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COPII-dependent transport from the endoplasmic reticulum Charles Barlowe The coat protein complex II (COPII) forms transport vesicles from the endoplasmic reticulum and segregates biosynthetic cargo from ER-resident proteins. Recent high-resolution structural studies on individual COPII subunits and on the polymerized coat reveal the molecular architecture of COPII vesicles. Other advances have shown that integral membrane accessory proteins act with the COPII coat to collect specific cargo molecules into ER-derived transport vesicles. Addresses Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA; e-mail: [email protected] Current Opinion in Cell Biology 2002, 14:417–422 0955-0674/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations COP coat protein complex GAP GTPase-activating protein
Introduction Coat protein complex II (COPII) coats are assembled on the surface of ER membranes by sequential binding of the Sar1p GTPase, followed by the Sec23p–Sec24p (Sec23/24p) complex and then the Sec13p–Sec31p (Sec13/31p) complex [1]. In brief outline (Figure 1), Sar1p is activated by the ER-localized Sec12p guanine nucleotide exchange factor, producing Sar1p–GTP in transient association with cargo to
be included in COPII vesicles. Activated Sar1p then recruits the Sec23/24p complex to ER membranes, forming stabilized Sar1p–cargo–Sec23/24p complexes [2–4]. Importantly, the Sec23p subunit possesses a Sar1p GTPase-activating protein (GAP) activity, and conversion to Sar1p–GDP discourages formation of ternary complexes. Therefore, some mode of GTPase regulation appears critical for cargo selection and for progression towards coat assembly. Finally, the Sec13/31p complex is recruited to these cargo complexes, which then drives membrane curvature and further stimulates the Sec23/24p GAP activity toward Sar1p [5]. As vesicles are formed, transport intermediates emerge from ER exit sites and migrate toward the Golgi complex [6]. Although one level of understanding has been reached, many questions remain concerning the molecular contacts made during COPII assembly, how these contacts are regulated, and how a wide variety of cargo molecules are collected into COPII vesicles. In this review, I will focus on recent advances in structural analyses of the COPII coat and on mechanisms by which distinct secretory cargo are selected for export.
Structure of the Sar1p GTPase Assembly of the COPII coat is connected to a GTPase cycle — a property shared among the other characterized intracellular coat complexes, clathrin and COPI [7]. Activated Sar1–GTP promotes COPII assembly, whereas GTP
Figure 1 Model for COPII cargo selection and budding from the ER. (a) The Sar1p GTPase is activated at exports sites by the guanine nucleotide exchange factor Sec12p. Transient Sar1p–cargo complexes form and are stabilized by binding of the Sec23/24p complex. (b) Ternary cargo complexes are collected by the Sec13/31p subunit. This leads to coat polymerization and membrane deformation. As Sec13/31p subunits polymerize, the Sec23p GAP activity is in position to stimulate GTP hydrolysis by Sar1p causing release of this GTPase from budding vesicles. The various types of cargo accommodated by COPII vesicles are shown in black.
(a) Sar1p GTPase activation, ternary complex formation Sec12p
ER lumen (b) Coat polymerization, membrane deformation
Sar1–GTP
ER lumen
Sec23–24p Sec13–31p
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hydrolysis leads to coat disassembly and recycles COPII subunits for subsequent rounds of budding. Many aspects of this cycle, including production of coated vesicles, can be reconstituted in defined reactions containing Sar1p, Sec23/24p, Sec13/31p, guanine nucleotides and artificial liposomes that approximate the phospholipid composition of ER membranes [8]. This minimal system has allowed for a detailed examination of the requirements for vesicle formation and of the dynamics of coat assembly [5]. However, the minimal COPII budding reactions with liposomes have a requirement for Sar1p that is locked in its activated state. This contrasts COPII budding reactions sustained with isolated ER membranes, where vesicle formation proceeds in the presence or absence of GTP hydrolysis [9]. What regulatory components are missing in the minimal system? How is the Sar1p GTPase regulated to ensure proper cargo inclusion and coat polymerization? To fully understand these mechanisms, structural information is imperative. Recently, the three-dimensional structure of mammalian Sar1p–GDP has been determined to 1.7 Å resolution [10••]. As with other GTPases for which structures have been determined, the GTP binding site of Sar1p is true to form [11] and shares many features with its ARF (ADP-ribosylation factor) relatives [12,13]. Although the Sar1p structure does not provide an instant picture of regulation, a guide is now in hand, and the subtle differences noted in the Sar1p structure are likely to hold many of its regulatory secrets. Notable variations in Sar1p include a conserved patch of bulky hydrophobic residues at the amino terminus (residues three to five), an amphipathic α helix (residues 15–19) in perpendicular orientation to the central β sheets, and a surface-exposed insert (residues 156–171) in the loop region connecting the α4 helix and β6 strand. Indeed, mutations in either of these regions result in loss of function when tested in assays that measure Sar1p activity. More specifically, mutation of the amino-terminal patch of hydrophobic residues in Sar1p inhibits membrane recruitment and Sec12p-dependent guanine nucleotide exchange activity. Alterations in the amphipathic α helix also inhibit membrane recruitment; however, in this instance Sec12p exchange activity was not affected, but Sec23/24p-mediated GAP activity was reduced. On the basis of these findings, it has been proposed that Sec12pdependent activation and membrane association act through the amino-terminal hydrophobic patch, whereas Sec23/24p recruitment and stabilization of Sar1–cargo complexes use the amphipathic α helix [10••]. Further studies will be required to confirm these regulatory contacts, and the structure/function analyses are underway. Ultimately, structural studies of defined ternary complexes consisting of cargo bound to activated Sar1p and Sec23/24p should provide a unique view into GTPase-catalyzed cargo capture.
A coat built of polygonal units The Sec23p–Sec24p heterodimer arrives after activated Sar1p is recruited to ER membranes in cargo complexes.
Earlier studies established that the Sec23p subunit possessed a Sar1p-specific GAP activity required for vesicle formation [14]. The Sec24p subunit has been assigned a role in cargo recognition, as diversity in this Sec23p partner underlies a capacity to export distinct secretory cargo from the ER [15]. Yeast cells are endowed with three Sec24p homologues and higher eukaryotes possess at least four isoforms [16]. Studies suggest that variation in the Sec24p subunit can provide COPII vesicles with broader specificity in the types of cargo recognised, as well as greater flexibility in vesicle size to accommodate larger molecules [17]. How can distinct Sec23p–Sec24p heterodimers operate with such high levels of sophistication and discrimination? Complementary structural studies from the Kirchhausen and Heuser laboratories are now guiding investigators on these questions [18••,19••]. High-resolution electron microscopy (EM) studies of the Sec23/24p complex document an extended bi-lobed structure (of 16–17 nm), described as ‘bone-like’ by the Kirchhausen group and a ‘bow tie’ by the Heuser group. Here, it seems that this COPII subunit has evolved a division-of-labor strategy, with the mass of Sec23p forming one lobe and the Sec24p subunit forming the other. On the basis of these structural considerations, one could envisage Sec23/24p surrounding a Sar1p–GTP–cargo complex, with Sec24p contacting cargo and Sec23p bound to Sar1p. But where does Sar1p–GTP rest on this ‘bone’, and how is the Sec23p GAP activity regulated? Again, structural studies on Sec23/24p in ternary complexes with Sar1p and cargo could be most informative. The Sec13/31p complex is the final COPII subunit recruited. It drives budding from liposomes or isolated ER membranes. Not only does this subunit induce membrane curvature, in defined reactions Sec13/31p addition also provides a burst of Sar1p–GTPase activity when added to pre-formed Sec23/24p–Sar1p–GTP complexes [5]. Earlier studies had indicated that the Sec31p subunit could bind to both the Sec23p and Sec24p subunits [20]. It has been suggested that Sec13/31p binding and the subsequent rearrangements during coat polymerization position the Sec23p GAP appropriately for full access to Sar1p [5]. Here again, structural studies will be crucial to test such models. EM studies of the Sec13/31p complex now reveal an apparently flexible structure of 24–30 nm in length with globular domains attached to both ends of an extended rod domain [18••,19••]. Furthermore, biochemical experiments indicate Sec13/31p is a heterotetramer consisting of two copies of Sec13p and two copies of Sec31p. Limited proteolysis of the Sec13/31p complex suggests the rod and globular domains at one end are composed of two Sec31p subunits, while the opposing globular domain is composed of two Sec13p subunits [18••]. Finally, the polymerized COPII structure with all subunits locked on was visualized by deep-etched rotary-shadowed imaging [19••]. In these images, a mesh-like architecture was clearly observed on
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Figure 2
Surface structure of COPII vesicles. Coated vesicles synthesized from ER membranes in the presence of GMP-PNP were adsorbed on the surface of mica flakes and viewed by deep-etch rotary-shadowing electron microscopy. The flexible Sec13/31p subunits that form polygonal ridges on
vesicles are traced in yellow. Panel widths are 175 nm. Micrograph courtesy of John Heuser and reproduced with permission from K Matsuoka et al. Proceedings of the National Academy of Sciences of the USA 2001, 98:13705–13709 (Copyright [2001] National Academy of Sciences USA).
the surfaces of COPII vesicles. Remarkably, the flexible structural unit of the Sec13/31p complex could be superimposed along the edges of this polygonal mesh (Figure 2).
subunits provides a cogent mechanism for selective export [2–4]. What signals are contained on export cargo for recognition by COPII? For some transmembrane secretory cargo, good progress has been realised. In studies of vesicular stomatitis virus G protein (VSV-G) and potassium-channel proteins, results indicate that a di-acidic EXD amino acid motif (where X can be any amino acid) present on their carboxy-terminal tail is necessary for ER export [21,22]. Interestingly, an overlapping tyrosine signal in this same VSV-G tail sequence is required for optimal ER-export rates [23], suggesting that multiple determinants might operate.
To confirm an outer layer arrangement for the Sec13/31p subunits, a lipid crosslinking strategy of COPII-coated membranes provides an independent line of evidence [19••]. Here, the authors find Sar1p and Sec23/24p proteins formed efficient crosslinked products, with phospholipid head groups on the surface of coated liposomes. In contrast, the Sec13/31p complex was inefficiently crosslinked to phospholipids, consistent with an outer layer arrangement distal to the Sar1p–Sec23/24p underlayer. Given these collective observations, the authors find comparisons to clathrin-coated vesicle formation irresistible, and for good reason. Parallels seem appropriate for COPII and clathrin coats, as small GTPases (Sar1p versus ARF) link adaptor complexes (Sec23/24p versus clathrin adaptor proteins) to specific vesicle cargo, and then an outer layer coat (Sec13/31p versus clathrin) clusters adaptor–cargo complexes and induces membrane curvature. However, a difference was noted in that clathrin-coated vesicles may be confined to a more rigid size and structure, whereas the flexibility of the outer-layer Sec13/31p may allow for a softer version of coated vesicles to accommodate a range of cargo sizes [19••].
Cargo selection by COPII
ER export of other integral membrane proteins that cycle between the ER and Golgi compartments, such as ERGIC53 and the p24 proteins, depends on a pair of hydrophobic residues (e.g. FF or LL) contained in their cytoplasmically exposed tail sequences [24,25]. Moreover, binding studies indicate a correlation between export potency and COPII-binding efficiency [26•,27]. Attachment of these hydrophobic signals to reporter molecules accelerates their transport efficiency, and, surprisingly, the exact composition and position of the hydrophobic pair seem unimportant [26•]. Indeed, database searches of other transmembrane proteins that are exported from the ER often contain these hydrophobic motifs, and in some instances are known to be required for ER export [28,29]. Notably, these types of hydrophobic motifs are extremely rare in ER-resident membrane proteins [26•].
Certain proteins are transported from the ER by a selective export mechanism that concentrates cargo into budding vesicles [1]. Experiments showing assembly of vesicle cargo molecules into ternary complexes with COPII
In other cases, accessory factors are required to direct transmembrane secretory proteins into COPII vesicles [30]. Erv14p, an integral membrane protein that cycles
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between the ER and Golgi complex, serves as an adaptor that links the transmembrane secretory protein Axl2p to the COPII vesicle coat [31]. This finding suggests that some integral membrane cargo may not possess adequate sorting signals for efficient ER export and will rely on COPII adaptors. Again, hydrophobic residues in a cytoplasmic loop of Erv14p are critical for its binding to COPII and for guiding ER export [31]. Therefore, the current information on ER-export signals suggests that there may not be a single stringent code but rather multiple signals exist that may be decoded by the COPII budding machinery. Perhaps further studies, focused on specific ER export signals and how they directly influence both packaging into COPII vesicles and formation of ternary cargo complexes, will lead to a more comprehensive model. While the process of transmembrane cargo selection into COPII vesicles is coming into focus, mechanisms underlying export of soluble secretory cargo from the ER are less clear. Evidence has been provided for receptor-mediated [2,32–34] and for bulk-flow or post-ER sorting mechanisms [35,36]. Some putative cargo receptors have been characterized [33,34]; however, a difficulty with the receptormediated model has been a shortage of receptors that could link the variety of soluble cargo to COPII coats. Genetic approaches in yeast have not yielded a set of ER export receptors; however, biochemical characterization of COPII vesicles may have identified additional components of this ER sorting machinery [37]. One of the recently identified proteins, Erv29p, is a multispanning membrane protein that cycles between the ER and Golgi compartments. Yeast strains that lack Erv29p accumulate certain soluble secretory proteins (e.g. glycopro-alpha factor and pro-carboxypeptidase Y) in the ER, whereas other soluble and integral membrane cargo are transported at the wild-type rate. In vitro assays indicate that Erv29p binds to the glyco-pro-alpha factor cargo and is required for its incorporation into COPII vesicles [38•,39•]. In addition to Erv29p, ERGIC53 and the p24 proteins are also thought to sort cargo into ER-derived transport vesicles [33,34]. These proteins cycle between the ER and Golgi compartments and presumably link cargo to coat. However, it is not at all clear how these cargo receptors recognise and bind their ligands in the ER and then release them upon arrival at the Golgi complex. These questions remain unanswered and advances in the selective export model may call for novel experimentation. Finally, it remains to be determined how prevalent the selective export mechanism operates for secretory cargo, because some soluble cargo (e.g. amylase and chymotypsinogen) are not concentrated into ER-derived transport vesicles [36].
example, inactivation of the Sar1p protein causes a block in COPII-dependent transport and has rapid and profound effects on Golgi structure [40]. Cell-cycle status and environmental conditions must also be dealt with at the level of ER export [41]. How is a proper balance struck? Apparently, additional layers of transcriptional and posttranscriptional regulation are imposed upon the ER-export machinery. A striking example of transcriptional control has been observed when mRNA levels were monitored under conditions of ER stress that activate the unfolded protein response pathway [42]. Specifically, SEC12, SEC13, SEC24 and ERV29 are upregulated upon activation of this pathway. This study and others [39•,43•] indicate that ER quality control and ER export machinery collaborate to maintain ER homeostasis. Post-translational regulation at the level of a kinase/phosphatase cycle also regulates the assembly of COPII coats on ER membranes [44,45]. A recent report suggests that a diacylglycerol kinase (DGKδ) protein somehow impinges on this kinase/phosphatase cycle [46]. Other environmental conditions affect transport through the early secretory pathway [47,48]; however, there is limited information with regards to how cellular conditions are sensed and then relayed to the transport machinery. Given the diversity in cell types and the variety of proteins that must pass through the ER, one can expect more surprises ahead.
Conclusions Structural studies on individual COPII subunits and on the polymerized coat are now providing the first molecular glimpses into transport vesicle formation. This structural information, coupled with refined assays to measure subreactions in COPII assembly and the ability to synthesize intermediates in the assembly pathway, provide a unique opportunity to discern mechanisms of GTPase-directed budding. Additional integral membrane components of the ER export machinery have been described and appear to expand the range of cargo that can be linked to COPII. Characterization of these components strengthens a model for receptor-mediated export of certain soluble secretory proteins from the ER. Questions on how this core export machinery is regulated in response to specific cellular circumstances remain at the frontier for cell biologists.
Acknowledgements I thank John Heuser and Randy Schekman for figures used in this review and I wish to thank Stefan Otte for his comments. Studies in my laboratory are supported by the National Institutes of Health (GM52549).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest
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24. Kappeler F, Klopfenstein DR, Foguet M, Paccaud JP, Hauri HP: The recycling of ERGIC53 in the early secretory pathway. ERGIC53 carries a cytosolic endoplasmic reticulum-exit determinant interacting with COPII. J Biol Chem 1997, 272:31801-31808. 25. Dominguez M, Dejgaard K, Fullekrug J, Dahan S, Fazel A, Paccaud JP, Thomas DY, Bergeron JJ, Nilsson T: gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COPI and II coatomer. J Cell Biol 1998; 140:751-765. 26. Nufer O, Guldbrandsen S, Degen M, Kappeler F, Paccaud J-P, Tani K, • Hauri H-P: Role of the cytoplasmic C-terminal amino acids of membrane proteins in ER export. J Cell Sci 2002, 115:619-628. The authors show that a variety of hydrophobic motifs on the cytoplasmic tail of a transmembrane protein can accelerate its transport to the Golgi complex. Moreover, a single carboxy-terminal valine reside in the –1 position can act as an efficient export signal. The authors note that these motifs are common among other type I membrane proteins that are transported from the ER. 27.
Belden WJ, Barlowe, C: Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex. J Biol Chem 2001, 276:43040-43048.
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38. Belden WJ, Barlowe C: Role of Erv29p in collecting soluble • secretory proteins into ER-derived transport vesicles. Science 2001, 294:1528-1531. A strong case for the receptor-mediated export model is provided for Erv29pdependent packing of glyco-pro-alpha factor into COPII vesicles. In vitro experimentation indicates a direct interaction between Erv29p and glyco-proalpha factor, and in vivo studies suggest a saturatable relationship between this receptor and its ligand. Although the study was conducted in yeast cells, Erv29p is found in all species examined, suggesting a general mechanism. 39. Caldwell SR, Hill KJ, Cooper AA: Degradation of endoplasmic • reticulum (ER) quality control substrates requires transport between the ER and Golgi. J Biol Chem 2001, 276:23296-23303. One of two important studies (see also Vashist et al. [2001] [43•]) showing a link between transport from the ER and ER quality control. Following the degradation of misfolded secretory proteins such as CPY*, the authors report that anterograde transport between the ER and Golgi is required for efficient turnover. Furthermore, other proteins that operate in transport through the early secretory pathway influence CPY* degradation rates, including Erv29p. The report is the first to document that several soluble secretory proteins accumulate in the ER of erv29 deletion strains. 40. Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J: Maintenance of Golgi structure and function depends on the integrity of ER export. J Cell Biol 2001, 155:557-570.
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42. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P: Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 2000, 101:249-258.
48. Rust RC, Landmann L, Gosert R, Tang BL, Hong W, Hauri HP, Egger D, Bienz K: Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol 2001, 75:9808-9818.
COPII-dependent transport from the endoplasmic reticulum Charles Barlowe The coat protein complex II (COPII) forms transport vesicles from the endoplasmic reticulum and segregates biosynthetic cargo from ER-resident proteins. Recent high-resolution structural studies on individual COPII subunits and on the polymerized coat reveal the molecular architecture of COPII vesicles. Other advances have shown that integral membrane accessory proteins act with the COPII coat to collect specific cargo molecules into ER-derived transport vesicles. Addresses Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA; e-mail: [email protected] Current Opinion in Cell Biology 2002, 14:417–422 0955-0674/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations COP coat protein complex GAP GTPase-activating protein
Introduction Coat protein complex II (COPII) coats are assembled on the surface of ER membranes by sequential binding of the Sar1p GTPase, followed by the Sec23p–Sec24p (Sec23/24p) complex and then the Sec13p–Sec31p (Sec13/31p) complex [1]. In brief outline (Figure 1), Sar1p is activated by the ER-localized Sec12p guanine nucleotide exchange factor, producing Sar1p–GTP in transient association with cargo to
be included in COPII vesicles. Activated Sar1p then recruits the Sec23/24p complex to ER membranes, forming stabilized Sar1p–cargo–Sec23/24p complexes [2–4]. Importantly, the Sec23p subunit possesses a Sar1p GTPase-activating protein (GAP) activity, and conversion to Sar1p–GDP discourages formation of ternary complexes. Therefore, some mode of GTPase regulation appears critical for cargo selection and for progression towards coat assembly. Finally, the Sec13/31p complex is recruited to these cargo complexes, which then drives membrane curvature and further stimulates the Sec23/24p GAP activity toward Sar1p [5]. As vesicles are formed, transport intermediates emerge from ER exit sites and migrate toward the Golgi complex [6]. Although one level of understanding has been reached, many questions remain concerning the molecular contacts made during COPII assembly, how these contacts are regulated, and how a wide variety of cargo molecules are collected into COPII vesicles. In this review, I will focus on recent advances in structural analyses of the COPII coat and on mechanisms by which distinct secretory cargo are selected for export.
Structure of the Sar1p GTPase Assembly of the COPII coat is connected to a GTPase cycle — a property shared among the other characterized intracellular coat complexes, clathrin and COPI [7]. Activated Sar1–GTP promotes COPII assembly, whereas GTP
Figure 1 Model for COPII cargo selection and budding from the ER. (a) The Sar1p GTPase is activated at exports sites by the guanine nucleotide exchange factor Sec12p. Transient Sar1p–cargo complexes form and are stabilized by binding of the Sec23/24p complex. (b) Ternary cargo complexes are collected by the Sec13/31p subunit. This leads to coat polymerization and membrane deformation. As Sec13/31p subunits polymerize, the Sec23p GAP activity is in position to stimulate GTP hydrolysis by Sar1p causing release of this GTPase from budding vesicles. The various types of cargo accommodated by COPII vesicles are shown in black.
(a) Sar1p GTPase activation, ternary complex formation Sec12p
ER lumen (b) Coat polymerization, membrane deformation
Sar1–GTP
ER lumen
Sec23–24p Sec13–31p
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hydrolysis leads to coat disassembly and recycles COPII subunits for subsequent rounds of budding. Many aspects of this cycle, including production of coated vesicles, can be reconstituted in defined reactions containing Sar1p, Sec23/24p, Sec13/31p, guanine nucleotides and artificial liposomes that approximate the phospholipid composition of ER membranes [8]. This minimal system has allowed for a detailed examination of the requirements for vesicle formation and of the dynamics of coat assembly [5]. However, the minimal COPII budding reactions with liposomes have a requirement for Sar1p that is locked in its activated state. This contrasts COPII budding reactions sustained with isolated ER membranes, where vesicle formation proceeds in the presence or absence of GTP hydrolysis [9]. What regulatory components are missing in the minimal system? How is the Sar1p GTPase regulated to ensure proper cargo inclusion and coat polymerization? To fully understand these mechanisms, structural information is imperative. Recently, the three-dimensional structure of mammalian Sar1p–GDP has been determined to 1.7 Å resolution [10••]. As with other GTPases for which structures have been determined, the GTP binding site of Sar1p is true to form [11] and shares many features with its ARF (ADP-ribosylation factor) relatives [12,13]. Although the Sar1p structure does not provide an instant picture of regulation, a guide is now in hand, and the subtle differences noted in the Sar1p structure are likely to hold many of its regulatory secrets. Notable variations in Sar1p include a conserved patch of bulky hydrophobic residues at the amino terminus (residues three to five), an amphipathic α helix (residues 15–19) in perpendicular orientation to the central β sheets, and a surface-exposed insert (residues 156–171) in the loop region connecting the α4 helix and β6 strand. Indeed, mutations in either of these regions result in loss of function when tested in assays that measure Sar1p activity. More specifically, mutation of the amino-terminal patch of hydrophobic residues in Sar1p inhibits membrane recruitment and Sec12p-dependent guanine nucleotide exchange activity. Alterations in the amphipathic α helix also inhibit membrane recruitment; however, in this instance Sec12p exchange activity was not affected, but Sec23/24p-mediated GAP activity was reduced. On the basis of these findings, it has been proposed that Sec12pdependent activation and membrane association act through the amino-terminal hydrophobic patch, whereas Sec23/24p recruitment and stabilization of Sar1–cargo complexes use the amphipathic α helix [10••]. Further studies will be required to confirm these regulatory contacts, and the structure/function analyses are underway. Ultimately, structural studies of defined ternary complexes consisting of cargo bound to activated Sar1p and Sec23/24p should provide a unique view into GTPase-catalyzed cargo capture.
A coat built of polygonal units The Sec23p–Sec24p heterodimer arrives after activated Sar1p is recruited to ER membranes in cargo complexes.
Earlier studies established that the Sec23p subunit possessed a Sar1p-specific GAP activity required for vesicle formation [14]. The Sec24p subunit has been assigned a role in cargo recognition, as diversity in this Sec23p partner underlies a capacity to export distinct secretory cargo from the ER [15]. Yeast cells are endowed with three Sec24p homologues and higher eukaryotes possess at least four isoforms [16]. Studies suggest that variation in the Sec24p subunit can provide COPII vesicles with broader specificity in the types of cargo recognised, as well as greater flexibility in vesicle size to accommodate larger molecules [17]. How can distinct Sec23p–Sec24p heterodimers operate with such high levels of sophistication and discrimination? Complementary structural studies from the Kirchhausen and Heuser laboratories are now guiding investigators on these questions [18••,19••]. High-resolution electron microscopy (EM) studies of the Sec23/24p complex document an extended bi-lobed structure (of 16–17 nm), described as ‘bone-like’ by the Kirchhausen group and a ‘bow tie’ by the Heuser group. Here, it seems that this COPII subunit has evolved a division-of-labor strategy, with the mass of Sec23p forming one lobe and the Sec24p subunit forming the other. On the basis of these structural considerations, one could envisage Sec23/24p surrounding a Sar1p–GTP–cargo complex, with Sec24p contacting cargo and Sec23p bound to Sar1p. But where does Sar1p–GTP rest on this ‘bone’, and how is the Sec23p GAP activity regulated? Again, structural studies on Sec23/24p in ternary complexes with Sar1p and cargo could be most informative. The Sec13/31p complex is the final COPII subunit recruited. It drives budding from liposomes or isolated ER membranes. Not only does this subunit induce membrane curvature, in defined reactions Sec13/31p addition also provides a burst of Sar1p–GTPase activity when added to pre-formed Sec23/24p–Sar1p–GTP complexes [5]. Earlier studies had indicated that the Sec31p subunit could bind to both the Sec23p and Sec24p subunits [20]. It has been suggested that Sec13/31p binding and the subsequent rearrangements during coat polymerization position the Sec23p GAP appropriately for full access to Sar1p [5]. Here again, structural studies will be crucial to test such models. EM studies of the Sec13/31p complex now reveal an apparently flexible structure of 24–30 nm in length with globular domains attached to both ends of an extended rod domain [18••,19••]. Furthermore, biochemical experiments indicate Sec13/31p is a heterotetramer consisting of two copies of Sec13p and two copies of Sec31p. Limited proteolysis of the Sec13/31p complex suggests the rod and globular domains at one end are composed of two Sec31p subunits, while the opposing globular domain is composed of two Sec13p subunits [18••]. Finally, the polymerized COPII structure with all subunits locked on was visualized by deep-etched rotary-shadowed imaging [19••]. In these images, a mesh-like architecture was clearly observed on
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Figure 2
Surface structure of COPII vesicles. Coated vesicles synthesized from ER membranes in the presence of GMP-PNP were adsorbed on the surface of mica flakes and viewed by deep-etch rotary-shadowing electron microscopy. The flexible Sec13/31p subunits that form polygonal ridges on
vesicles are traced in yellow. Panel widths are 175 nm. Micrograph courtesy of John Heuser and reproduced with permission from K Matsuoka et al. Proceedings of the National Academy of Sciences of the USA 2001, 98:13705–13709 (Copyright [2001] National Academy of Sciences USA).
the surfaces of COPII vesicles. Remarkably, the flexible structural unit of the Sec13/31p complex could be superimposed along the edges of this polygonal mesh (Figure 2).
subunits provides a cogent mechanism for selective export [2–4]. What signals are contained on export cargo for recognition by COPII? For some transmembrane secretory cargo, good progress has been realised. In studies of vesicular stomatitis virus G protein (VSV-G) and potassium-channel proteins, results indicate that a di-acidic EXD amino acid motif (where X can be any amino acid) present on their carboxy-terminal tail is necessary for ER export [21,22]. Interestingly, an overlapping tyrosine signal in this same VSV-G tail sequence is required for optimal ER-export rates [23], suggesting that multiple determinants might operate.
To confirm an outer layer arrangement for the Sec13/31p subunits, a lipid crosslinking strategy of COPII-coated membranes provides an independent line of evidence [19••]. Here, the authors find Sar1p and Sec23/24p proteins formed efficient crosslinked products, with phospholipid head groups on the surface of coated liposomes. In contrast, the Sec13/31p complex was inefficiently crosslinked to phospholipids, consistent with an outer layer arrangement distal to the Sar1p–Sec23/24p underlayer. Given these collective observations, the authors find comparisons to clathrin-coated vesicle formation irresistible, and for good reason. Parallels seem appropriate for COPII and clathrin coats, as small GTPases (Sar1p versus ARF) link adaptor complexes (Sec23/24p versus clathrin adaptor proteins) to specific vesicle cargo, and then an outer layer coat (Sec13/31p versus clathrin) clusters adaptor–cargo complexes and induces membrane curvature. However, a difference was noted in that clathrin-coated vesicles may be confined to a more rigid size and structure, whereas the flexibility of the outer-layer Sec13/31p may allow for a softer version of coated vesicles to accommodate a range of cargo sizes [19••].
Cargo selection by COPII
ER export of other integral membrane proteins that cycle between the ER and Golgi compartments, such as ERGIC53 and the p24 proteins, depends on a pair of hydrophobic residues (e.g. FF or LL) contained in their cytoplasmically exposed tail sequences [24,25]. Moreover, binding studies indicate a correlation between export potency and COPII-binding efficiency [26•,27]. Attachment of these hydrophobic signals to reporter molecules accelerates their transport efficiency, and, surprisingly, the exact composition and position of the hydrophobic pair seem unimportant [26•]. Indeed, database searches of other transmembrane proteins that are exported from the ER often contain these hydrophobic motifs, and in some instances are known to be required for ER export [28,29]. Notably, these types of hydrophobic motifs are extremely rare in ER-resident membrane proteins [26•].
Certain proteins are transported from the ER by a selective export mechanism that concentrates cargo into budding vesicles [1]. Experiments showing assembly of vesicle cargo molecules into ternary complexes with COPII
In other cases, accessory factors are required to direct transmembrane secretory proteins into COPII vesicles [30]. Erv14p, an integral membrane protein that cycles
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between the ER and Golgi complex, serves as an adaptor that links the transmembrane secretory protein Axl2p to the COPII vesicle coat [31]. This finding suggests that some integral membrane cargo may not possess adequate sorting signals for efficient ER export and will rely on COPII adaptors. Again, hydrophobic residues in a cytoplasmic loop of Erv14p are critical for its binding to COPII and for guiding ER export [31]. Therefore, the current information on ER-export signals suggests that there may not be a single stringent code but rather multiple signals exist that may be decoded by the COPII budding machinery. Perhaps further studies, focused on specific ER export signals and how they directly influence both packaging into COPII vesicles and formation of ternary cargo complexes, will lead to a more comprehensive model. While the process of transmembrane cargo selection into COPII vesicles is coming into focus, mechanisms underlying export of soluble secretory cargo from the ER are less clear. Evidence has been provided for receptor-mediated [2,32–34] and for bulk-flow or post-ER sorting mechanisms [35,36]. Some putative cargo receptors have been characterized [33,34]; however, a difficulty with the receptormediated model has been a shortage of receptors that could link the variety of soluble cargo to COPII coats. Genetic approaches in yeast have not yielded a set of ER export receptors; however, biochemical characterization of COPII vesicles may have identified additional components of this ER sorting machinery [37]. One of the recently identified proteins, Erv29p, is a multispanning membrane protein that cycles between the ER and Golgi compartments. Yeast strains that lack Erv29p accumulate certain soluble secretory proteins (e.g. glycopro-alpha factor and pro-carboxypeptidase Y) in the ER, whereas other soluble and integral membrane cargo are transported at the wild-type rate. In vitro assays indicate that Erv29p binds to the glyco-pro-alpha factor cargo and is required for its incorporation into COPII vesicles [38•,39•]. In addition to Erv29p, ERGIC53 and the p24 proteins are also thought to sort cargo into ER-derived transport vesicles [33,34]. These proteins cycle between the ER and Golgi compartments and presumably link cargo to coat. However, it is not at all clear how these cargo receptors recognise and bind their ligands in the ER and then release them upon arrival at the Golgi complex. These questions remain unanswered and advances in the selective export model may call for novel experimentation. Finally, it remains to be determined how prevalent the selective export mechanism operates for secretory cargo, because some soluble cargo (e.g. amylase and chymotypsinogen) are not concentrated into ER-derived transport vesicles [36].
example, inactivation of the Sar1p protein causes a block in COPII-dependent transport and has rapid and profound effects on Golgi structure [40]. Cell-cycle status and environmental conditions must also be dealt with at the level of ER export [41]. How is a proper balance struck? Apparently, additional layers of transcriptional and posttranscriptional regulation are imposed upon the ER-export machinery. A striking example of transcriptional control has been observed when mRNA levels were monitored under conditions of ER stress that activate the unfolded protein response pathway [42]. Specifically, SEC12, SEC13, SEC24 and ERV29 are upregulated upon activation of this pathway. This study and others [39•,43•] indicate that ER quality control and ER export machinery collaborate to maintain ER homeostasis. Post-translational regulation at the level of a kinase/phosphatase cycle also regulates the assembly of COPII coats on ER membranes [44,45]. A recent report suggests that a diacylglycerol kinase (DGKδ) protein somehow impinges on this kinase/phosphatase cycle [46]. Other environmental conditions affect transport through the early secretory pathway [47,48]; however, there is limited information with regards to how cellular conditions are sensed and then relayed to the transport machinery. Given the diversity in cell types and the variety of proteins that must pass through the ER, one can expect more surprises ahead.
Conclusions Structural studies on individual COPII subunits and on the polymerized coat are now providing the first molecular glimpses into transport vesicle formation. This structural information, coupled with refined assays to measure subreactions in COPII assembly and the ability to synthesize intermediates in the assembly pathway, provide a unique opportunity to discern mechanisms of GTPase-directed budding. Additional integral membrane components of the ER export machinery have been described and appear to expand the range of cargo that can be linked to COPII. Characterization of these components strengthens a model for receptor-mediated export of certain soluble secretory proteins from the ER. Questions on how this core export machinery is regulated in response to specific cellular circumstances remain at the frontier for cell biologists.
Acknowledgements I thank John Heuser and Randy Schekman for figures used in this review and I wish to thank Stefan Otte for his comments. Studies in my laboratory are supported by the National Institutes of Health (GM52549).
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