Silencing of Mammalian Sar1 Isoforms Reveals COPII‐Independent Protein Sorting and Transport

The Sar1 GTPase coordinates the assembly of coat protein complex‐II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER‐to‐Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII‐mediated transport in mammalian cells, we used small interfering RNA (siRNA)‐mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER‐to‐Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen‐I is inhibited. These data provide proof‐of‐principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII .     

Mechanisms of COPII vesicle formation and protein sorting

The evolutionarily conserved coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). COPII coat is responsible for direct capture of cargo proteins and for the physical deformation of the ER membrane that drives the COPII vesicle formation. In addition to coat proteins, recent data have indicated that the Ras-like small GTPase Sar1 plays multiple roles, such as COPII coat recruitment, cargo sorting, and completion of the final fission. In the present review, we summarize current knowledge of COPII-mediated vesicle formation from the ER, as well as highlighting non-canonical roles of COPII components.     

Sar1 GTPase Activity Is Regulated by Membrane Curvature

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.     

Identification of a Site in Sar1 Involved in the Interaction with the Cytoplasmic Tail of Glycolipid Glycosyltransferases

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1^D198A) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1^D189A also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.     

Persistence of Golgi matrix distribution exhibits the same dependence on Sar1p activity as a Golgi glycosyltransferase

We investigated the relative distributional persistence of Golgi 'matrix' proteins and glycosyltransferases to an endoplasmic reticulum exit block induced by expression of a GDP-restricted Sar1p. HeLa cells were microinjected with plasmid encoding the GDP-restricted mutant (T39N) of Sar1p to block endoplasmic reticulum exit and then scored for the distribution of GM130 (Golgi m atrix protein of 130  kDa), a cis located golgin; p27, a member of the p24 family of proteins; giantin, a protein that interacts indirectly with GM130; and the Golgi glycosyltransferase, N-acetylgalactosaminyltransferase-2 (GalNAcT2). All of these proteins lost their compact, juxtanuclear distribution and displayed characteristics of endoplasmic reticulum/cytoplasmic accumulation with the same dependence on plasmid concentration. The kinetics of redistribution of GM130 and GalNAcT2 were identical. Expression of Sar1pT39N displaced the COPII coat protein Sec13p from endoplasmic reticulum exit sites consistent with disruption of these sites. This occurred without disturbing the overall distribution of endoplasmic reticulum membrane. Furthermore, the reassembly of a juxtanuclear Golgi matrix as assayed by the distribution of GM130 following washout of the Golgi disrupting drug, brefeldin A, was blocked by microinjected Sar1pT39N plasmids. We conclude that the persistence, i.e. stability and maintenance, of Golgi matrix distribution and its reassembly following drug disruption are exquisitely dependent on Sar1p activity.     

Carbon tetrachloride suppresses ER-Golgi transport by inhibiting COPII vesicle formation on the ER membrane in the RLC-16 hepatocyte cell line

Carbon tetrachloride (CCl₄) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl₄ inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl₄ on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl₄ suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl₄ treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl₄-induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl₄ inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.     

Sec23 Homolog Nel1 Is a Novel GTPase-activating Protein for Sar1 but Does Not Function as a Subunit of the Coat Protein Complex II (COPII) Coat

The coat protein complex II (COPII) generates transport carriers from the endoplasmic reticulum (ER) under the control of the small GTPase Sar1. Sec23 is well known as a structural component of the COPII coat and as a GTPase-activating protein (GAP) for Sar1. Here, we showed that Saccharomyces cerevisiae contains a novel Sec23 paralog, Nel1, which appears not to function as a subunit of the COPII coat. Nel1 does not associate with any of the COPII components, but it exhibits strong Sar1 GAP activity. We also demonstrated that the chromosomal deletion of NEL1 leads to a significant growth defect in the temperature-sensitive sar1D32G background, suggesting a possible functional link between these proteins. In contrast to Sec23, which is predominantly localized at ER exit sites on the ER membrane, a major proportion of Nel1 is localized throughout the cytosol. Our findings highlight a possible role of Nel1 as a novel GAP for Sar1.     

Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells

Arf GTPases are known to be key regulators of vesicle budding in various steps of membrane traffic in yeast and animal cells. We cloned the Arabidopsis Arf1 homologue, AtArf1, and examined its function. AtArf1 complements yeast arf1 arf2 mutants and its GFP-fusion is localized to the Golgi apparatus in plant cells like its animal counterpart. The expression of dominant negative mutants of AtArf1 in tobacco and Arabidopsis cultured cells affected the localization of co-expressed GFP-tagged proteins in a variety of ways. AtArf1 Q71L and AtArf1 T31N, GTP- and GDP-fixed mutants, respectively, changed the localization of a cis-Golgi marker, AtErd2-GFP, from the Golgi apparatus to the endoplasmic reticulum but not that of GFP-AtRer1B or GFP-AtSed5. GFP-AtRer1B and GFP-AtSed5 were accumulated in aberrant structures of the Golgi by AtArf1 Q71L. A soluble vacuolar protein, sporamin-GFP, was also located to the ER by AtArf1 Q71L. These results indicate that AtArf1 play roles in the vesicular transport between the ER and the Golgi and in the maintenance of the normal Golgi organization in plant cells.     

Mon1a protein acts in trafficking through the secretory apparatus

Mon1a was originally identified as a modifier gene of vesicular traffic, as a mutant Mon1a allele resulted in increased localization of cell surface proteins, whereas reduced levels of Mon1a showed decreased secretory activity. Here we show that Mon1a affects different steps in the secretory pathway including endoplasmic reticulum-to-Golgi traffic. siRNA-dependent reduction of Mon1a levels resulted in a delay in the reformation of the Golgi apparatus after Brefeldin A treatment. Endoglycosidase H treatment of ts045VSVG-GFP confirmed that knockdown of Mon1a delayed endoplasmic reticulum-to-Golgi trafficking. Reductions in Mon1a also resulted in delayed trafficking from Golgi to the plasma membrane. Immunoprecipitation and mass spectrometry analysis showed that Mon1a associates with dynein intermediate chain. Reductions in Mon1a or dynein altered steady state Golgi morphology. Reductions in Mon1a delayed formation of ERGIC-53-positive vesicles, whereas reductions in dynein did not affect vesicle formation. These data provide strong evidence for a role for Mon1a in anterograde trafficking through the secretory apparatus.     

Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1p^dn) blocking ER export. Sar1p^dn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1p^dn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.     

The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the “K loop,” which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.     

Inhibition of Golgi-apparatus function by brefeldin A in maize coleoptiles and its consequences on auxin-mediated growth,cell-wall extensibility and secretion of cell-wall proteins

Brefeldin A (BFA), a fungal metabolite causing dysfunction of the Golgi apparatus in plant and animal cells, was used to investigate the role of secretory processes at the plasma membrane in auxin-mediated elongation growth of maize (Zea mays L.) coleoptiles. In abraded coleoptile segments BFA produced, within less than 30 min, a decrease in the incorporation of [³H]leucine into tightly bound cell-wall proteins, accompanied by an increased incorporation into the intracellular pool of putative cell-wall glycoproteins. Total protein synthesis was not affected. Electron micrographs revealed striking morphological changes in dictyosomes (especially vesiculation of trans-cisternae), accumulation of Golgi vesicles and dilation of the endoplasmic reticulum. These effects are taken as indication that BFA interferes with the secretion of cell-wall components. Elongation growth of coleoptile segments in the presence and absence of auxin was inhibited by 80% in 20 mg·l^–1 BFA. If BFA was applied to segments growing in the presence of auxin, maximum inhibition was reached after about 30 min, indicating that the growth response depends on an uninterrupted supply of a cell-wall or plasma-membrane component (wall-loosening factor) delivered by the secretory pathway. After its secretion, this factor has a rather short growth-effective life time. The inhibition of auxin-mediated growth by BFA was accompanied by an elimination of auxin-induced cell-wall extensibility and by an inhibition of auxin-induced proton excretion. Fusicoccin-induced proton excretion was similarly affected by BFA. It is concluded that both the wall-loosening process underlying elongation growth as well as proton excretion depend on an intact secretory pathway from the Golgi apparatus to the cell wall; however, a causal relationship between these processes is not warranted by the data.Abbreviations BFA brefeldin A - FC fusicoccin - TCA trichloroacetic acid - WLF wall-loosening factor Supported by Deutsche Forschungsgemeinschaft (SFB 206). We thank Ms. B. Huvermann and Mrs. C. Plachy for conducting growth and proton excretion measurements.     

Transport of the GlcNAc-1-phosphotransferase α/β-Subunit Precursor Protein to the Golgi Apparatus Requires a Combinatorial Sorting Motif

The Golgi-resident N-acetylglucosamine-1-phosphotransferase (PT) complex is composed of two α-, β-, and γ-subunits and represents the key enzyme for the biosynthesis of mannose 6-phosphate recognition marker on soluble lysosomal proteins. Mutations in the PT complex cause the lysosomal storage diseases mucolipidosis II and III. A prerequisite for the enzymatic activity is the site-1 protease-mediated cleavage of the PT α/β-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT α/β-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT α/β-subunit precursor protein are exported whereas coexpressed separate α- and β-subunits failed to reach the cis-Golgi compartment. Mutational analyses revealed combinatorial, non-exchangeable dileucine and dibasic motifs located in a defined sequence context in the cytosolic N- and C-terminal domains that are required for efficient ER exit and subsequent proteolytic activation of the α/β-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT α/β-subunit precursor protein is inhibited indicating its transport in coat protein complex II-coated vesicles. Expression studies of missense mutations identified in mucolipidosis III patients that alter amino acids in the N- and C-terminal domains demonstrated that the substitution of a lysine residue in close proximity to the dileucine sorting motif impaired ER-Golgi transport and subsequent activation of the PT α/β-subunit precursor protein. The data suggest that the oligomeric type III membrane protein PT complex requires a combinatorial sorting motif that forms a tertiary epitope to be recognized by distinct sites within the coat protein complex II machinery.     

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [³H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.     

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.     

Endomembranes and vesicle trafficking

Over the past year extensive analyses of the accumulated data on the structural and functional organisation of the endomembrane system and vesicular trafficking in higher plants have shown it to be far more complex than previously anticipated. The availability of molecular tools combined with new opportunities to visualise endomembrane dynamics in vivo will allow better understanding of the fundamental processes underlying the complexity of endomembrane behaviour and vesicular trafficking.     

A Novel Membrane Protein Complex on the Endoplasmic Reticulum and Early Golgi Compartments in the Yeast Saccharomyces cerevisiae

A novel membrane protein, Yml067c in the systematic ORF name, was discovered as a component of immunoisolated vesicles of the early Golgi compartment of the yeast Saccharomyces cerevisiae (Cho et al., FEBS Lett. 469, 151-154 (2000)). Conserved sequences having sequence similarity to Yml067c were widely distributed in the eukaryotes and one of them, Yal042w, was found in the Saccharomyces genome database. In the yeast cell, Yml067c and Yal042w were found to form a heterooligomeric complex by immunoprecipitation of their tagged derivatives from the detergent-solubilized membrane. Cell fractionation and indirect immunofluorescent staining indicated that the majority of these proteins were localized on the ER membrane. Therfore, the Yml067c-Yal042w complex should shuttle between the ER and the early Golgi compartment as well as the p24-family proteins.