scholarly journals TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import

2021 ◽  
Author(s):  
Chanhong Kim ◽  
Jun Fang ◽  
Bingqi Li ◽  
Lih-Jen Chen ◽  
Vivek Dogra ◽  
...  
Keyword(s):  
Protein Import ◽  
Genetic Interaction ◽  
Plant Stress ◽  
Plastid Division ◽  
Gain Of Function ◽  
Mutant Proteins ◽  
Ubiquitin E3 Ligase ◽  
Transit Peptides ◽  
Plastid Protein ◽  
Mutant Phenotypes

The chloroplast translocons TOC75 and TIC236 are homologs of the bacterial translocation and assembly module (Tam) A and TamB involved in protein export. Here, we unveil a TIC236-allied component, the chloroplast outer membrane protein CRUMPLED LEAF (CRL), absence of which impairs plastid division and induces autoimmune responses in Arabidopsis thaliana. A forward genetic screen aimed at finding crl suppressors revealed multiple TIC236 gain-of-function mutations (TIC236GFs). Despite the low sequence identity between TIC236 and bacterial TamB, each mutated TIC236GF residue is conserved in TamB. Consistently, a tic236-knockdown mutant exhibited multiple lesion phenotypes similar to crl, indicating a shared functionality of CRL and TIC236. Ensuing reverse genetic analyses revealed genetic interaction between CRL and SP1, a RING-type ubiquitin E3 ligase, as well as with the plastid protease FTSH11, which function in TOC and TIC protein turnover, respectively. Loss of either SP1 or FTSH11 rescued crl mutant phenotypes to varying degrees due to increased translocon levels. Consistent with impaired plastid division exhibited by both crl and tic236-knockdown mutants, CRL interacts with the transit peptides of proteins essential in plastid division, and TIC236GF mutant proteins reinforce their import via increased TIC236 stability. Overall, our data shed new light on the links between plastid division, plant stress response and plastid protein import. We have also isolated and characterized the first GF mutants exhibiting increased protein import efficiency, which may inspire chloroplast engineering for agricultural advancement.

scholarly journals Xrp1 genetically interacts with the ALS-associated FUS orthologue caz and mediates its toxicity

2018 ◽  
Vol 217 (11) ◽  
pp. 3947-3964 ◽  
Author(s):  
Moushami Mallik ◽  
Marica Catinozzi ◽  
Clemens B. Hug ◽  
Li Zhang ◽  
Marina Wagner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Span ◽  
Motor Neurons ◽  
Gene Expression Regulation ◽  
Genetic Interaction ◽  
Motor Deficits ◽  
Chromatin Binding ◽  
Mutant Phenotypes ◽  
Gene Expression Dysregulation ◽  
Lateral Sclerosis

Cabeza (caz) is the single Drosophila melanogaster orthologue of the human FET proteins FUS, TAF15, and EWSR1, which have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In this study, we identified Xrp1, a nuclear chromatin-binding protein, as a key modifier of caz mutant phenotypes. Xrp1 expression was strongly up-regulated in caz mutants, and Xrp1 heterozygosity rescued their motor defects and life span. Interestingly, selective neuronal Xrp1 knockdown was sufficient to rescue, and neuronal Xrp1 overexpression phenocopied caz mutant phenotypes. The caz/Xrp1 genetic interaction depended on the functionality of the AT-hook DNA-binding domain in Xrp1, and the majority of Xrp1-interacting proteins are involved in gene expression regulation. Consistently, caz mutants displayed gene expression dysregulation, which was mitigated by Xrp1 heterozygosity. Finally, Xrp1 knockdown substantially rescued the motor deficits and life span of flies expressing ALS mutant FUS in motor neurons, implicating gene expression dysregulation in ALS-FUS pathogenesis.

scholarly journals Retrograde signalling in a virescent mutant triggers an anterograde delay of chloroplast biogenesis that requires GUN1 and is essential for survival

2020 ◽  
Vol 375 (1801) ◽  
pp. 20190400 ◽  
Author(s):  
Naresh Loudya ◽  
Tolulope Okunola ◽  
Jia He ◽  
Paul Jarvis ◽  
Enrique López-Juez
Keyword(s):  
Protein Import ◽  
Early Stage ◽  
Plastid Development ◽  
Plastid Genomes ◽  
Expression Of Genes ◽  
Fitness Value ◽  
Retrograde Signalling ◽  
Value Loss ◽  
Elevated Expression ◽  
Plastid Protein

Defects in chloroplast development are ‘retrograde-signalled’ to the nucleus, reducing synthesis of photosynthetic or related proteins. The Arabidopsis cue8 mutant manifests virescence, a slow-greening phenotype, and is defective at an early stage in plastid development. Greening cotyledons or early leaf cells of cue8 exhibit immature chloroplasts which fail to fill the available cellular space. Such chloroplasts show reduced expression of genes of photosynthetic function, dependent on the plastid-encoded polymerase (PEP), while the expression of genes of housekeeping function driven by the nucleus-encoded polymerase (NEP) is elevated, a phenotype shared with mutants in plastid genetic functions. We attribute this phenotype to reduced expression of specific PEP-controlling sigma factors, elevated expression of RPOT (NEP) genes and maintained replication of plastid genomes (resulting in densely coalesced nucleoids in the mutant), i.e. it is due to an anterograde nucleus-to-chloroplast correction, analogous to retention of a juvenile plastid state. Mutants in plastid protein import components, particularly those involved in housekeeping protein import, also show this ‘retro-anterograde’ correction. Loss of CUE8 also causes changes in mRNA editing. The overall response has strong fitness value: loss of GUN1, an integrator of retrograde signalling, abolishes elements of it (albeit not others, including editing changes), causing bleaching and eventual seedling lethality upon cue8 gun1 . This highlights the adaptive significance of virescence and retrograde signalling. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

Pex5p stabilizes Pex14p: a study using a newly isolated pex5 CHO cell mutant, ZPEG101

2012 ◽  
Vol 449 (1) ◽  
pp. 195-207 ◽  
Author(s):  
Ryuichi Natsuyama ◽  
Kanji Okumoto ◽  
Yukio Fujiki
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cell Mutant ◽  
Peroxisomal Membrane ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Signal Type ◽  
Mutant Phenotypes

Pex5p [PTS (peroxisome-targeting signal) type 1 receptor] plays an essential role in peroxisomal matrix protein import. In the present study, we isolated a novel PEX5-deficient CHO (Chinese-hamster ovary) cell mutant, termed ZPEG101, showing typical peroxisomal import defects of both PTS1 and PTS2 proteins. ZPEG101 is distinct from other known pex5 CHO mutants in its Pex5p expression. An undetectable level of Pex5p in ZPEG101 results in unstable Pex14p, which is due to inefficient translocation to the peroxisomal membrane. All of the mutant phenotypes of ZPEG101 are restored by expression of wild-type Pex5pL, a longer form of Pex5p, suggesting a role for Pex5p in sustaining the levels of Pex14p in addition to peroxisomal matrix protein import. Complementation analysis using various Pex5p mutants revealed that in the seven pentapeptide WXXXF/Y motifs in Pex5pL, known as the multiple binding sites for Pex14p, the fifth motif is an auxiliary binding site for Pex14p and is required for Pex14p stability. Furthermore, we found that Pex5p–Pex13p interaction is essential for the import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. Therefore ZPEG101 with no Pex5p would be a useful tool for investigating Pex5p function and delineating the mechanisms underlying peroxisomal matrix protein import.

scholarly journals The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton.

1996 ◽  
Vol 134 (1) ◽  
pp. 117-132 ◽  
Author(s):  
V Moreau ◽  
A Madania ◽  
R P Martin ◽  
B Winson
Keyword(s):  
Actin Cytoskeleton ◽  
Genetic Interaction ◽  
Lucifer Yellow ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Gene Encoding ◽  
Membrane Growth ◽  
Mutant Phenotypes ◽  
Mutant Cells

Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2-H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament-associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis.

scholarly journals The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

2017 ◽  
Vol 292 (17) ◽  
pp. 6952-6964 ◽  
Author(s):  
Mónica Zufferey ◽  
Cyrille Montandon ◽  
Véronique Douet ◽  
Emilie Demarsy ◽  
Birgit Agne ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
The Novel ◽  
Plastid Protein

scholarly journals A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris

1998 ◽  
Vol 140 (4) ◽  
pp. 807-820 ◽  
Author(s):  
Ype Elgersma ◽  
Minetta Elgersma-Hooisma ◽  
Thibaut Wenzel ◽  
J. Michael McCaffery ◽  
Marilyn G. Farquhar ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Protein Import ◽  
Subcellular Location ◽  
Chondrodysplasia Punctata ◽  
Rhizomelic Chondrodysplasia Punctata ◽  
Methanol Metabolism ◽  
Mutant Proteins ◽  
Peroxisomal Targeting ◽  
Signal Type ◽  
Model Protein

Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence.

scholarly journals Major changes in plastid protein import and the origin of the Chloroplastida

2019 ◽  
Author(s):  
Michael Knopp ◽  
Sriram G. Garg ◽  
Maria Handrich ◽  
Sven B. Gould
Keyword(s):  
Protein Import ◽  
Light Stress ◽  
High Light ◽  
Rna Seq ◽  
High Light Stress ◽  
Dual Targeting ◽  
Plastid Protein ◽  
Pigment Profile ◽  
Microscopy Data ◽  
Green Lineage

AbstractWhile core components of plastid protein import (Toc and Tic) and the principle of using N-terminal targeting sequences (NTS) are conserved, lineage-specific differences are known. Rhodophytes and glaucophytes carry a conserved NTS motif, which was lost in the green lineage that also added novel proteins to Toc and Tic. Here we compare the components of plastid protein import and generated RNA-Seq, pigment profile and trans-electron microscopy data based on high-light stress from representatives of the three archaeplastidal groups. In light of plastid protein targeting, we compare the response to high-light stress of archaeplastidal representatives based on RNA-Seq, pigment profile and trans-electron microscopy data. Like land plants, the chlorophyte Chlamydomonas reinhardtii displays a broad respond to high-light stress, not observed to the same degree in the glaucophyte Cyanophora paradoxa or the rhodophyte Porphyridium purpureum. We find that only the green lineage encodes a conserved duplicate of the outer plastid membrane protein channel Oep80, namely Toc75 and suggest that the ability to respond to high-light stress entailed evolutionary changes in protein import, including the departure from phenylalanine-based targeting and the introduction of a green-specific Toc75 next to other import components unique to Chloroplastida. One consequence of relaxed NTS specificity was the origin of dual-targeting of plastid derived proteins to mitochondria and vice versa, using a single ambiguous NTS. Changes in the plastid protein import enabled the green lineage to import proteins at a more efficient rate, including those required for high-light stress response, a prerequisite for the colonization of land.High-lightsLoss of Phe-based N-terminal targeting sequences (NTS) triggered the origin of dual-targeting using a single ambiguous NTSThe Chloroplastida evolved a green-specific Toc75 for high throughput import, next to a universal and ancient Omp85 present in all ArchaeplastidaA broad response to high-light stress appears unique to ChloroplastidaRelaxation of functional constraints allowed a broader modification of the green Toc/Tic machineryCritical changes in plastid targeting enabled the origin and success of the Chloroplastida and their later conquer of land

scholarly journals Altering Polycomb Repressive Complex 2 activity partially suppresses ddm1 mutant phenotypes in Arabidopsis

2019 ◽  
Author(s):  
Martin Rougée ◽  
Leandro Quadrana ◽  
Jérôme Zervudacki ◽  
Vincent Colot ◽  
Lionel Navarro ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Arabidopsis Thaliana ◽  
Genetic Interaction ◽  
Genomic Analysis ◽  
Flowering Plant ◽  
Polycomb Repressive Complex ◽  
Polycomb Repressive Complex 2 ◽  
Covalent Modifications ◽  
Silencing Pathways ◽  
Mutant Phenotypes

AbstractIn plants and mammals, DNA methylation is a hallmark of transposable element (TE) sequences that contributes to their epigenetic silencing. In contrast, histone H3 lysine 27 trimethylation (H3K27me3), which is deposited by the Polycomb Repressive Complex 2 (PRC2), is a hallmark of repressed genes. Nevertheless, there is a growing body of evidence for a functional interplay between these pathways. In particular, many TE sequences acquire H3K27me3 when they lose DNA methylation and it has been proposed that PRC2 can serve as a back-up silencing system for hypomethylated TEs. Here, we describe in the flowering plant Arabidopsis thaliana the gain of H3K27m3 at hundreds of TEs in the mutant ddm1, which is defective in the maintenance of DNA methylation specifically over TE and other repeat sequences. Importantly, we show that this gain essentially depends on CURLY LEAF (CLF), which is one of two otherwise partially redundant H3K27 methyltransferases active in vegetative tissues. Finally, our results challenge the notion that PRC2 can be a compensatory silencing system for hypomethylated TEs, as the complete loss of H3K27me3 in ddm1 clf double mutant plants was not associated with further reactivation of TE expression nor with a burst of transposition. Instead, and surprisingly, ddm1 clf plants exhibited less activated TEs, and a chromatin recompaction as well as hypermethylation of linker DNA compared to ddm1. Thus, we have described an unexpected genetic interaction between DNA methylation and Polycomb silencing pathways, where a mutation in PRC2 does not aggravate the molecular phenotypes linked to TE hypomethylation in ddm1 but instead partially suppresses them.Author summaryEpigenetic marks are covalent modifications of the DNA or its associated proteins (Histones) that impact gene expression in a heritable manner without changing DNA sequence. In plants and mammals, DNA methylation and trimethylation of Lysine 27 of Histone 3 (H3K27me3) are two conserved, major epigenetic systems that mediate the transcriptional silencing of transposons (invasive mobile genetic elements) and of developmental genes respectively. However, in the absence of DNA methylation, H3K27me3 marks can be recruited to transposons, suggesting that the two silencing systems can be compensatory. To test this hypothesis, we analyzed a compound DNA methylation and H3K27me3 mutant of the plant model Arabidopsis thaliana (importantly, mammals harboring equivalent mutations would not be viable). First, this approach allowed us to gain mechanistic insights into the recruitment of H3K27me3 at transposons. Furthermore, we also showed that transposon silencing release in the DNA methylation mutant was not enhanced, contrary to our initial hypothesis, but, surprisingly, partially suppressed by a mutation in H3K27me3 deposition. Thus, our genomic analysis revealed an unexpected and antagonistic genetic interaction between two major silencing pathways whose interplay is at the heart of many biological processes, including cancer.

scholarly journals The HD-ZIP IV transcription factor Tril regulates fruit spine density through gene dosage effects in cucumber

2020 ◽  
Vol 71 (20) ◽  
pp. 6297-6310
Author(s):  
Hui Du ◽  
Gang Wang ◽  
Jian Pan ◽  
Yue Chen ◽  
Tingting Xiao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Genetic Interaction ◽  
Gene Dosage ◽  
Expression Patterns ◽  
Tissue Expression ◽  
Spine Density ◽  
Gene Dosage Effect ◽  
Gene Dosage Effects ◽  
Transgenic Lines ◽  
Mutant Phenotypes

Abstract Trichomes and fruit spines are important traits that directly affect the appearance quality and commercial value of cucumber (Cucumis sativus). Tril (Trichome-less), encodes a HD-Zip IV transcription factor that plays a crucial role in the initiation of trichomes and fruit spines, but little is known about the details of the regulatory mechanisms involved. In this study, analysis of tissue expression patterns indicated that Tril is expressed and functions in the early stages of organ initiation and development. Expression of Tril under the control of its own promoter (the TrilPro::Tril-3*flag fragment) could partly rescue the mutant phenotypes of tril, csgl3 (cucumber glabrous 3, an allelic mutant of tril), and fs1 (few spines 1, a fragment substitution in the Tril promoter region), providing further evidence that Tril is responsible for the initiation of trichomes and fruit spines. In lines with dense spine, fs1-type lines, and transgenic lines of different backgrounds containing the TrilPro::Tril-3*flag foreign fragment, spine density increased in conjunction with increases in Tril expression, indicating that Tril has a gene dosage effect on fruit spine density in cucumber. Numerous Spines (NS) is a negative regulatory factor of fruit spine density. Characterization of the molecular and genetic interaction between Tril and NS/ns demonstrated that Tril functions upstream of NS with respect to spine initiation. Overall, our results reveal a novel regulatory mechanism governing the effect of Tril on fruit spine development, and provide a reference for future work on breeding for physical quality in cucumber.

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