Arabidopsis mannan synthase CSLA9 and glucan synthase CSLC4 have opposite orientations in the Golgi membrane

Several proteins encoded by the cellulose synthase-like (CSL) gene family are known to be processive glycan synthases involved in the synthesis of cell-wall polysaccharides. These include CSLA proteins, which synthesize β-(1→4)-linked mannans found in the walls of many plant species, and CSLC proteins, which are thought to synthesize the β-(1→4)-linked glucan backbone of xyloglucan, an abundant polysaccharide in the primary walls of many plants. CSLA and CSLC proteins are predicted to have multiple membrane spans, and their products (mannan and xyloglucan) accumulate in the Golgi lumen. Knowing where these proteins are located in the cell and how they are orientated in the membrane is important for understanding many aspects of mannan and xyloglucan biosynthesis. In this study, we investigate the subcellular localization and membrane protein topology of CSLA9 and CSLC4, the members of these two families that are most highly expressed in Arabidopsis. CSLA9 and CSLC4 are found predominantly in Golgi membranes, based on co-localization with the known ER/Golgi marker ERD2-YFP. The topology of epitope-tagged proteins was examined using protease protection experiments. Experiments were designed to determine the positions of both the protein termini and the active loop of the CSL proteins investigated. The topology of CSLA9 is characterized by an odd number of transmembrane domains (probably five) and an active site that faces the Golgi lumen. In contrast, CSLC4 has an even number of transmembrane domains (probably six) and an active site that faces the cytosol. The implications of these topologies on various aspects of hemicellulose biosynthesis are discussed.     

Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.     

Building the wall: genes and enzyme complexes for polysaccharide synthases

The complete sequence of the Arabidopsis genome has revealed a total of 40 cellulose synthase (CesA) and cellulose synthase-like (Csl) genes. Recent studies suggest that each CESA polypeptide contains only one catalytic center, and that two or more polypeptides from different genes might be needed to form a functional cellulose synthase complex.     

Genetic dissection of plant cell-wall biosynthesis

The plant cell wall is a complex structure consisting of a variety of polymers including cellulose, xyloglucan, xylan and polygalacturonan. Biochemical and genetic analysis has made it possible to clone genes encoding cellulose synthases (CesA). A comparison of the predicted protein sequences in the Arabidopsis genome indicates that 30 divergent genes with similarity to CesAs exist. It is possible that these cellulose synthase-like (Csl) proteins do not contribute to cellulose synthesis, but rather to the synthesis of other wall polymers. A major challenge is, therefore, to assign biological function to these genes. In an effort to address this issue we have systematically identified T-DNA or transposon insertions in 17 Arabidopsis Csls. Phenotypic characterization of "knock-out" mutants includes the determination of spectroscopic profile differences in mutant cell walls from wild-type plants by Fourier-transform IR microscopy. A more precise characterization includes cell wall fractionation followed by neutral sugar composition analysis by anionic exchange chromatography.     

A cellulose synthase-like protein involved in hyphal tip growth and morphological differentiation in streptomyces

Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing beta-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslA(Sc)) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslA(Sc) replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslA(Sc). cslA(Sc) was constitutively transcribed, and an enhanced green fluorescent protein-CslA(Sc) fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslA(Sc) interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslA(Sc) membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development.     

Arabidopsis Sec21p and Sec23p homologs. Probable coat proteins of plant COP-coated vesicles

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.     

Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles

Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.     

Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase

The Arabidopsis genome project has recently reported sequences with similarity to members of the terpene synthase (TPS) gene family of higher plants. Surprisingly, several Arabidopsis terpene synthase-like sequences (AtTPS) share the most identity with TPS genes that participate in secondary metabolism in terpenoid-accumulating plant species. Expression of a putative Arabidopsis terpene synthase gene, designated AtTPS03, was demonstrated by amplification of a 392-bp cDNA fragment using primers designed to conserved regions of plant terpene synthases. Using the AtTPS03 fragment as a hybridization probe, a second AtTPS cDNA, designated AtTPS10, was isolated from a jasmonate-induced cDNA library. The partial AtTPS10 cDNA clone contained an open reading frame of 1665 bp encoding a protein of 555 amino acids. Functional expression of AtTPS10 in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate (C(10)) into the acyclic monoterpenes beta-myrcene and (E)-beta-ocimene and small amounts of cyclic monoterpenes. Based on sequence relatedness, AtTPS10 was classified as a member of the TPSb subfamily of angiosperm monoterpene synthases. Sequence comparison of AtTPS10 with previously cloned monoterpene synthases suggests independent events of functional specialization of terpene synthases during the evolution of terpenoid secondary metabolism in gymnosperms and angiosperms. Functional characterization of the AtTPS10 gene was prompted by the availability of Arabidopsis genome sequences. Although Arabidoposis has not been reported to form terpenoid secondary metabolites, the unexpected expression of TPS genes belonging to the TPSb subfamily in this species strongly suggests that terpenoid secondary metabolism is active in the model system Arabidopsis.     

Biosynthesis of plant cell wall polysaccharides - a complex process

Cellulose, a major component of plant cell walls, is made by dynamic complexes that move within the plasma membrane while depositing cellulose directly into the wall. On the other hand, matrix polysaccharides are made in the Golgi and delivered to the wall via secretory vesicles. Several Golgi proteins that are involved in glucomannan and xyloglucan biosynthesis have been identified, including some glycan synthases that show sequence similarity to the cellulose synthase proteins and several glycosytransferases that add sidechains to the polysaccharide backbones. Recent progress in identifying the proteins needed for polysaccharide biosynthesis should lead to an improved understanding of the molecular details of these complex processes, and eventually to an ability to manipulate them in an effort to generate plants that have improved properties for human uses.     

Topology of the maize mixed linkage (1->3),(1->4)-beta-d-glucan synthase at the Golgi membrane

Mixed-linkage (1-->3),(1-->4)-beta-d-glucan is a plant cell wall polysaccharide composed of cellotriosyl and cellotetraosyl units, with decreasingly smaller amounts of cellopentosyl, cellohexosyl, and higher cellodextrin units, each connected by single (1-->3)-beta-linkages. (1-->3),(1-->4)-beta-Glucan is synthesized in vitro with isolated maize (Zea mays) Golgi membranes and UDP-[(14)C]d-glucose. The (1-->3),(1-->4)-beta-glucan synthase is sensitive to proteinase K digestion, indicating that part of the catalytic domain is exposed to the cytoplasmic face of the Golgi membrane. The detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid] (CHAPS) also lowers (1-->3),(1-->4)-beta-glucan synthase activity. In each instance, the treatments selectively inhibit formation of the cellotriosyl units, whereas synthesis of the cellotetraosyl units is essentially unaffected. Synthesis of the cellotriosyl units is recovered when a CHAPS-soluble factor is permitted to associate with Golgi membranes at synthesis-enhancing CHAPS concentrations but lost if the CHAPS-soluble fraction is replaced by fresh CHAPS buffer. In contrast to other known Golgi-associated synthases, (1-->3),(1-->4)-beta-glucan synthase behaves as a topologic equivalent of cellulose synthase, where the substrate UDP-glucose is consumed at the cytosolic side of the Golgi membrane, and the glucan product is extruded through the membrane into the lumen. We propose that a cellulose synthase-like core catalytic domain of the (1-->3),(1-->4)-beta-glucan synthase synthesizes cellotetraosyl units and higher even-numbered oligomeric units and that a separate glycosyl transferase, sensitive to proteinase digestion and detergent extraction, associates with it to add the glucosyl residues that complete the cellotriosyl and higher odd-numbered units, and this association is necessary to drive polymer elongation.     

Sphingomyelin organization is required for vesicle biogenesis at the Golgi complex

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D ‐ceramide‐C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D ‐ceramide‐C6‐treated HeLa cells revealed an increase in the levels of C6‐sphingomyelin, C6‐glucosylceramide, and diacylglycerol. D ‐ceramide‐C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D ‐ceramide‐C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6‐sphingomyelin prevented liquid‐ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.     

The cytoplasmic localization of the catalytic site of CSLF6 supports a channeling model for the biosynthesis of mixed‐linkage glucan

Mixed‐linkage glucan (MLG) is a significant cell wall carbohydrate in grasses and an important carbon source for human consumption and biofuel production. MLG biosynthesis depends on the biochemical activity of membrane spanning glucan synthases encoded by the CSLH and CSLF cellulose synthase‐like gene families. CSLF proteins are the best characterized to date but relatively little information is known about their topology with respect to the biosynthetic membranes. In this study, we report on the topology of CSLF6 protein derived from the model grass species Brachypodium distachyon (BdCSLF6) when it is expressed in heterologous systems. Using live cell imaging and immuno‐electron microscopy analyses of tobacco epidermal cells expressing BdCSLF6, we demonstrate that a functional yellow fluorescent protein (YFP) fusion of BdCSLF6 is localized to the Golgi apparatus and that the Golgi localization of BdCSLF6 is sufficient for MLG biosynthesis. By implementing protease protection assays of BdCSLF6 expressed in the yeast Pichia pastoris, we also demonstrate that the catalytic domain, the N‐terminus and the C‐ terminus of the protein are exposed in the cytosol. Furthermore, we found that BdCSLF6 is capable of producing MLG not only in tobacco cells but also in Pichia, which generally does not produce MLG. Together, these results support the conclusion that BdCSLF6 can produce both of the linkages present in the (1,3;1,4)‐β‐d ‐glucan chain of MLG and that the product is channelled at the Golgi into the secretory pathway for deposition into the cell wall.     

Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes

In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis- Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro.

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.     

Pausing of Golgi Bodies on Microtubules Regulates Secretion of Cellulose Synthase Complexes in Arabidopsis

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by plasma membrane–bound complexes containing cellulose synthase proteins (CESAs). Here, we establish a role for the cytoskeleton in intracellular trafficking of cellulose synthase complexes (CSCs) through the in vivo study of the green fluorescent protein (GFP)-CESA3 fusion protein in Arabidopsis thaliana hypocotyls. GFP-CESA3 localizes to the plasma membrane, Golgi apparatus, a compartment identified by the VHA-a1 marker, and, surprisingly, a novel microtubule-associated cellulose synthase compartment (MASC) whose formation and movement depend on the dynamic cortical microtubule array. Osmotic stress or treatment with the cellulose synthesis inhibitor CGA 325''615 induces internalization of CSCs in MASCs, mimicking the intracellular distribution of CSCs in nongrowing cells. Our results indicate that cellulose synthesis is coordinated with growth status and regulated in part through CSC internalization. We find that CSC insertion in the plasma membrane is regulated by pauses of the Golgi apparatus along cortical microtubules. Our data support a model in which cortical microtubules not only guide the trajectories of CSCs in the plasma membrane, but also regulate the insertion and internalization of CSCs, thus allowing dynamic remodeling of CSC secretion during cell expansion and differentiation.     

Cellulose biosynthesis: current views and evolving concepts

* AIMS: To outline the current state of knowledge and discuss the evolution of various viewpoints put forth to explain the mechanism of cellulose biosynthesis. * SCOPE: Understanding the mechanism of cellulose biosynthesis is one of the major challenges in plant biology. The simplicity in the chemical structure of cellulose belies the complexities that are associated with the synthesis and assembly of this polysaccharide. Assembly of cellulose microfibrils in most organisms is visualized as a multi-step process involving a number of proteins with the key protein being the cellulose synthase catalytic sub-unit. Although genes encoding this protein have been identified in almost all cellulose synthesizing organisms, it has been a challenge in general, and more specifically in vascular plants, to demonstrate cellulose synthase activity in vitro. The assembly of glucan chains into cellulose microfibrils of specific dimensions, viewed as a spontaneous process, necessitates the assembly of synthesizing sites unique to most groups of organisms. The steps of polymerization (requiring the specific arrangement and activity of the cellulose synthase catalytic sub-units) and crystallization (directed self-assembly of glucan chains) are certainly interlinked in the formation of cellulose microfibrils. Mutants affected in cellulose biosynthesis have been identified in vascular plants. Studies on these mutants and herbicide-treated plants suggest an interesting link between the steps of polymerization and crystallization during cellulose biosynthesis. * CONCLUSIONS: With the identification of a large number of genes encoding cellulose synthases and cellulose synthase-like proteins in vascular plants and the supposed role of a number of other proteins in cellulose biosynthesis, a complete understanding of this process will necessitate a wider variety of research tools and approaches than was thought to be required a few years back.     

Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesicles in Dictyostelium cells

Golvesin is a new protein associated with membranes of the Golgi apparatus and post-Golgi vesicles in Dictyostelium cells. An internal hydrophobic sequence of 24 amino-acid residues is responsible for anchoring golvesin to the membranes of these organelles. In an attempt to visualize organelle dynamics in vivo, we have used specific antibody and other labels to localize golvesin-green fluorescent protein (GFP) constructs to different cellular compartments. With a GFP tag at its N-terminus, golvesin shows the same localization as the untagged protein. It is transferred to two post-Golgi compartments, the endosomal and contractile vacuole systems. Endosomes are decorated with GFP-golvesin within less than 10 min of their internalisation, and keep the label during the acidic phase of the pathway. Blockage of the C-terminus with GFP causes entrapment of the protein in the Golgi apparatus, indicating that a free C-terminus is required for transfer of golvesin to any of the post-Golgi compartments. The C-terminally tagged golvesin proved to be a reliable Golgi marker in Dictyostelium cells revealing protrusion of Golgi tubules at peak velocities of 3 to 4 microm x s(-1). The fusion protein is retained in Golgi vesicles during mitosis, visualizing Golgi disassembly and reorganization in line with cytokinesis.     

A structure-based mechanism for Arf1-dependent recruitment of coatomer to membranes

Budding of COPI-coated vesicles from Golgi membranes requires an Arf family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the γ- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P(2)-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules.