scholarly journals Endosidin20 targets cellulose synthase catalytic domain to inhibit cellulose biosynthesis

2020 ◽  
Author(s):  
Lei Huang ◽  
Xiaohui Li ◽  
Weiwei Zhang ◽  
Nolan Ung ◽  
Nana Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cellulose Synthase ◽  
Catalytic Site ◽  
Cellulose Synthesis ◽  
Spatiotemporal Resolution ◽  
Chemical Genetic ◽  
Biochemical Assays

AbstractCellulose is synthesized by rosette structured cellulose synthase (CESA) complexes (CSCs), each of which is composed of multiple units of CESAs in three different isoforms. CSCs rely on vesicle trafficking for delivery to the plasma membrane where they catalyze cellulose synthesis. Although the rosette structured CSCs were observed decades ago, it remains unclear what amino acids in plant CESA that directly participate in cellulose catalytic synthesis. It is also not clear how the catalytic activity of CSCs influences their efficient transport at the subcellular level. Here we report characterization of the small molecule Endosidin20 (ES20) and present evidence that it represents a new CESA inhibitor. We show data from chemical genetic analyses, biochemical assays, structural modeling, and molecular docking to support our conclusion that ES20 targets the catalytic site of Arabidopsis CESA6. Further, chemical genetic analysis reveals important amino acids that potentially form the catalytic site of plant CESA6. Using high spatiotemporal resolution live-cell imaging, we found that inhibition of CSC catalytic activity by inhibitor treatment, or by creating missense mutation at amino acids in the predicted catalytic site, causes reduced efficiency in CSC transport to the plasma membrane. Our results show that the catalytic activity of plant CSCs is integrated with subcellular trafficking dynamics.One sentence summaryEndosidin20 targets cellulose synthase at the catalytic site to inhibit cellulose synthesis and the inhibition of catalytic activity reduces cellulose synthase complex delivery to the plasma membrane.

scholarly journals A mutation in the catalytic domain of cellulose synthase 6 halts its transport to the Golgi apparatus

2019 ◽  
Vol 70 (21) ◽  
pp. 6071-6083 ◽  
Author(s):  
Sungjin Park ◽  
Bo Song ◽  
Wei Shen ◽  
Shi-You Ding
Keyword(s):  
Golgi Apparatus ◽  
Catalytic Domain ◽  
Cellulose Synthase ◽  
Cellulose Synthesis ◽  
Normal Transport

D395N in the catalytic domain of CESA6 interrupts its normal transport to the Golgi, which hampers its function in cellulose synthesis.

scholarly journals Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

1993 ◽  
Vol 13 (4) ◽  
pp. 2420-2431 ◽  
Author(s):  
D C Huang ◽  
C J Marshall ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cell Transformation ◽  
Negative Regulator ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
3T3 Fibroblasts ◽  
C Terminus

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

scholarly journals Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity

2005 ◽  
Vol 391 (2) ◽  
pp. 285-289 ◽  
Author(s):  
Nandita S. Raikwar ◽  
Rosario F. Bowen ◽  
Mark A. Deeg
Keyword(s):  
Catalytic Activity ◽  
Phospholipase D ◽  
Catalytic Domain ◽  
Computer Modelling ◽  
Critical Role ◽  
Biochemical Properties ◽  
Catalytic Site ◽  
Wild Type ◽  
Histidine Residues ◽  
Hydrolysis Of

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.

scholarly journals Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4335
Author(s):  
Gerasimos Daras ◽  
Dimitris Templalexis ◽  
Fengoula Avgeri ◽  
Dikran Tsitsekian ◽  
Konstantina Karamanou ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Economic Value ◽  
Cellulose Synthase ◽  
Protein Isoforms ◽  
Research Progress ◽  
Cellulose Synthesis ◽  
Biotic Stresses

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.

scholarly journals TRANVIA (TVA) facilitates cellulose synthase trafficking and delivery to the plasma membrane

2021 ◽  
Vol 118 (30) ◽  
pp. e2021790118
Author(s):  
Tamara Vellosillo ◽  
José R. Dinneny ◽  
Chris R. Somerville ◽  
David W. Ehrhardt
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Cellulose Synthase ◽  
Specific Protein ◽  
Primary Cell Wall ◽  
Cellulose Synthesis ◽  
Cellulose Content ◽  
Enhanced Sensitivity ◽  
Intracellular Compartments ◽  
Secretory System

Cellulose is synthesized at the plasma membrane by cellulose synthase (CESA) complexes (CSCs), which are assembled in the Golgi and secreted to the plasma membrane through the trans-Golgi network (TGN) compartment. However, the molecular mechanisms that guide CSCs through the secretory system and deliver them to the plasma membrane are poorly understood. Here, we identified an uncharacterized gene, TRANVIA (TVA), that is transcriptionally coregulated with the CESA genes required for primary cell wall synthesis. The tva mutant exhibits enhanced sensitivity to cellulose synthesis inhibitors; reduced cellulose content; and defective dynamics, density, and secretion of CSCs to the plasma membrane as compared to wild type. TVA is a plant-specific protein of unknown function that is detected in at least two different intracellular compartments: organelles labeled by markers for the TGN and smaller compartments that deliver CSCs to the plasma membrane. Together, our data suggest that TVA promotes trafficking of CSCs to the plasma membrane by facilitating exit from the TGN and/or interaction of CSC secretory vesicles with the plasma membrane.

scholarly journals Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation

2018 ◽  
Vol 115 (27) ◽  
pp. E6366-E6374 ◽  
Author(s):  
Yoichiro Watanabe ◽  
Rene Schneider ◽  
Sarah Barkwill ◽  
Eliana Gonzales-Vigil ◽  
Joseph L. Hill ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Expansion ◽  
Secondary Wall ◽  
Transition Period ◽  
Cellulose Synthase ◽  
Wall Thickening ◽  
Cellulose Synthesis ◽  
Primary Wall ◽  
Dynamic Behaviors

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function

1993 ◽  
Vol 13 (4) ◽  
pp. 2420-2431
Author(s):  
D C Huang ◽  
C J Marshall ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cell Transformation ◽  
Negative Regulator ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
3T3 Fibroblasts ◽  
C Terminus

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

scholarly journals A G protein-coupled receptor-like module regulates Cellulose Synthase secretion from the endomembrane system in Arabidopsis

2020 ◽  
Author(s):  
Heather E. McFarlane ◽  
Daniela Mutwil-Anderwald ◽  
Jana Verbančič ◽  
Kelsey L. Picard ◽  
Timothy E. Gookin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Protein Complex ◽  
Cellulose Synthase ◽  
Cell Wall Integrity ◽  
Cellulose Synthesis ◽  
Endomembrane System ◽  
G Protein Coupled

AbstractCellulose synthesis is essential for plant morphology, water transport and defense, and provides raw material for biomaterials and fuels. Cellulose is produced at the plasma membrane by Cellulose Synthase (CESA) protein complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked from the Golgi apparatus and trans-Golgi Network (TGN) to the plasma membrane. Since CESA enzymes are only active in the plasma membrane, control of CSC secretion is a critical step in the regulation of cellulose synthesis. However, the regulatory framework for CSC secretion is not clarified. In this study, we identify members of a family of seven transmembrane domain-containing proteins (7TMs) as important for cellulose production during cell wall integrity stress. 7TM proteins are often associated with guanine nucleotide-binding protein (G) protein signalling and mutants in several of the canonical G protein complex components phenocopied the 7tm mutant plants. Unexpectedly, the 7TM proteins localized to the Golgi apparatus/TGN where they interacted with the G protein complex. Here, the 7TMs and G proteins regulated CESA trafficking, but did not affect general protein secretion. Furthermore, during cell wall stress, 7TMs’ localization was biased towards small CESA-containing vesicles, specifically associated with CSC trafficking. Our results thus outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses upon exposure to cell wall integrity stress.

scholarly journals CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane

2018 ◽  
Vol 115 (15) ◽  
pp. E3578-E3587 ◽  
Author(s):  
Xiaoyu Zhu ◽  
Shundai Li ◽  
Songqin Pan ◽  
Xiaoran Xin ◽  
Ying Gu
Keyword(s):  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
De Novo ◽  
Cellulose Synthase ◽  
Specific Protein ◽  
Cellulose Synthesis ◽  
Docking Site ◽  
Multiple Components ◽  
Exocyst Complex

Cellulose synthesis occurs exclusively at the plasma membrane by cellulose synthase complexes (CSCs). Therefore, delivery of CSCs to discrete sites at the plasma membrane is critical for cellulose synthesis. Despite their significance, the delivery of CSCs is poorly understood. Here we used proteomics approaches, functional genetics, and live cell imaging to show that the de novo secretion of CSCs is mediated by cooperation among cellulose synthase interactive 1 (CSI1), the plant-specific protein PATROL1, and exocyst complex in Arabidopsis thaliana. We propose that CSI1 plays a role in marking the docking site, which allows CSCs-containing vesicles access to the plasma membrane through its interaction with microtubules. PATROL1 assists in exocytosis by its interaction with multiple components, including CSI1, CSCs, and exocyst subunits. Both PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membrane. By monitoring the exocyst complex, PATROL1, CSI1, and CSCs dynamics in real time, we present a timeline of events for exocytosis of CSCs. Our findings provide unique insights into the evolution of exocytosis in eukaryotes.

scholarly journals Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

2021 ◽  
Vol 118 (11) ◽  
pp. e2024015118
Author(s):  
Zhu Qiao ◽  
Edwin R. Lampugnani ◽  
Xin-Fu Yan ◽  
Ghazanfar Abbas Khan ◽  
Wuan Geok Saw ◽  
...  
Keyword(s):  
Amino Acids ◽  
Arabidopsis Thaliana ◽  
Catalytic Domain ◽  
Specific Region ◽  
Cellulose Synthesis ◽  
Uridine Diphosphate ◽  
In Planta ◽  
Core Domain ◽  
Cellulose Synthases ◽  
Insight Into

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

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