The transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif ((607)RI(608)) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine transporters and the more distantly related GABA transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.

Target of rapamycin complex 2 (TORC2) is a widely conserved serine/threonine protein kinase. In the yeast Saccharomyces cerevisiae, TORC2 is essential, playing a key role in plasma membrane homeostasis. In this role, TORC2 regulates diverse processes, including sphingolipid synthesis, glycerol production and efflux, polarization of the actin cytoskeleton, and endocytosis. The major direct substrate of TORC2 is the AGC-family kinase Ypk1. Ypk1 connects TORC2 signaling to actin polarization and to endocytosis via the flippase kinases Fpk1 and Fpk2. Here, we report that Fpk1 mediates TORC2 signaling to control actin polarization, but not endocytosis, via aminophospholipid flippases. To search for specific targets of these flippase kinases, we exploited the fact that Fpk1 prefers to phosphorylate Ser residues within the sequence RXS(L/Y)(D/E), which is present ∼90 times in the yeast proteome. We observed that 25 of these sequences are phosphorylated by Fpk1 in vitro We focused on one sequence hit, the Ark/Prk-family kinase Akl1, as this kinase previously has been implicated in endocytosis. Using a potent ATP-competitive small molecule, CMB4563, to preferentially inhibit TORC2, we found that Fpk1-mediated Akl1 phosphorylation inhibits Akl1 activity, which, in turn, reduces phosphorylation of Pan1 and of other endocytic coat proteins and ultimately contributes to a slowing of endocytosis kinetics. These results indicate that the regulation of actin polarization and endocytosis downstream of TORC2 is signaled through separate pathways that bifurcate at the level of the flippase kinases.

Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R) gene, a central determinant of black (eumelanin) vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1RD) and Recessive Red (MC1Re). A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1RD. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA), a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele.

The vector-borne, protistan parasite Trypanosoma brucei is the only known eukaryote with a multifunctional RNA polymerase I that, in addition to ribosomal genes, transcribes genes encoding the parasite's major cell-surface proteins-the variant surface glycoprotein (VSG) and procyclin. In the mammalian bloodstream, antigenic variation of the VSG coat is the parasite's means to evade the immune response, while procyclin is necessary for effective establishment of trypanosome infection in the fly. Moreover, the exceptionally high efficiency of mono-allelic VSG expression is essential to bloodstream trypanosomes since its silencing caused rapid cell-cycle arrest in vitro and clearance of parasites from infected mice. Here we describe a novel protein complex that recognizes class I promoters and is indispensable for class I transcription; it consists of a dynein light chain and six polypeptides that are conserved only among trypanosomatid parasites. In accordance with an essential transcriptional function of the complex, silencing the expression of a key subunit was lethal to bloodstream trypanosomes and specifically affected the abundance of rRNA and VSG mRNA. The complex was dubbed class I transcription factor A.

Intracellular trafficking is critical for delivering molecules and organelles to their proper destinations to carry out normal cellular functions. Disruption of intracellular trafficking has been implicated in the pathogenesis of various neurodegenerative disorders. In addition, a number of genes involved in vesicle/organelle trafficking are also essential for pigmentation, and loss of those genes is often associated with mouse coat-color dilution and human hypopigmentary disorders. Hence, we postulated that screening for mouse mutants with both neurological defects and coat-color dilution will help identify additional factors associated with intracellular trafficking in neuronal cells. In this study, we characterized a mouse mutant with a unique N-ethyl-N-nitrosourea (ENU)-induced mutation, named nur17. nur17 mutant mice exhibit both coat-color dilution and ataxia due to Purkinje cell degeneration in the cerebellum. By positional cloning, we identified that the nur17 mouse carries a T-to-C missense mutation in archain 1 (Arcn1) gene which encodes the delta subunit of the coat protein I (COPI) complex required for intracellular trafficking. Consistent with this function, we found that intracellular trafficking is disrupted in nur17 melanocytes. Moreover, the nur17 mutation leads to common characteristics of neurodegenerative disorders such as abnormal protein accumulation, ER stress, and neurofibrillary tangles. Our study documents for the first time the physiological consequences of the impairment of the ARCN1 function in the whole animal and demonstrates a direct association between ARCN1 and neurodegeneration.

A yeast plasma membrane protein, Chs3p, transits to the mother-bud neck from a reservoir comprising the trans-Golgi network (TGN) and endosomal system. Two TGN/endosomal peripheral proteins, Chs5p and Chs6p, and three Chs6p paralogues form a complex that is required for the TGN to cell surface transport of Chs3p. The role of these peripheral proteins has not been clear, and we now provide evidence that they create a coat complex required for the capture of membrane proteins en route to the cell surface. Sec7p, a Golgi protein required for general membrane traffic and functioning as a nucleotide exchange factor for the guanosine triphosphate (GTP)-binding protein Arf1p, is required to recruit Chs5p to the TGN surface in vivo. Recombinant forms of Chs5p, Chs6p, and the Chs6p paralogues expressed in baculovirus form a complex of approximately 1 MD that binds synthetic liposomes in a reaction requiring acidic phospholipids, Arf1p, and the nonhydrolyzable GTPgammaS. The complex remains bound to liposomes centrifuged on a sucrose density gradient. Thin section electron microscopy reveals a spiky coat structure on liposomes incubated with the full complex, Arf1p, and GTPgammaS. We termed the novel coat exomer for its role in exocytosis from the TGN to the cell surface. Unlike other coats (e.g., coat protein complex I, II, and clathrin/adaptor protein complex), the exomer does not form buds or vesicles on liposomes.

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

A RT-PCR assay developed to amplify the full coat protein (CP) gene of apple stem pitting virus (ASPV) was evaluated using 180 Greek apple and pear samples and showed a broad detection range. This method was used to investigate the presence of ASPV in quince in Greece and showed a high incidence of 52%. The sequences of 14 isolates from various hosts with a distinct RFLP profile were determined. ASPV population genetics and the factors driving ASPV evolution were analyzed using the Greek ASPV sequences, novel sequences from Brazilian apple trees and Chinese botanical Pyrus species, and homologous sequences retrieved from GenBank. Fourteen variant types of Greek, Brazilian and botanical isolates, which differ in CP gene length and presence of indels, were identified. In addition, these analyses showed high intra- and inter-group variation among isolates from different countries and hosts, indicating the significant variability present in ASPV. Recombination events were detected in four isolates originating from Greek pear and quince and two from Brazilian apples. In a phylogenetic analysis, there was a tendency for isolates to cluster together based on CP gene length, the isolation host, and the detection method applied. Although there was no strict clustering based on geographical origin, most isolates from a given country tended to regroup in specific clusters. Interestingly, it was found that the phylogeny was correlated to the type, position, and pattern of indels, which represent hallmarks of specific lineages and indicate their possible role in virus diversification, rather than the CP size itself. Evidence of recombination between isolates from botanical and cultivated species and the clustering of isolates from botanical species and isolates from cultivated species suggest the existence of a possible undetermined transmission mechanism allowing the exchange of ASPV isolates between the cultivated and wild/ornamental hosts.

The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic whose mechanisms are unknown. HIV-1 Nef hijacks AP-1 to sequester major histocompatibility complex class I (MHC-I), evading immune detection. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin and determined its structure. The coat assembles via Arf1 dimer interfaces. AP-1-positive tubules are enriched in cells upon clathrin knockdown. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef sequesters MHC-I. Coat contact residues are conserved across Arf isoforms and the Arf-dependent AP complexes AP-1, AP-3, and AP-4. Thus, AP complexes can self-assemble with Arf1 into tubular coats without clathrin or other scaffolding factors. The AP-1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats as an intermediate in clathrin vesicle formation from internal membranes and as an MHC-I sequestration mechanism in HIV-1 infection.

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

Ancestral coatomer element 1 (ACE1) proteins assemble latticework coats for COPII vesicles and the nuclear pore complex. The ACE1 protein Sec31 and Sec13 make a 2:2 tetramer that forms the edge element of the COPII outer coat. In this study, we report that the COPII accessory protein Sec16 also contains an ACE1. The 165-kD crystal structure of the central domain of Sec16 in complex with Sec13 was solved at 2.7-A resolution. Sec16 and Sec13 also make a 2:2 tetramer, another edge element for the COPII system. Domain swapping at the ACE1-ACE1 interface is observed both in the prior structure of Sec13-Sec31 and in Sec13-Sec16. A Sec31 mutant in which domain swapping is prevented adopts an unprecedented laminated structure, solved at 2.8-A resolution. Our in vivo data suggest that the ACE1 element of Sec31 can functionally replace the ACE1 element of Sec16. Our data support Sec16 as a scaffold for the COPII system and a template for the Sec13-Sec31 coat.

The kinetoplastids are an important group of protozoa from the Excavata supergroup, and cause numerous diseases with wide environmental, economic and ecological impact. Trypanosoma brucei, the causative agent of human African trypanosomiasis, expresses a dense variant surface glycoprotein (VSG) coat, facilitating immune evasion via rapid switching and antigenic variation. Coupled to VSG switching is efficient clathrin-mediated endocytosis (CME), which removes anti-VSG antibody from the parasite surface. While the precise molecular basis for an extreme CME flux is unknown, genes encoding the AP2 complex, central to CME in most organisms, are absent from T. brucei, suggesting a mechanistic divergence in trypanosome CME. Here we identify the AP complex gene cohorts of all available kinetoplastid genomes and a new Trypanosoma grayi genome. We find multiple secondary losses of AP complexes, but that loss of AP2 is restricted to T. brucei and closest relatives. Further, loss of AP2 correlates precisely with the presence of VSG genes, supporting a model whereby these two adaptations may function synergistically in immune evasion.

Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6 cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein-like family.

Direct cell-to-cell spreading of Listeria monocytogenes requires the bacteria to induce actin-based finger-like membrane protrusions in donor host cells that are endocytosed through caveolin-rich membrane invaginations by adjacent receiving cells. An actin shell surrounds these endocytic sites; however, its structure, composition, and functional significance remain elusive. Here, we show that the formin mDia1, but surprisingly not the Arp2/3 complex, is enriched at the membrane invaginations generated by L. monocytogenes during HeLa and Jeg-3 cell infections. Electron microscopy reveals a band of linear actin filaments that run along the longitudinal axis of the invagination membrane. Mechanistically, mDia1 expression is vital for the assembly of this F-actin shell. mDia1 is also required for the recruitment of Filamin A, a caveola-associated F-actin cross-linking protein, and caveolin-1 to the invaginations. Importantly, mixed-cell infection assays show that optimal caveolin-based L. monocytogenes cell-to-cell spreading correlates with the formation of the linear actin filament-containing shell by mDia1. IMPORTANCE Listeria monocytogenes spreads from one cell to another to colonize tissues. This cell-to-cell movement requires the propulsive force of an actin-rich comet tail behind the advancing bacterium, which ultimately distends the host plasma membrane into a slender bacterium-containing membrane protrusion. These membrane protrusions induce a corresponding invagination in the membrane of the adjacent host cell. The host cell that receives the protrusion utilizes caveolin-based endocytosis to internalize the structures, and filamentous actin lines these membrane invaginations. Here, we set out to determine the structure and function of this filamentous actin "shell." We demonstrate that the formin mDia1, but not the Arp2/3 complex, localizes to the invaginations. Morphologically, we show that this actin is organized into linear arrays and not branched dendritic networks. Mechanistically, we show that the actin shell is assembled by mDia1 and that mDia1 is required for efficient cell-to-cell transfer of L. monocytogenes.

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.

A safe and broadly effective direct acting anti-hepatitis C virus (HCV) agent that can withstand the viral mutation is needed. In this study, human single chain antibody variable fragments (HuscFvs) to conserved non-structural protein-5A (NS5A) of HCV were produced by phage display technology. Recombinant NS5A was used as bait for fishing-out the protein bound-phages from the HuscFv-phage display library. NS5A-bound HuscFvs produced by five phage transfected-E. coli clones were linked molecularly to nonaarginine (R9) for making them cell penetrable (become transbodies). The human monoclonal transbodies inhibited HCV replication in the HCVcc infected human hepatic cells and also rescued the cellular antiviral immune response from the viral suppression. Computerized simulation verified by immunoassays indicated that the transbodies used several residues in their multiple complementarity determining regions (CDRs) to form contact interface with many residues of the NS5A domain-I which is important for HCV replication complex formation and RNA binding as well as for interacting with several host proteins for viral immune evasion and regulation of cellular physiology. The human monoclonal transbodies have high potential for testing further as a new ramification of direct acting anti-HCV agent, either alone or in combination with their cognates that target other HCV proteins.

Autochthonous cattle breeds are genetic resources that, in many cases, have been fixed for inheritable exterior phenotypes useful to understand the genetic mechanisms affecting these breed-specific traits. Reggiana and Modenese are two closely related autochthonous cattle breeds mainly raised in the production area of the well-known Protected Designation of Origin Parmigiano-Reggiano cheese, in the North of Italy. These breeds can be mainly distinguished for their standard coat colour: solid red in Reggiana and solid white with pale shades of grey in Modenese. In this study we genotyped with the GeneSeek GGP Bovine 150k single nucleotide polymorphism (SNP) chip almost half of the extant cattle populations of Reggiana (n = 1109 and Modenese (n = 326) and used genome-wide information in comparative FST analyses to detect signatures of selection that diverge between these two autochthonous breeds. The two breeds could be clearly distinguished using multidimensional scaling plots and admixture analysis. Considering the top 0.0005% FST values, a total of 64 markers were detected in the single-marker analysis. The top FST value was detected for the melanocortin 1 receptor (MC1R) gene mutation, which determines the red coat colour of the Reggiana breed. Another coat colour gene, agouti signalling protein (ASIP), emerged amongst this list of top SNPs. These results were also confirmed with the window-based analyses, which included 0.5-Mb or 1-Mb genome regions. As variability affecting ASIP has been associated with white coat colour in sheep and goats, these results highlighted this gene as a strong candidate affecting coat colour in Modenese breed. This study demonstrates how population genomic approaches designed to take advantage from the diversity between local genetic resources could provide interesting hints to explain exterior traits not yet completely investigated in cattle.

ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. We identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system, and show that these regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. We found that COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets.

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.