Surface structure of the COPII-coated vesicle

The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p > Sec13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated vesicles. Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31. Partially uncoated profiles revealed strands of Sec13/31p stripped from the vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat.     

Specific interaction of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II component Sec24p of endoplasmic reticulum-derived transport vesicles

The generation of transport vesicles at the endoplasmic reticulum (ER) depends on cytosolic proteins, which, in the form of subcomplexes (Sec23p/Sec24p; Sec13p/Sec31p) are recruited to the ER membrane by GTP-bound Sar1p and form the coat protein complex II (COPII). Using affinity chromatography and two-hybrid analyses, we found that the essential COPII component Sec24p, but not Sec23p, binds to the cis-Golgi syntaxin Sed5p. Sec24p/Sed5p interaction in vitro was not dependent on the presence of [Sar1p.GTP]. The binding of Sec24p to Sed5p is specific; none of the other seven yeast syntaxins bound to this COPII component. Whereas the interaction site of Sec23p is within the N-terminal half of the 926-aa-long Sec24p (amino acid residues 56-549), Sed5p binds to the N- and C-terminal halves of the protein. Destruction by mutagenesis of a potential zinc finger within the N-terminal half of Sec24p led to a nonfunctional protein that was still able to bind Sec23p and Sed5p. Sec24p/Sed5p binding might be relevant for cargo selection during transport-vesicle formation and/or for vesicle targeting to the cis-Golgi.     

Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.     

Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes

Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.     

The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells

The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells.     

Crystal structure of the Sec18p N-terminal domain

Yeast Sec18p and its mammalian orthologue N-ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two beta-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled.     

Specific SNARE complex binding mode of the Sec1/Munc-18 protein, Sec1p

The Sec1/Munc-18 (SM) family of proteins is required for vesicle fusion in eukaryotic cells and has been linked to the membrane-fusion proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SM proteins may activate the target-membrane SNARE, syntaxin, for assembly into the fusogenic SNARE complex. In support of an activation role, SM proteins bind directly to their cognate syntaxins. An exception is the yeast Sec1p, which does not bind the yeast plasma-membrane syntaxin, Sso1p. This exception could be explained if the SM interaction motif were blocked by the highly stable closed conformation of Sso1p. We tested the possibility of a latent binding motif using sso1 mutants in yeast and reconstituted the Sec1p binding specificity observed in vivo with purified proteins in vitro. Our results indicate there is no latent binding motif in Sso1p. Instead, Sec1p binds specifically to the ternary SNARE complex, with no detectable binding to the binary t-SNARE complex or any of the three individual SNAREs in their uncomplexed forms. We propose that vesicle fusion requires a specific interaction between the SM protein and the ternary SNARE complex.     

Crystal structure of the Sec4p.Sec2p complex in the nucleotide exchanging intermediate state

Vesicular transport during exocytosis is regulated by Rab GTPase (Sec4p in yeast), which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. Here, we report the crystal structure of the Sec2p GEF domain in a complex with the nucleotide-free Sec4p at 2.7 A resolution. Upon complex formation, the Sec2p helices approach each other, flipping the side chain of Phe-109 toward Leu-104 and Leu-108 of Sec2p. These three residues provide a hydrophobic platform to attract the side chains of Phe-49, Ile-53, and Ile-55 in the switch I region as well as Phe-57 and Trp-74 in the interswitch region of Sec4p. Consequently, the switch I and II regions are largely deformed, to create a flat hydrophobic interface that snugly fits the surface of the Sec2p coiled coil. These drastic conformational changes disrupt the interactions between switch I and the bound guanine nucleotide, which facilitates the GDP release. Unlike the recently reported 3.3 A structure of the Sec4p.Sec2p complex, our structure contains a phosphate ion bound to the P-loop, which may represent an intermediate state of the nucleotide exchange reaction.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners

Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPase Sec4p. Sec2p is highly phosphorylated, and we have explored the role of phosphorylation in the regulation of its function. We have identified three phosphosites and demonstrate that phosphorylation regulates the interaction of Sec2p with its binding partners Ypt32p, Sec15p, and phosphatidyl-inositol-4-phosphate. In its nonphosphorylated form, Sec2p binds preferentially to the upstream Rab, Ypt32p-GTP, thus forming a Rab guanine nucleotide exchange factor cascade that leads to the activation of the downstream Rab, Sec4p. The nonphosphorylated form of Sec2p also binds to the Golgi-associated phosphatidyl-inositol-4-phosphate, which works in concert with Ypt32p-GTP to recruit Sec2p to Golgi-derived secretory vesicles. In contrast, the phosphorylated form of Sec2p binds preferentially to Sec15p, a downstream effector of Sec4p and a component of the exocyst tethering complex, thus forming a positive-feedback loop that prepares the secretory vesicle for fusion with the plasma membrane. Our results suggest that the phosphorylation state of Sec2p can direct a switch in its regulatory binding partners that facilitates maturation of the secretory vesicle and helps to promote the directionality of vesicular transport.Rab GTP-binding proteins serve as key regulators of membrane traffic. They act by recruiting a wide variety of effectors that together can direct the major steps of vesicular traffic, including vesicle budding, delivery, tethering, and fusion of the vesicle with the acceptor compartment (1). They function as molecular switches that toggle from a cytosolic, inactive conformation (GDP-bound) to a membrane-associated, active conformation (GTP-bound). This fundamental characteristic enables them to recruit effectors only when associated with a specific membrane domain. Importantly, this switch is under control of guanine nucleotide exchange factors (GEFs), making these proteins critical determinants for the localization of active Rab proteins (reviewed in ref. 2).Sec4p is a Rab protein that associates specifically with secretory vesicles traveling from the Golgi to sites of polarized growth in yeast. It plays an essential role at this stage of the secretory pathway by recruiting at least three different effectors. In its GTP-bound form, Sec4p binds directly to Myo2p, a type V myosin, on secretory vesicles, to promote their transport along polarized actin cables, to Sec15p, a component of the exocyst complex that tethers secretory vesicles to the plasma membrane in preparation for exocytic fusion, and to Sro7p, an Lgl family member shown to regulate exocytic SNARE function (3–5). The ability of Sec4p to interact with these effectors depends upon its activation by its GEF, Sec2p (6).Sec2p is a large protein of 759 amino acids divided into several domains. The N-terminal region (amino acids 1–160) forms a long, homodimeric coiled-coil domain. Importantly, this domain contains the active site that catalyzes the Sec4p GDP-to-GTP exchange reaction (6, 7). The remainder of Sec2p interacts with a variety of other components that together control its cellular localization and thereby regulate its exchange activity. Sec2p tagged with GFP is concentrated at exocytic sites including prebud sites, the tips of small buds, and at the mother–daughter neck (8). Sec2p is brought to these sites by riding on secretory vesicles as they are delivered by the type V myosin, Myo2p, on polarized actin cables.A key observation was that localization of Sec2p to exocytic sites requires its interaction with the Golgi-associated Rab, Ypt32p, in its GTP-bound state (9). This finding suggested the existence of a Rab GEF cascade mechanism in which one Rab, Ypt32p, in its activated state recruits Sec2p, which in turn activates the next Rab on the pathway, Sec4p. This mechanism has been conserved through evolution. A Rab GEF cascade involving the corresponding mammalian homologs Rab11–Rabin8–Rab8 has been shown to coordinate ciliogenesis, impairment of which has been implicated in numerous genetic disorders (10). Other examples of Rab GEF cascades have been reported, establishing the widespread use of a mechanism that likely ensures both continuity and directionality in membrane traffic pathways (reviewed in ref. 11; 12, 13).Sec2p also serves as a key component in another important regulatory circuit. Sec2p binds directly to Sec15p, an effector of Sec4p and a subunit of the exocyst-tethering complex (14). The formation of this GEF/Rab/effector complex potentially constitutes a positive-feedback loop that could lead to the creation of a membrane microdomain marked by a high concentration of Sec4-GTP and exocyst complex. Importantly, the Sec15p-binding site in Sec2p overlaps with the Ypt32p-binding site just downstream of the catalytic domain (amino acids 160–258), and Sec15p and Ypt32p compete against each other for binding to Sec2p (14). The implication is that Sec2p can be involved in either a GEF cascade or a GEF–effector positive-feedback loop but not both at the same time. A downstream region (amino acids 450–508) negatively regulates the binding of Sec15p to Sec2p by an auto-inhibitory mechanism. Truncation or point mutations within this domain lead to increased binding to Sec15p, and limited proteolysis analysis suggests that these Sec2p mutants are in a more accessible, open, conformation (14).Recently, we found that the phosphoinositide phosphatidyl-inositol-4-phosphate [PI(4)P] plays an important role in regulating the association of Sec2p with its alternate binding partners, Ypt32-GTP and Sec15p (15). PI(4)P is highly enriched on Golgi membranes and works in concert with Ypt32p-GTP to recruit Sec2p onto Golgi-derived vesicles by interacting with Sec2p through three positively charged patches. Interestingly, the interaction with PI(4)P keeps Sec2p in an auto-inhibited conformation that prevents its association with Sec15p on nascent secretory vesicles. However, the level of PI(4)P appears to drop before the secretory vesicles are concentrated at exocytic sites. This drop in PI(4)P concentration relieves the auto-inhibition, allowing a switch in Sec2p binding partners from Ypt32p to Sec15p and results in the formation of the Sec2p/Sec4p/Sec15p GEF–effector complex that promotes tethering of the vesicle to the plasma membrane in preparation for exocytic fusion.Several studies have suggested that, after vesicle tethering, Sec2p must be released from the exocyst complex so that it will be available to engage in additional rounds of vesicular traffic (8, 14). Sec2p mutants that are not auto-inhibited by the 58-amino acid domain cannot recycle and, even in their cytosolic form, remain bound to Sec15p. The nature of the signal that normally triggers the release of Sec2p from Sec15p has not yet been addressed.Previously, we have shown that Sec2p is highly phosphorylated in vivo (8). Here we explore the role of phosphorylation in the regulation of Sec2p function. In this study, we identify previously unknown phosphosites within the Sec15p/Ypt32p-GTP–binding region of Sec2p. We show that phosphorylation promotes the interaction of Sec2p with Sec15p and inhibits binding to Ypt32p-GTP and PI(4)P. Our findings suggest that Sec2p must cycle between a phosphorylated and a nonphosphorylated state for optimal localization and efficient vesicular transport. We propose that Sec2p is phosphorylated as secretory vesicles mature to promote its interaction with the exocyst complex. Then, the vesicle is tethered to the plasma membrane, Sec2p is dephosphorylated and released into the cytoplasm to facilitate new rounds of vesicular transport.     

Sterols block binding of COPII proteins to SCAP,thereby controlling SCAP sorting in ER

Sterols inhibit their own synthesis in mammalian cells by blocking the vesicular endoplasmic reticulum-to-Golgi transport of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), a sterol-sensing protein that escorts SREBPs. Unable to reach the Golgi, SREBPs are not processed by Golgi-resident proteases, and they fail to activate genes required for cholesterol synthesis. The current studies were designed to reveal whether sterols block SCAP movement by inhibiting synthesis of special vesicles dedicated to SCAP, or whether sterols block SCAP incorporation into common coat protein (COP)II-coated vesicles. Through immunoisolation, we show that SCAP-containing vesicles, formed in vitro, also contain vesicular stomatitis virus glycoprotein (VSVG) protein, a classic marker of COPII-coated vesicles. Sterols selectively block incorporation of SCAP into these vesicles without blocking incorporation of VSVG protein. We show that the mammalian vesicular budding reaction can be reconstituted by recombinant yeast COPII proteins that support incorporation of SCAP as well as VSVG into vesicles. Sterols block SCAP incorporation into vesicles by blocking Sar1-dependent binding of the COPII proteins Sec 23/24 to SCAP. These studies demonstrate feedback control of a biosynthetic pathway by the regulated binding of COPII proteins to an endoplasmic reticulum-to-Golgi transport protein.     

Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18

Sec17 [soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17^L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17^F21S,M22S, with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17^F21S,M22S, interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.Intracellular vesicular traffic between organelles is the basis of cell growth, hormone secretion, and neurotransmission. At each step of exocytic and endocytic trafficking, membranes dock and fuse, mixing their lipids and luminal contents while keeping them separate from the cytosol. Families of proteins, conserved from yeast to humans, mediate docking and fusion. Fusion requires Rab family GTPases and “effector” proteins that bind to a Rab in its active, GTP-bound state (1). Among the effectors are large, organelle-specific tethering complexes. Fusion requires SNARE proteins and their chaperones. SNAREs (2) are proteins that can “snare” (bind to) each other, in cis (when anchored to the same membrane) or in trans (when anchored to apposed, tethered membranes). SNAREs have a conserved “SNARE domain” with a characteristic heptad repeat. SNAREs are categorized as R-SNAREs if they have a central arginyl residue, or Qa-, Qb-, or Qc-SNAREs with a central glutamyl residue (3). SNAREs form RQaQbQc quaternary cis- or trans-SNARE complexes, which bind SNARE chaperones, including the Sec1/Munc18 family of SNARE binding proteins, and Sec18/NSF (N-ethylmaleimide–sensitive factor), an AAA family ATPase that drives SNARE complex disassembly (4). Sec17/α-SNAP (soluble NSF attachment protein) is a cochaperone to Sec18 that enhances its rate of SNARE complex disassembly (5).We study fusion with yeast vacuoles. The homotypic fusion of vacuoles has been studied extensively through genetic identification of vacuole morphology (vam) mutants (6) and vacuole protein sorting (vps) mutants (7), through a colorimetric assay of the fusion of isolated vacuoles (8), and more recently through the fusion of proteoliposomes reconstituted with defined, purified proteins and lipids (9–11). Sec17, Sec18, and ATP catalyze the first stage of vacuole fusion, in which cis-SNARE complexes are disassembled (12). Tethering is then supported by the Rab Ypt7 and the large, multisubunit tethering complex termed HOPS (13). Vps33, one of the HOPS subunits, is the vacuolar SM (Sec1/mUNC-18 family) protein. HOPS has direct affinity for vacuolar SNAREs (14–16), and helps to catalyze SNARE complex assembly and the subsequent fusion (17).During in vitro fusion incubations, most Sec17 is released from vacuoles during cis-SNARE complex disassembly (12). However, a few percent of the vacuolar SNAREs form trans-SNARE complexes (18), and Sec17 is a major constituent of these complexes (19). Furthermore, although Sec17 and Sec18 can disassemble trans-SNARE complexes (19) and will block fusion events in which tethering is supplied by an unphysiological agent such as polyethylene glycol (13), Sec17 and Sec18 work synergistically with HOPS to promote fusion (9, 20). This synergy is even seen when the SNAREs are initially disposed with the R-SNARE on one set of proteoliposomes and the three Q-SNAREs on the others (9), a condition that per se does not require cis-SNARE complex disassembly by Sec18. Finally, added Sec17 restores fusion to vacuoles where fusion is blocked by a defined C-terminal truncation in the SNARE domain of the Qc-SNARE Vam7, in the apparent absence of ATP or Sec18 activity (21). These observations prompted us to reevaluate the roles of Sec17 and Sec18 in the fusion pathway.We now exploit proteoliposomes bearing purified vacuolar Rab and SNAREs to reinvestigate the roles of Sec17 and Sec18. Generous amounts of Sec18 alone can disassemble cis-SNARE complexes, allowing proteoliposomes bearing all four vacuolar SNAREs to fuse at a moderate rate. The rate of fusion can be stimulated by wild-type Sec17, as expected, but also by a Sec17 mutant that has greatly diminished capacity to stimulate Sec18. This suggested that Sec17 could act in ways other than through Sec18 stimulation. We therefore examined fusion incubations where the SNAREs are disposed on complementary proteoliposomes such that Sec18 is not required at all. We find that Sec17 can trigger rapid fusion of proteoliposomes that are already joined by trans-SNARE associations.     

Mso1p: A yeast protein that functions in secretion and interacts physically and genetically with Sec1p

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1–1 mutation. We now describe a third suppressor of sec1–1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.     

Sec16B is involved in the endoplasmic reticulum export of the peroxisomal membrane biogenesis factor peroxin 16 (Pex16) in mammalian cells

Sec16 plays a key role in the formation of coat protein II vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: Sec16A, which is a longer primary ortholog of yeast Sec16, and Sec16B, which is a shorter distant ortholog. Previous studies have shown that Sec16B, as well as Sec16A, defines ER exit sites, where coat protein II vesicles are formed in mammalian cells. Here, we reveal an unexpected role of Sec16B in the biogenesis of mammalian peroxisomes. When overexpressed, Sec16B was targeted to the entire ER, whereas Sec16A was mostly cytosolic. Concomitant with the overexpression of Sec16B, peroxisomal membrane biogenesis factors peroxin 3 (Pex3) and Pex16 were redistributed from peroxisomes to Sec16B-positive ER membranes. Knockdown of Sec16B but not Sec16A by RNAi affected the morphology of peroxisomes, inhibited the transport of Pex16 from the ER to peroxisomes, and suppressed expression of Pex3. These phenotypes were significantly reversed by the expression of RNAi-resistant Sec16B. Together, our results support the view that peroxisomes are formed, at least partly, from the ER and identify a factor responsible for this process.Most eukaryotic cells contain peroxisomes, which are single membrane-bound organelles that function in various metabolic pathways, including the β-oxidation of fatty acids, biosynthesis of plasmalogens and bile acids, and hydrogen peroxide metabolism (1). To perform this variety of functions, peroxisomes are highly dynamic; their number, size, and function change in response to cellular conditions. In addition, unlike mitochondria, peroxisomes can be formed through de novo synthesis as well as through the growth and division of preexisting peroxisomes (2, 3).Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and posttranslationally imported to peroxisomes (4). This import pathway includes the recognition of two distinct peroxisomal targeting signals (PTS1 and PTS2) by peroxin 5 (Pex5) and Pex7, respectively, followed by translocation across the membrane through the import machinery, including Pex14 and Really Interesting New Gene peroxins (5, 6). The import pathway for peroxisomal membrane proteins (PMPs), on the other hand, is believed to be independent of that used by matrix proteins. Genetic phenotype complementation analysis of yeast and mammalian mutants devoid of peroxisome membranes revealed that Pex3, Pex16, and Pex19 are essential for PMP import (references in ref. 7). Pex3 is a PMP import receptor (8), and Pex19 is a chaperone and import receptor for most PMPs (9). Pex16 appears to function as a Pex3-Pex19 receptor in mammals (7) and as a negative regulator of peroxisome fission in yeast Yarrowia lipolytica (10) but is absent in Saccharomyces cerevisiae (11).Although compelling evidence suggests that PMPs are transported directly from the cytosol to peroxisomes (7–9, 12), recent work has suggested that some PMPs, including the PMP import receptors Pex3 and Pex16, seem to be, at least partly, transported from the endoplasmic reticulum (ER) en route to peroxisomes (13). In addition, several lines of evidence suggest that the ER participates in the de novo formation of peroxisomes (13–20). A very recent study involving a yeast cell-free system revealed that ER-peroxisome carriers are formed in a Pex19-dependent manner (21).In this report, we show that Sec16B plays an important role in the transport of Pex16 from the ER to peroxisomes in mammalian cells. Sec16 was first characterized in yeast S. cerevisiae as a 240-kDa peripheral membrane protein that interacts with coat protein II (COPII) coat components and facilitates their assembly and vesicle budding (22–25). In yeast Pichia pastoris, Sec16 defines ER exit sites (ERESs) (26), special domains where COPII-coated vesicles are formed (27). There are two mammalian orthologs, Sec16A (250 kDa) and Sec16B (117 kDa) (also referred to as Sec16L and Sec16S, respectively) (28–30). Sec16A, which is localized in cup-like structures in ERESs (31), appears to be the primary Sec16 ortholog because its molecular mass is similar to that of Sec16 in yeast (22) and Drosophila (32). Sec16B, which appears to be conserved in vertebrates, is also localized in ERESs, but its function has not been fully examined in the context of membrane trafficking. Our results suggest that Sec16B may participate in the formation of new peroxisomes derived from the ER.     

A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in "cis" and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking.     

The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway

The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals that the dome-shaped Trs85 subunit associates primarily with the Trs20 subunit of TRAPPI. We further demonstrate that TRAPPIII binds the coat protein complex (COP) II coat subunit Sec23. The COPII coat facilitates the budding and targeting of ER-derived vesicles with their acceptor compartment. We provide evidence that COPII-coated vesicles and the ER-Golgi fusion machinery are needed for macroautophagy. Our results imply that TRAPPIII binds to COPII vesicles at the phagophore assembly site and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. These events are conserved in yeast to mammals.Macroautophagy is a highly conserved catabolic process that uses a specialized membrane trafficking pathway to target proteins and organelles for degradation (1). Defects in this process have been linked to a variety of human diseases, including neurodegenerative diseases such as Parkinson’s disease (2). Macroautophagy is induced by a variety of physiological stresses and begins with the expansion of a cup-shaped nucleating membrane called the phagophore, or isolation membrane. As the phagophore expands, it engulfs intracellular proteins and membranes that are marked for degradation. This expanding membrane eventually closes to become an autophagosome, a double-membrane structure that seals its contents from the cytosol and delivers it to the lysosome or vacuole for degradation. A central unanswered question in the autophagy field is the mechanism by which the phagophore forms and matures into an autophagosome. Although it was once thought that the phagophore assembles de novo, recent evidence suggests it forms from a preexisting compartment. Compartments on the secretory pathway, including the endoplasmatic reticulum (ER) and Golgi complex, have been invoked in phagophore assembly (3, 4).A collection of ATG (autophagy-related) genes, the products of which regulate autophagy, were identified in the yeast Saccharomyces cerevisiae (1). Many of the Atg proteins needed for macroautophagy in yeast are shared with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that transports certain hydrolases into the vacuole. Both pathways require the sequestration of cargo within a double-membrane structure; however, only the macroautophagy pathway is conserved in higher eukaryotes (5). When autophagy is induced, ATG gene products assemble at the phagophore assembly site (PAS) in a hierarchical manner. The scaffold protein complex that organizes this site is the Atg17 complex (6, 7).Previous studies have shown that the transport protein particle (TRAPP) III complex, an autophagy-specic guanine nucleotide exchange factor (GEF) for the Rab GTPase Ypt1, is recruited to the PAS (8) by Atg17 (9). At the PAS, TRAPPIII activates Ypt1 (8), which then recruits its downstream effector, the serine/threonine Atg1 kinase, to the PAS (9). TRAPPIII is one of three multimeric GEFs, called TRAPPI, TRAPPII, and TRAPPIII, that activate Ypt1 on different trafficking pathways (10). TRAPPI, an elongated complex ∼18 nm in length (11, 12), binds to ER-derived COPII-coated vesicles via an interaction between the TRAPPI subunit Bet3 and the coat subunit Sec23 (11, 13). The TRAPPIII complex contains the same six subunits present in TRAPPI plus one unique subunit, Trs85, that targets this complex to the PAS (8, 9). Here, we describe the single-particle electron microscopy (EM) structure of TRAPPIII from S. cerevisiae. TRAPPIII (23 nm) is longer than TRAPPI with Trs85 capping one end of the complex. As in TRAPPI, the two Bet3 subunits in TRAPPIII are solvent accessible and are available for interaction with Sec23. This observation suggests that TRAPPIII may play a role in tethering COPII-coated vesicles at the PAS. Consistent with this proposal, we find that COPII vesicles accumulate at the PAS when autophagy is blocked. Additionally, we show that mutations in components of the ER-Golgi trafficking machinery, which mediate COPII vesicle fusion, disrupt autophagy. Finally, we find that COPII vesicles accumulate at or near ER-mitochondria contact sites when COS-7 cells are starved. ER-mitochondria contact sites are where autophagosomes form in mammalian cells (14).     

Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2.

The clathrin-associated protein complex 2 (AP-2 complex) is a group of proteins associated with clathrin-coated vesicles and believed to interact with cytoplasmic domains of receptors found in the plasma membrane. AP-2 was purified as an assembly of several polypeptide chains (alpha, beta, AP50, and AP17), of which only the alpha and beta chains (100-115 kDa) show significant heterogeneity. We have obtained cDNA clones for two distinct rat brain beta chains. We have also studied the domain organization of bovine brain AP-2 complexes by selective proteolysis. Results of these studies show that the alpha and beta chains have a similar two-domain organization. Their amino-terminal domains are relatively invariant whereas their carboxyl-terminal domains are variable in both sequence and length. We propose that the variable domains select receptors for inclusion in coated vesicles.     

ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function

Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component β-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.     

Identification of yeast component A: reconstitution of the geranylgeranyltransferase that modifies Ypt1p and Sec4p.

Members of a large family of small GTP-binding proteins, termed Rabs in mammalian cells or Ypt and Sec4 in yeast, regulate vesicular traffic in all eukaryotic cells. These proteins are able to bind to membranes because they are modified by the type II geranylgeranyltransferase (GGTase-II), a multisubunit complex. Component A, encoded by the choroideremia gene in humans, is an escort protein that brings Rabs to component B, the catalytic alpha/beta heterodimer. Mutations in the catalytic subunits of the yeast GGTase-II (Bet2p/Mad2p) disrupt the membrane attachment of Ypt1p and Sec4p and this in turn blocks membrane traffic. In mammalian cells, deletions in choroideremia lead only to retinal degeneration, even though GGTase-II activity is defective. The yeast MRS6 gene encodes a protein that is approximately 30% identical to the choroideremia gene product. Here we show that the addition of recombinant Mrs6p to bacterially expressed Bet2p (beta subunit) and Mad2p (alpha subunit) reconstitutes GGTase-II activity in vitro, demonstrating that Mrs6p is yeast component A. Like Bet2p and Mad2p, Mrs6p is required for the membrane attachment of Ypt1p and Sec4p in vivo. In contrast to what has been observed before for the loss of function of the choroideremia gene, the depletion of Mrs6p from yeast cells blocks vesicular transport. Thus, these findings suggest that there is one essential escort protein in yeast, while more than one may exist in mammalian cells.     

Hsp90 enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore

Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. Here, we investigate how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro. Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p.