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Molecular Mechanism of the Specificity of Protein Import into chloroplasts and Mitochondria in Plant Cells

Molecular Plant 2019年第7期12卷 951-966页

作者： Dong Wook Lee Sumin Lee Junho Lee Seungjin Woo Md.Abdur Razzak Alessandro Vitale Inhwan Hwang Division of Integrative Biosciences and Biotechnology Pohang University of Science and Technology Pohang 37673 Korea Department of Life Sciences Pohang University of Science and Technology Pohang 37673 Korea Istituto di Biologia e Biotecnologia Agraria Consiglio Nazionale Delle Ricerche Milano Italy

Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.

关键词： transit peptide presequence protein import into chloroplasts and mitochondria N-terminal specificity domain C-terminal common translocation domain import specificity determination

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Na_(2)CO_(3)-responsive Photosynthetic and ROS Scavenging Mechanisms in chloroplasts of Alkaligrass Revealed by Phosphoproteomics

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Genomics, Proteomics & Bioinformatics 2020年第3期18卷 271-288页

作者： Jinwei Suo Heng Zhang Qi Zhao Nan Zhang Yongxue Zhang Ying Li Baohua Song Juanjuan Yu Jianguo Cao Tai Wang Ji Luo Lihai Guo Jun Ma Xumin Zhang Yimin She Lianwei Peng Weimin Ma Siyi Guo Yuchen Miao Sixue Chen Zhi Qin Shaojun Dai Development Center of Plant Germplasm Resources College of Life and Environmental SciencesShanghai Normal UniversityShanghai 200234China Alkali Soil Natural Environmental Science Center Northeast Forestry UniversityKey Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil FieldMinistry of EducationHarbin 150040China State Key Laboratory of Subtropical Silviculture Zhejiang A&F UniversityHangzhou 311300China Institute of Botany Chinese Academy of SciencesBeijing 100093China AB Sciex Asia Pacific Application Support Center Shanghai 200233China Shanghai Center for Plant Stress Biology Chinese Academy of SciencesShanghai 201602China School of Life Sciences Fudan UniversityShanghai 200433China Institute of Plant Stress Biology State Key Laboratory of Cotton BiologyDepartment of BiologyHenan UniversityKaifeng 475004China Department of Biology Genetics InstitutePlant Molecular and Cellular Biology ProgramInterdisciplinary Center for Biotechnology ResearchUniversity of FloridaGainesvilleFL 32610USA

Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to plants.To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,physiological and diverse quantitative proteomics analyses of alkaligrass(Puccinellia tenuiflora)under Na_(2)CO_(3)stress were conducted.In addition,Western blot,real-time PCR,and transgenic techniques were applied to validate the proteomic results and test the functions of the Na_(2)CO_(3)-responsive proteins.A total of 104 and 102 Na_(2)CO_(3)-responsive proteins were identified in leaves and chloroplasts,respectively.In addition,84 Na_(2)CO_(3)-responsive phosphoproteins were identified,including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts,which are crucial for the regulation of photosynthesis,ion transport,signal transduction,and energy homeostasis.A full-length Pt FBA encoding an alkaligrass chloroplastic fructosebisphosphate aldolase(FBA)was overexpressed in wild-type cells of cyanobacterium Synechocystis sp.Strain PCC 6803,leading to enhanced Na_(2)CO_(3)tolerance.All these results indicate that thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photosystem(PS)Ⅱ,PSI activity,and ROS homeostasis were altered in response to Na_(2)CO_(3)stress,which help to improve our understanding of the Na_(2)CO_(3)-responsive mechanisms in halophytes.

关键词： chloroplasts Na_(2)CO_(3)stress ROS scavenging Phosphoproteomics Puccinellia tenuiflora

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Determinants of the Specif icity of Protein Targeting to chloroplasts or Mitochondria

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Molecular Plant 2019年第7期12卷 893-895页

作者： Lucas McKinnon Steven M.Theg Department of Plant Biology University of California Davis Davis CA USA

chloroplasts and mitochondria are both thought to have arisen through primary endosymbiosis (Kutschera and Niklas, 2005). As each organelle evolved, most of its genome was transferred to the host nucleus resulting in the vast majority of its proteome being nuclear encoded and synthesized on cytosolic ribosomes (Shi and Theg, 2013).

关键词： chloroplasts Mitochondria

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Ion Channels in Plant Bioenergetic Organelles, chloroplasts and Mitochondria： From Molecular Identification to Function

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Molecular Plant 2016年第3期9卷 371-395页

作者： Luca Carraretto Enrico Teardo Vanessa Checchetto Giovanni Finazzi Nobuyuki Uozumi Ildiko Szabo Department of Biology University of Padova Padova 35121 Italy CNR Institute of Neuroscience University of Padova Padova 35121 Italy UMR 5168 Laboratoire de Physiologie Cellulaire Vegetale (LPCV) CNRS/UJF / INRA / CEA Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV) CEA Grenoble 38054 Grenoble France Department of Siomolecular Engineering Graduate Schoo~ of Engineering Tohoku University Aobayama 6-6-07 Sendai 980-8579 Japan

Recent technical advances in electrophysiological measurements, organelle-targeted fluorescence imaging, and organelle proteomics have pushed the research of ion transport a step forward in the case of the plant bioenergetic organelles, chloroplasts and mitochondria, leading to the molecular identification and functional characterization of several ion transport systems in recent years. Here we focus on channels that mediate relatively high-rate ion and water flux and summarize the current knowledge in this field, focusing on targeting mechanisms, proteomics, electrophysiology, and physiological function. In addition, since chloroplasts evolved from a cyanobacterial ancestor, we give an overview of the information available about cyanobacterial ion channels and discuss the evolutionary origin of chloroplast channels. The recent molecular identification of some of these ion channels allowed their physiological functions to be studied using genetically modified Arabidopsis plants and cyanobacteria. The view is emerging that alteration of chloroplast and mitochondrial ion homeostasis leads to organelle dysfunction, which in turn significantly affects the energy metabolism of the whole organism. Clear-cut identification of genes encoding for chan- nels in these organelles, however, remains a major challenge in this rapidly developing field. Multiple stra- tegies including bioinformatics, cell biology, electrophysiology, use of organelle-targeted ion-sensitive probes, genetics, and identification of signals eliciting specific ion fluxes across organelle membranes should provide a better understanding of the physiological role of organellar channels and their contribution to signaling pathways in plants in the future.

关键词： ion channels chloroplasts mitochondria cyanobacteria endosymbiosis plant physiology

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Dual-Targeting of Arabidopsis 6-Phosphogluconolactonase 3 （PGL3）to chloroplasts and Peroxisomes Involves Interaction with Trx m2 in the Cytosol

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Molecular Plant 2014年第1期7卷 252-255页

作者： Christian Holscher Tanja Meyer Antje von Schaewen Institut for Biologie und Biotechnologie der Pflanzen Westfalische Wilhelms-Universitat Munster Schlossgarten 3 48149 Munster Germany

The oxidative pentose-phosphate pathway （OPPP） represents a central branch of cellular metabolism emanating from glucose-6-phosphate （G6P） to provide reductive power （NADPH） and sugar phosphates for anabolic biosyntheses. In plant cells, the oxidative OPPP branch is found in the cytosol and in plastids, consisting of glucose-6-phosphate dehydrogenase （G6PD）, 6-phosphogluconolactonase （PGL）, and 6-phosphogluconate dehydrogenase （6PGD）. These enzymes are encoded by small gene families in the nuclear genome, which, in Arabidopsis, comprise six G6PD, five 6-PGL, and three 6-PGD isoforms （Kruger and von Schaewen, 2003）. Specific targeting motifs at the C-terminus of 6-PGL and 6-PGD isoforms suggested that the OPPP may also occur in peroxisomes （Reumann et al., 2004）.

关键词： Cytosol C-Terminus nuclear genome Plastid Peroxisomes 6-phosphogluconolactonase Gene families Plant cells chloroplasts Glucosephosphate Dehydrogenase Dual Glucose-6-Phosphate Arabidopsis sugar phosphates

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On the Impact of Precursor Unfolding during Protein Import into chloroplasts

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Molecular Plant 2010年第3期3卷 499-508页

作者： Maike Ruprecht Tihana Bionda Takehiro Sato Maik S. Sommer Toshiya Endo Enrico Schleiff Goethe University Cluster of Excellence Macromolecular Complexes Centre of Membrane Proteomics Department of Biosciences Molecular Cell Biology of Plants Max-von-Laue Str. 9 D-60438 Frankfurt Germany Department of Chemistry Graduate School of Science and Institute for Advanced Research Nagoya University Chikusa-ku Nagoya 464-8602 Japan Present address: Department of Molecular and Cellular Medicine Texas A&M University System Health Science Center College Station TX 77843-11114 USA

Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a posttranslational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa （OE33）. Our results suggest that p（OE33）titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.

关键词： chloroplasts import precursor protein titin energetic of translocation.

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Effects of Ce^(3+) on improvement of spectral characteristics and function of chloroplasts damaged by linolenic acid in spinach

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Journal of Rare Earths 2009年第2期27卷 288-293页

作者：刘晓晴迮玉官刘超周敏李娜段艳梅殷四涛洪法水 College of Life Sciences Suzhou UniversitySuzhou 215123China

Linolenic acid has great effects on the structure and function of chloroplast. We studied the effects of Ce3＋ on the improvement of chloroplast spectral characteristics and oxygen evolution damaged by linolenic acid in spinach. Results showed that Ce3＋ could decrease the light absorption increased by linolenic acid and promote the distribution of excitation energy to PS II and alleviate the decrease of PS Ⅱ fluo- rescence yield caused by linolenic acid. The linolenic acid treatments in various concentrations reduced the oxygen-evolving rate of chloroplasts, but the rate was accelerated since adding Ce3＋.

关键词： Ce3＋ linolenic acid chloroplasts spectral characteristic O2-evolving rare earths

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Maize ZmFDR3 localized in chloroplasts is involved in iron transport

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Science China(Life Sciences) 2009年第9期52卷 864-871页

作者： HAN JianHui1,SONG XiuFang1,LI Peng1,YANG HuiJun2 & YIN LiPing1 1 College of Life Science,Capital Normal University,Beijing 100048,China 2 Department of Biological Sciences,University of South Carolina,Columbia,SC 29208,USA College of Life Science Capital Normal University Beijing 100048 China

Iron is an essential nutrient for plant metabolism such that Fe-limited plants display chlorosis and suffer from reduced photosynthetic efficiency. Differential display previously identified genes whose expression was elevated in Fe-deficient maize roots. Here,we describe the functional characterization of one of the genes identified in the screen,ZmFDR3 (Zea maize Fe-deficiency-related). Heterologous functional complementation assays using a yeast iron uptake mutant showed that ZmFDR3 functions in iron transport. ZmFDR3 contains a domain found in FliN-proteins of the type III secretion system and is predicted to localize to the thylakoid of plastids. Fluorescence immunocytochemistry showed that ZmFDR3 is localized in the plastids of roots,stems and leaves,with high expression found in guard cell chloroplasts. Transgenic tobacco expressing a 35S-ZmFDR3 construct contains elevated iron content,displays well arranged thylakoid membranes and has photosynthetic indices that are higher than those of the wild type. Together,these results suggest that ZmFDR3 functions in chloroplast iron transport.

关键词： ZmFDR3 iron transport iron content chloroplasts

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Metabolic engineering of photorespiration by transforming multiple genes into chloroplasts of rice and potato

Metabolic engineering of photorespiration by transforming mu...

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从植物科学到农业发展——2012全国植物生物学大会

作者： Boran Shen,Zhen Yao,Han He,Xinxiang Peng~* State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,South China Agricultural University,Guangzhou,China.

Environmental stresses,such as drought and high temperature,are increasingly limiting factors for rice production,thus generating resistant engineering plants is of practical significance.Our project is to create a shortcut pathway for photorespiration inside the chloroplast of rice,and such modification would lead to a direct release of the photorespiratory CO2 within the chloroplast thereby forming a novel CO2 concentrating mechanism.Three genes,i.***.glycolate dehydrogenase(GDH) from Synecocystis sp, glyoxylate carboligase(GCL) and tartronic semialdehyde reductase(TSR) from E.coli,were fused to the rice rbcS chloroplast targeting peptide(TP) at N-term and cloned into a multiple gene expression vector pYL1305.The constructed plasmid was then transformed into rice and potato.Eight independent lines of rice were obtained,and RT-PCR analysis showed that the three genes were appreciably expressed at the mRNA level.However,for the time being the proteins and enzymatic activities failed to be detected.By using the protoplast transient expression technology,fusion proteins,i.e.GFP-TP,TP-GDH-GFP, TP-GCL-GFP,TP-TSR-GFP,were respectively transformed into rice leaf protoplasts. Fluorescence observation demonstrated that,while GFP-TP could be well localized into the chloroplast,the other fusion genes were expressed mainly outside the chloroplast.The results implicate that rice may be able to prevent heterologous proteins from getting into the chloroplast.We are currently focusing on understanding the possible mechanisms.

关键词： multigene transformation photorespiration chloroplasts rice potato

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Both metaxin and Tom20 together with two mitochondria-specific motifs support mitochondrial targeting of dual-targeting AtSufE1

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Journal of Integrative Plant Biology 2022年第8期64卷 1596-1613页

作者： Seungjin Woo Byeongho Moon Inhwan Hwang Department of Life Sciences Pohang University of Science and TechnologyPohang 790‐784Korea

Plant cells have two endosymbiotic organelles,chloroplasts,and mitochondria.These organelles perform specific functions that depend on organelle-specific proteins.The majority of chloroplast and mitochondrial proteins are specifically imported by the transit peptide and presequence,respectively.However,a significant number of proteins are also dually targeted to these two organelles.Currently,it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria.In this study,the mechanism underlying mitochondrial targeting of dual targeting At Suf E1 in Arabidopsis was elucidated.The N-terminal fragment containing 80 residues of AtSufE1(AtSufE1N80)was sufficient to confer dual targeting of reporter protein,At SufE1N80:GFP,in protoplasts.Two sequence motifs,two arginine residues at 15and 21positions,and amino acid(aa)sequence motif AKTLLLRPLK from the 31to 40aa position,were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting.The sequence motif PSEVPFRRT from the 41 stto 50 thaa position constitutes a common motif for targeting to both chloroplasts and mitochondria.For mitochondrial import of At Suf E1:N80,Metaxin played a critical role.In addition,Bi FC and protein pull-down experiments showed that At Suf E1N80 specifically interacts with import receptors,Metaxin and Tom20.The interaction of At Suf E1N80 with Metaxin was required for the interaction with Tom20.Based on these results,we propose that mitochondrial targeting of dual-targeting At Suf E1 is mediated by both mitochondria-specific and common sequence motifs in the signal sequence through the interaction with import receptors,Metaxin and Tom20,in a successive manner.

关键词： AtSufE1 chloroplasts common motif dual targeting metaxin mitochondria mitochondria-specific motif Tom20

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