a small soluble protein that accelerates PC2 prohormone-converting enzyme export by
stabilizing the transport-competent conformation (Muller et al. 1997). In addition to serving as
a cargo capture receptor for some glycoproteins, ERGIC-53 is an assembly workbench during
oxidative oligomerization of IgM molecules (Cortini & Sitia 2010).
Maturation Sensing
Some receptors serve as sensors that distinguish between intermediates and the final cargo products. This seems to be the case for specific p24 proteins, which are lectins that bind to the fully
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modified glycan in GPI-anchored proteins and accelerate transport from the ER (Manzano-Lopez
et al. 2015). It is also possible that certain fully folded cargo display surface features that continue
to interact with ER-resident proteins such that receptors are needed to bind to these surfaces for
efficient partitioning into ER exit sites and incorporation into COPII vesicles.
Inhibition of Premature Ligand Association or Enzyme Activity
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The function of some export factors is to prevent their clients from expressing their function either
in the ER or en route to the target organelle. The activity to be suppressed can be proteolytic or
can involve association with other proteins. An example is the invariant chain of MHC class II
antigens. The invariant chain associates with newly synthesized MHC class II alpha-beta-dimers
and helps them undergo folding while preventing premature peptide binding (Castellino et al.
1997). The invariant chain then escorts antigens via the Golgi complex to endosomes.
Release from Hydrophobic Mismatch
As discussed above, the ER membrane bilayer is too thin for the TMDs of many proteins destined
for export to thicker downstream membranes (Ronchi et al. 2008). Erv14 (Cornichon, CNIH4)
is an example of an escort for such cargo (Herzig et al. 2012).
Masking of Retrieval Sequences
First identified in GABABR1 receptors, a C-terminal RXR motif provides a retrieval signal that
interacts with COPI and drives membrane protein retrieval to the ER. Transport is inhibited by
masking of the signals during oligomerization and by interaction with 14-3-3 proteins (Smith et al.
2011).
Creating Membrane Microdomains in ERES that Promote Bulk Flow
Two consequences of collecting cargo receptors and their cargo in the ERES may be to accelerate
vesicle formation and to generate a membrane microdomain with properties that allow inclusion
of cargo that by itself is devoid of export signals (Borgese 2016). In other words, the cargo capture
process may promote bulk flow.
SUMMARY
The data that we review above make evident that there is no single mechanism behind the exceptional specificity and efficiency observed in cargo transport in the early secretory pathway.
Although cargo capture is important and necessary in many different ways, it operates together
and in parallel with retention, retrieval, and bulk flow. Hundreds of proteins are involved in providing selectivity to cargo movements by either preventing or promoting the inclusion of cargo in
carrier vesicles. In addition to supporting protein maturation, the proteostasis network in the ER,
with its multitude of molecular chaperones and folding factors and its cell type–dependent adjustments, is a major determinant in selective export. Different proteins and cell types have evolved
to exploit the rich opportunities provided by a complex and flexible system in many imaginative
and sophisticated ways.
Unfortunately, the data available on quantitative aspects of the different pathways are limited.
Reliable understanding of the magnitude, rates, and efficiencies of vesicle transport is largely
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lacking. Hard data are also missing about surface areas and volumes of the compartments involved,
about the concentrations and distribution of molecules, and about changes induced by external
stimuli and physiological states. The biophysical properties of luminal environments of the ER
and the ERES remain poorly understood. The possibility that partitioning of proteins between
liquid phases in the ER lumen plays a role in retention, and that the domain structure within the
membrane defines access of membrane proteins to the ERES, needs to be pursued. The role of
tubular membrane elements and retrieval mechanisms from the ERGIC and the Golgi complex
is also incompletely analyzed.
It is time to step back and address some of the basic, general topics again by using powerful new
technologies that allow quantitative analysis of complex processes in live cells. There is a lot to be
learned. How are organelle surface area and volume maintained in the face of the rapid fluxes? Is
most of the cargo handled by cargo capture, or is cargo capture limited to clientele with special
requirements? Is the secretory mechanism in professional secretory cells different from that in cells
with a more modest secretory program? How is the export of specific proteins regulated? Such
topics are highly relevant for understanding cell function and for apprehending and controlling a
variety of diseases and disorders caused by defects in protein expression and secretion.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank Maya Schuldiner and Ineke Braakman for comments on this review. C.B. acknowledges
support from the National Institutes of Health. A.H. acknowledges support from the European
Research Council and the Swiss National Science Foundation.
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