scholarly journals Structural and functional insights into the mechanism of action of plant borate transporters

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Savvas Saouros ◽  
Thotegowdanapalya C. Mohan ◽  
Cristina Cecchetti ◽  
Silke Lehmann ◽  
Joseph D. Barrit ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Plant Growth And Development ◽  
Anion Exchanger 1 ◽  
Core Domain ◽  
Domain Organisation ◽  
Terminal Amino ◽  
Related Proteins ◽  
Lower Expression

AbstractBoron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are homologues of the human SLC4 family of transporters, which includes well studied mammalian transporters such as the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based (i) on known disease causing mutations of hAE1 (S466R, A500R) and (ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of borate efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease-causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3, lacking the 30 C-terminal amino acid residues. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.

scholarly journals Structural and functional insights into the mechanism of action of plant boron transporters

2020 ◽  
Author(s):  
Savvas Saouros ◽  
Thotegowdanapalya C Mohan ◽  
Cristina Cecchetti ◽  
Silke Lehmann ◽  
Joseph D Barritt ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Growth And Development ◽  
Loss Of Function ◽  
Plant Growth And Development ◽  
Anion Exchanger 1 ◽  
Core Domain ◽  
Domain Organisation ◽  
Terminal Amino ◽  
Related Proteins ◽  
Lower Expression

Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are members of the SLC4 family of transporters and thus homologues of well studied mammalian transporters including the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) mutants based i) on known disease causing mutations of hAE1 (S466R, A500R) and ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The mutants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of boron efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes both in soil and on plates under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3. The construct used to obtain this structure lacks the 30 C-terminal amino acids but is otherwise unmodified. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.

scholarly journals Actin-Related Protein 4 Interacts with PIE1 and Regulates Gene Expression in Arabidopsis

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 520
Author(s):  
Wenfeng Nie ◽  
Jinyu Wang
Keyword(s):  
Gene Expression ◽  
Plant Growth ◽  
Chromatin Remodeling ◽  
Leaf Size ◽  
Early Flowering ◽  
Physical Interaction ◽  
Loss Of Function ◽  
Single Nucleotide Change ◽  
Chromatin Remodeling Complex ◽  
Related Proteins

As essential structural components of ATP-dependent chromatin-remodeling complex, the nucleolus-localized actin-related proteins (ARPs) play critical roles in many biological processes. Among them, ARP4 is identified as an integral subunit of chromatin remodeling complex SWR1, which is conserved in yeast, humans and plants. It was shown that RNAi mediated knock-down of Arabidopsis thaliana ARP4 (AtARP4) could affect plant development, specifically, leading to early flowering. However, so far, little is known about how ARP4 functions in the SWR1 complex in plant. Here, we identified a loss-of-function mutant of AtARP4 with a single nucleotide change from glycine to arginine, which had significantly smaller leaf size. The results from the split luciferase complementation imaging (LCI) and yeast two hybrid (Y2H) assays confirmed its physical interaction with the scaffold and catalytic subunit of SWR1 complex, photoperiod-independent early flowering 1 (PIE1). Furthermore, mutation of AtARP4 caused altered transcription response of hundreds of genes, in which the number of up-regulated differentially expressed genes (DEGs) was much larger than those down-regulated. Although most DEGs in atarp4 are related to plant defense and response to hormones such as salicylic acid, overall, it has less overlapping with other swr1 mutants and the hta9 hta11 double-mutant. In conclusion, our results reveal that AtARP4 is important for plant growth and such an effect is likely attributed to its repression on gene expression, typically at defense-related loci, thus providing some evidence for the coordination of plant growth and defense, while the regulatory patterns and mechanisms are distinctive from other SWR1 complex components.

scholarly journals Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 715
Author(s):  
Tamara Tomanić ◽  
Claire Martin ◽  
Holly Stefen ◽  
Esmeralda Parić ◽  
Peter Gunning ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Growth Cones ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Primary Mouse ◽  
Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

Importance of Terminal Amino Acid Residues to the Transport of Oligopeptides across the Caco-2 Cell Monolayer

2017 ◽  
Vol 65 (35) ◽  
pp. 7705-7712 ◽  
Author(s):  
Long Ding ◽  
Liying Wang ◽  
Zhipeng Yu ◽  
Sitong Ma ◽  
Zhiyang Du ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Monolayer ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Terminal Amino

scholarly journals Tamm–Horsfall urinary glycoprotein. The chemical composition

1970 ◽  
Vol 120 (2) ◽  
pp. 417-424 ◽  
Author(s):  
A. P. Fletcher ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Amino Acid ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Amino Acid Residues ◽  
Carbohydrate Composition ◽  
Thiol Groups ◽  
Terminal Amino Acid ◽  
Cystine Content ◽  
Terminal Amino ◽  
Urinary Glycoprotein

1. A revised amino acid and carbohydrate composition of human Tamm–Horsfall glycoprotein is presented. 2. No significant differences were obtained in the amino acid composition of Tamm–Horsfall glycoprotein isolated from patients with cystic fibrosis. 3. The glycoprotein was shown to possess a high half-cystine content of 1 per 11–12 amino acid residues, which has been confirmed by performic acid oxidation and S-alkylation with iodoacetate and iodoacetamide. No thiol groups were detected in the glycoprotein. 4. Treatment of the glycoprotein with 0.5m-sodium hydroxide at 4°C for 2 days did not release heterosaccharide material, which suggests that the predominant carbohydrate–protein linkages present are not of the O-glycosidic type. 5. No N-terminal amino acid was detected in the glycoprotein.

scholarly journals The Methionine 549 and Leucine 552 Residues of Friedelin Synthase from Maytenus ilicifolia Are Important for Substrate Binding Specificity

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6806
Author(s):  
Bruna F. Mazzeu ◽  
Tatiana M. Souza-Moreira ◽  
Andrew A. Oliveira ◽  
Melissa Remlinger ◽  
Lidiane G. Felippe ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activities ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enzyme Specificity ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Pentacyclic Triterpene ◽  
Oxidosqualene Cyclases ◽  
Maytenus Ilicifolia

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

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