Major Changes in Plastid Protein Import and the Origin of the Chloroplastida.
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ABSTRACT:
SUBMITTER: Knopp M
PROVIDER: S-EPMC7038456 | biostudies-literature | 2020 Mar
REPOSITORIES: biostudies-literature
Publications
Major Changes in Plastid Protein Import and the Origin of the Chloroplastida.
Knopp Michael M Garg Sriram G SG Handrich Maria M Gould Sven B SB
iScience 20200208 3
Core components of plastid protein import and the principle of using N-terminal targeting sequences are conserved across the Archaeplastida, but lineage-specific differences exist. Here we compare, in light of plastid protein import, the response to high-light stress from representatives of the three archaeplastidal groups. Similar to land plants, Chlamydomonas reinhardtii displays a broad response to high-light stress, not observed to the same degree in the glaucophyte Cyanophora paradoxa or th ...[more]
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Project description:Aim: To identify regulatory factors that control: (1) chloroplast protein importand (2) chloroplast-to-nucleus signalling. This project is a joint proposal from the Jarvis lab which is interested in chloroplast protein import [1] and the Moller lab which is interested in plastid-to-nucleus signalling [2]. Background: The majority of chloroplast proteins are encoded in the nucleus and imported post-translationally into chloroplasts. The abundance of chloroplast proteins may therefore be regulated at multiple levels. It is well documented that the nuclear gene expression is responsive to (largely unknown) signals from the chloroplast [23] and evidence is now emerging that protein import is also a regulated process [1]. Protein import into chloroplasts is mediated by protein complexes in the outer and inner envelope membranes called Toc and Tic respectively. Biochemical studies of pea chloroplasts identified several Toc/Tic components. These proteins are mechanistic or structural components of the import apparatus. Arabidopsis homologues of the pea Toc/Tic proteins were identified by the AGI. Pea Toc34 is represented in Arabidopsis by two genes "Toc33 and Toc34" and pea Toc75 is represented by three genes. These different Tocs have different expression patterns and are proposed to have different precursor protein recognition specificities. The factors that regulate Toc expression in concert with the needs of plastids in developmentally different cells are unknown. Proposal: Two Arabidopsis mutants will be analysed. The ppi1 mutant is null for the putative precursor protein receptor Toc33 [1]and the ppi3 mutant is null for a putative component of the protein import channel Toc75-IV (on chromosome IV). ppi1 plants are yellow-green in appearance but remarkably healthy and grow only slightly more slowly than wild type. By contrast ppi3 plants are indistinguishable from wild type by eye although analysis of the mutant's chloroplast proteome is beginning to reveal some differences (K. Lilley personal communication). Gene expression changes in ppi1 are likely to be quite extensive. Retardation of chloroplast development in ppi1 will activate retrograde signalling pathways so that many nuclear photosynthetic genes are down-regulated. Changes in the expression of photosynthetic genes and of the genes responsible for mediating these responses may therefore be observed. Any regulatory and signalling genes identified will be of interest to the Moller lab. The expression of factors that regulate Toc/Tic gene expression may also be altered in ppi1. It should be possible to distinguish these factors from those involved in the general control of chloroplast gene expression by comparing the results from the two mutants. Genes affected in both mutants are more likely to be involved in regulating chloroplast import since it is unlikely that widespread changes in gene expression will be observed in ppi3. Changes in the expression of factors that regulate import post-translationallyand of the Toc/Tic genes themselves (many are on the RNA) may also be observed. References: 1. Jarvis P. et al. (1998) Science 282: 100-103. 2. Moller S.G. et al. (2001) Genes Dev. 15:90-103. 3. Jarvis P. (2001) Curr. Biol. 11: R307-R310.
2022-11-21 | E-NASC-35 | biostudies-arrayexpress
Project description:Aim: To identify regulatory factors that control: (1) chloroplast protein importand (2) chloroplast-to-nucleus signalling. This project is a joint proposal from the Jarvis lab which is interested in chloroplast protein import [1] and the Moller lab which is interested in plastid-to-nucleus signalling [2]. Background: The majority of chloroplast proteins are encoded in the nucleus and imported post-translationally into chloroplasts. The abundance of chloroplast proteins may therefore be regulated at multiple levels. It is well documented that the nuclear gene expression is responsive to (largely unknown) signals from the chloroplast [23] and evidence is now emerging that protein import is also a regulated process [1]. Protein import into chloroplasts is mediated by protein complexes in the outer and inner envelope membranes called Toc and Tic respectively. Biochemical studies of pea chloroplasts identified several Toc/Tic components. These proteins are mechanistic or structural components of the import apparatus. Arabidopsis homologues of the pea Toc/Tic proteins were identified by the AGI. Pea Toc34 is represented in Arabidopsis by two genes "Toc33 and Toc34" and pea Toc75 is represented by three genes. These different Tocs have different expression patterns and are proposed to have different precursor protein recognition specificities. The factors that regulate Toc expression in concert with the needs of plastids in developmentally different cells are unknown. Proposal: Two Arabidopsis mutants will be analysed. The ppi1 mutant is null for the putative precursor protein receptor Toc33 [1]and the ppi3 mutant is null for a putative component of the protein import channel Toc75-IV (on chromosome IV). ppi1 plants are yellow-green in appearance but remarkably healthy and grow only slightly more slowly than wild type. By contrast ppi3 plants are indistinguishable from wild type by eye although analysis of the mutant's chloroplast proteome is beginning to reveal some differences (K. Lilley personal communication). Gene expression changes in ppi1 are likely to be quite extensive. Retardation of chloroplast development in ppi1 will activate retrograde signalling pathways so that many nuclear photosynthetic genes are down-regulated. Changes in the expression of photosynthetic genes and of the genes responsible for mediating these responses may therefore be observed. Any regulatory and signalling genes identified will be of interest to the Moller lab. The expression of factors that regulate Toc/Tic gene expression may also be altered in ppi1. It should be possible to distinguish these factors from those involved in the general control of chloroplast gene expression by comparing the results from the two mutants. Genes affected in both mutants are more likely to be involved in regulating chloroplast import since it is unlikely that widespread changes in gene expression will be observed in ppi3. Changes in the expression of factors that regulate import post-translationallyand of the Toc/Tic genes themselves (many are on the RNA) may also be observed. References: 1. Jarvis P. et al. (1998) Science 282: 100-103. 2. Moller S.G. et al. (2001) Genes Dev. 15:90-103. 3. Jarvis P. (2001) Curr. Biol. 11: R307-R310. Experiment Overall Design: Number of plants pooled:
2008-06-14 | E-GEOD-6164 | biostudies-arrayexpress
Project description:Aim: To identify regulatory factors that control: (1) chloroplast protein importand (2) chloroplast-to-nucleus signalling. This project is a joint proposal from the Jarvis lab which is interested in chloroplast protein import [1] and the Moller lab which is interested in plastid-to-nucleus signalling [2]. Background: The majority of chloroplast proteins are encoded in the nucleus and imported post-translationally into chloroplasts. The abundance of chloroplast proteins may therefore be regulated at multiple levels. It is well documented that the nuclear gene expression is responsive to (largely unknown) signals from the chloroplast [23] and evidence is now emerging that protein import is also a regulated process [1]. Protein import into chloroplasts is mediated by protein complexes in the outer and inner envelope membranes called Toc and Tic respectively. Biochemical studies of pea chloroplasts identified several Toc/Tic components. These proteins are mechanistic or structural components of the import apparatus. Arabidopsis homologues of the pea Toc/Tic proteins were identified by the AGI. Pea Toc34 is represented in Arabidopsis by two genes "Toc33 and Toc34" and pea Toc75 is represented by three genes. These different Tocs have different expression patterns and are proposed to have different precursor protein recognition specificities. The factors that regulate Toc expression in concert with the needs of plastids in developmentally different cells are unknown. Proposal: Two Arabidopsis mutants will be analysed. The ppi1 mutant is null for the putative precursor protein receptor Toc33 [1]and the ppi3 mutant is null for a putative component of the protein import channel Toc75-IV (on chromosome IV). ppi1 plants are yellow-green in appearance but remarkably healthy and grow only slightly more slowly than wild type. By contrast ppi3 plants are indistinguishable from wild type by eye although analysis of the mutant's chloroplast proteome is beginning to reveal some differences (K. Lilley personal communication). Gene expression changes in ppi1 are likely to be quite extensive. Retardation of chloroplast development in ppi1 will activate retrograde signalling pathways so that many nuclear photosynthetic genes are down-regulated. Changes in the expression of photosynthetic genes and of the genes responsible for mediating these responses may therefore be observed. Any regulatory and signalling genes identified will be of interest to the Moller lab. The expression of factors that regulate Toc/Tic gene expression may also be altered in ppi1. It should be possible to distinguish these factors from those involved in the general control of chloroplast gene expression by comparing the results from the two mutants. Genes affected in both mutants are more likely to be involved in regulating chloroplast import since it is unlikely that widespread changes in gene expression will be observed in ppi3. Changes in the expression of factors that regulate import post-translationallyand of the Toc/Tic genes themselves (many are on the RNA) may also be observed. References: 1. Jarvis P. et al. (1998) Science 282: 100-103. 2. Moller S.G. et al. (2001) Genes Dev. 15:90-103. 3. Jarvis P. (2001) Curr. Biol. 11: R307-R310. Keywords: strain_or_line_design
2007-01-08 | GSE6164 | GEO
Project description:SignificanceAlthough plastid division is critical for plant development, how components of the plastid division machinery (PDM) are imported into plastids remains unexplored. A forward genetic screen to identify suppressors of a crumpled leaf (crl) mutant deficient in plastid division led us to find dominant gain-of-function (GF) mutations in TIC236, which significantly increases the import of PDM components and completely rescues crl phenotypes. The defective plastid division phenotypes in crl and tic236-knockdown mutants and CRL-TIC236 association in a functional complex indicate that the CRL-TIC236 module is vital for plastid division. Hence, we report the first GF translocon mutants and unveil CRL as a novel functional partner of TIC236 for PDM import.
| S-EPMC8931380 | biostudies-literature
Project description:Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD) complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL), an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.IMPORTANCE Apicomplexan parasites are responsible for important human diseases. There are no effective vaccines for use in humans, and drug treatment faces multiple challenges, including emerging resistance, lack of efficacy across the lifecycle, and adverse drug effects. The apicoplast is a promising target for novel treatments: this chloroplast-like organelle is derived from an algal symbiont, is absent from the host, and is essential for parasite growth and pathogenesis. We use Toxoplasma gondii as a model to study the apicoplast due to its strong genetic tools and established functional assays. We identify a plastid ubiquitin-like protein (PUBL) which is a novel ubiquitin-like protein and demonstrate its importance and that of the motor protein CDC48AP for apicoplast protein import. These findings broaden our understanding of the evolution and mechanistic workings of a unique parasite organelle and may lead to new opportunities for treatments against important human pathogens.
| S-EPMC5487736 | biostudies-literature
Project description:A 16-kDa plastid envelope protein was identified by chemical crosslinking that interacts with the precursor of NADPH:protochlorophyllide oxdidoreductase A (pPORA) during its posttranslational import into isolated barley chloroplasts. Protein purification and subsequent protein sequencing showed that the 16-kDa protein is an ortholog of a previously identified outer plastid envelope protein, Oep16. A protein of identical size was present in barley etioplasts and interacted with pPORA. Similar 16-kDa protein-dependent crosslink products of pPORA were detected in wheat, pea, and Arabidopsis chloroplasts. Database analyses revealed that the 16-kDa protein belongs to a family of preprotein and amino acid transporters found in free-living bacteria and endosymbiotic mitochondria and chloroplasts. Antibodies raised against the 16-kDa protein inhibited import of pPORA, highlighting its role in protein import.
| S-EPMC357075 | biostudies-literature
Project description:Plastids are semiautonomous organelles that contain only limited coding information in their own DNA. Because most of their genome was transferred to the nucleus after their endosymbiotic origin, plastids must import the major part of their protein constituents from the cytosol. The exact role of cytosolic targeting factors in the regulation of plastid protein import has not been determined. Here, we report that the nucleus-encoded NADPH:protochlorophyllide (Pchlide) oxidoreductase A plastid precursor (pPORA) can use two different plastid import pathways that differ by the requirements for cytosolic 14:3:3 proteins and Hsp70. pPORA synthesized in a wheat germ lysate segregated into different precursor fractions. While import of free pPORA and only Hsp70-complexed pPORA was Pchlide-dependent and involved the previously identified Pchlide-dependent translocon, 14:3:3 protein- and Hsp70-complexed pPORA was transported into Pchlide-free chloroplasts through the Toc75-containing standard translocon at the outer chloroplast membrane/translocon at the inner chloroplast membrane machinery. A 14:3:3 protein binding site was identified in the mature region of the (35)S-pPORA, which governed 14:3:3 protein- and Hsp70-mediated, Pchlide-independent plastid import. Collectively, our results reveal that the import of pPORA into the plastids is tightly regulated and involves different cytosolic targeting factors and plastid envelope translocon complexes.
| S-EPMC1895985 | biostudies-literature
Project description:Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans, whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome (B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans. How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.
| S-EPMC3294442 | biostudies-literature
Project description:The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis.
| S-EPMC5409464 | biostudies-literature
Project description:Protein acetylation is a key co- and post-translational modification. However, how different types of acetylation respond to environmental stress is still unknown. To address this, we investigated the role of a member of the newly discovered family of plastid acetyltransferases (GNAT2), which features both lysine- and N-terminal acetyltransferase activities. Our study aimed to provide a holistic multi-omics acetylation-dependent view of plant acclimation to short-term light changes. We found that both the yield and coverage of the N-terminal acetylome remained unchanged in WT and gnat2-KO backgrounds after 2 h of exposure to high light or darkness. Similarly, no differences in transcriptome or adenylate energy charge were observed between the genotypes under the tested light conditions. In contrast, the lysine acetylome proved to be sensitive to the changes in light conditions, especially in the gnat2 background. This suggests unique strategies of plant acclimation for quick responses to environmental changes involving lysine, but not N-terminal, GNAT2-mediated acetylation activity.
| S-EPMC11546460 | biostudies-literature