Eukaryotic Cell最新文献

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Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3'-Phosphoadenosine 5'-Phosphosulfate and ATP. 溶组织内阿米巴线粒体载体家族通过交换3'-磷酸腺苷- 5'-硫酸磷和ATP连接线粒体和细胞质途径的证据。
Eukaryotic Cell Pub Date : 2015-11-01 Epub Date: 2015-09-18 DOI: 10.1128/EC.00130-15
Fumika Mi-ichi, Akira Nozawa, Hiroki Yoshida, Yuzuru Tozawa, Tomoyoshi Nozaki
{"title":"Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3'-Phosphoadenosine 5'-Phosphosulfate and ATP.","authors":"Fumika Mi-ichi,&nbsp;Akira Nozawa,&nbsp;Hiroki Yoshida,&nbsp;Yuzuru Tozawa,&nbsp;Tomoyoshi Nozaki","doi":"10.1128/EC.00130-15","DOIUrl":"https://doi.org/10.1128/EC.00130-15","url":null,"abstract":"<p><p>Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3'-phosphoadenosine 5'-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00130-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34019100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 16
Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei? 血流型布氏锥虫未折叠蛋白反应途径?
Eukaryotic Cell Pub Date : 2015-11-01 Epub Date: 2015-08-28 DOI: 10.1128/EC.00118-15
Calvin Tiengwe, Abigail E N A Brown, James D Bangs
{"title":"Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?","authors":"Calvin Tiengwe,&nbsp;Abigail E N A Brown,&nbsp;James D Bangs","doi":"10.1128/EC.00118-15","DOIUrl":"https://doi.org/10.1128/EC.00118-15","url":null,"abstract":"<p><p>The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00118-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33960578","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 15
Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy. 利用固态核磁共振波谱分析曲霉菌生物膜胞外基质
Eukaryotic Cell Pub Date : 2015-11-01 Epub Date: 2015-07-10 DOI: 10.1128/EC.00050-15
Courtney Reichhardt, Jose A G Ferreira, Lydia-Marie Joubert, Karl V Clemons, David A Stevens, Lynette Cegelski
{"title":"Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy.","authors":"Courtney Reichhardt, Jose A G Ferreira, Lydia-Marie Joubert, Karl V Clemons, David A Stevens, Lynette Cegelski","doi":"10.1128/EC.00050-15","DOIUrl":"10.1128/EC.00050-15","url":null,"abstract":"<p><p>Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621319/pdf/zek1064.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33997458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus. 烟曲霉靶向基因破坏CRISPR/Cas9系统的建立
Eukaryotic Cell Pub Date : 2015-11-01 Epub Date: 2015-08-28 DOI: 10.1128/EC.00107-15
Kevin K Fuller, Shan Chen, Jennifer J Loros, Jay C Dunlap
{"title":"Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus.","authors":"Kevin K Fuller,&nbsp;Shan Chen,&nbsp;Jennifer J Loros,&nbsp;Jay C Dunlap","doi":"10.1128/EC.00107-15","DOIUrl":"https://doi.org/10.1128/EC.00107-15","url":null,"abstract":"<p><p>Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00107-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33960576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 177
Articles of Significant Interest Selected from This Issue by the Editors 由本刊编辑从本刊精选的重要文章
Eukaryotic Cell Pub Date : 2015-10-26 DOI: 10.1128/EC.00148-15
{"title":"Articles of Significant Interest Selected from This Issue by the Editors","authors":"","doi":"10.1128/EC.00148-15","DOIUrl":"https://doi.org/10.1128/EC.00148-15","url":null,"abstract":"African trypanosomes (Trypanosoma brucei) dwell in the bloodstream of infected mammalian hosts in continuous exposure to the adaptive immune response. They rely on an extremely high rate of endocytosis in order to clear antibodies bound to the cell surface. However, endocytosis occurs at only a single point on the plasma membrane—an invagination containing the base of the flagellum, termed the flagellar pocket. Coiled around the neck of the flagellar pocket on its cytoplasmic face is a fishhook-shaped cytoskeletal complex containing the protein TbMORN1. Morriswood and Schmidt (p. 1081–1093) show that depletion of TbMORN1 results in a striking phenotype in which protein access to the flagellar pocket is apparently impaired, suggesting that the complex may function as a kind of molecular valve.","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73195258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Differences in Host Innate Responses among Coccidioides Isolates in a Murine Model of Pulmonary Coccidioidomycosis. 小鼠肺球孢子菌病模型中不同球孢子菌分离株宿主先天反应的差异。
Eukaryotic Cell Pub Date : 2015-10-01 Epub Date: 2015-08-14 DOI: 10.1128/EC.00122-15
Eric R G Lewis, Victoria R David, Adina L Doyle, Khadijeh Rajabi, Jeffrey A Kiefer, Patrick Pirrotte, Bridget M Barker
{"title":"Differences in Host Innate Responses among Coccidioides Isolates in a Murine Model of Pulmonary Coccidioidomycosis.","authors":"Eric R G Lewis,&nbsp;Victoria R David,&nbsp;Adina L Doyle,&nbsp;Khadijeh Rajabi,&nbsp;Jeffrey A Kiefer,&nbsp;Patrick Pirrotte,&nbsp;Bridget M Barker","doi":"10.1128/EC.00122-15","DOIUrl":"https://doi.org/10.1128/EC.00122-15","url":null,"abstract":"<p><p>Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi and the causative agents of coccidioidomycosis, a mycosis endemic to certain semiarid regions in the Americas. The most common route of infection is by inhalation of airborne Coccidioides arthroconidia. Once a susceptible host inhales the conidia, a transition to mature endosporulated spherules can occur within the first 5 days of infection. For this study, we examined the host response in a murine model of coccidioidomycosis during a time period of infection that has not been well characterized. We collected lung tissue and bronchoalveolar lavage fluid (BALF) from BALB/c mice that were infected with a C. immitis pure strain, a C. immitis hybrid strain, or a C. posadasii strain as well as uninfected mice. We compared the host responses to the Coccidioides strains used in this study by assessing the level of transcription of selected cytokine genes in lung tissues and characterized host and fungal proteins present in BALF. Host response varied depending on the Coccidioides strain that was used and did not appear to be overly robust. This study provides a foundation to begin to dissect the host immune response early in infection, to detect abundant Coccidioides proteins, and to develop diagnostics that target these early time points of infection. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00122-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33924438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 14
Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens. 真菌、微藻和地衣的酶体超微结构和进化。
Eukaryotic Cell Pub Date : 2015-10-01 Epub Date: 2015-08-07 DOI: 10.1128/EC.00106-15
Jae-Hyeok Lee, John E Heuser, Robyn Roth, Ursula Goodenough
{"title":"Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens.","authors":"Jae-Hyeok Lee,&nbsp;John E Heuser,&nbsp;Robyn Roth,&nbsp;Ursula Goodenough","doi":"10.1128/EC.00106-15","DOIUrl":"https://doi.org/10.1128/EC.00106-15","url":null,"abstract":"<p><p>Eisosomes are among the few remaining eukaryotic cellular differentations that lack a defined function(s). These trough-shaped invaginations of the plasma membrane have largely been studied in Saccharomyces cerevisiae, in which their associated proteins, including two BAR domain proteins, have been identified, and homologues have been found throughout the fungal radiation. Using quick-freeze deep-etch electron microscopy to generate high-resolution replicas of membrane fracture faces without the use of chemical fixation, we report that eisosomes are also present in a subset of red and green microalgae as well as in the cysts of the ciliate Euplotes. Eisosome assembly is closely correlated with both the presence and the nature of cell walls. Microalgal eisosomes vary extensively in topology and internal organization. Unlike fungi, their convex fracture faces can carry lineage-specific arrays of intramembranous particles, and their concave fracture faces usually display fine striations, also seen in fungi, that are pitched at lineage-specific angles and, in some cases, adopt a broad-banded patterning. The conserved genes that encode fungal eisosome-associated proteins are not found in sequenced algal genomes, but we identified genes encoding two algal lineage-specific families of predicted BAR domain proteins, called Green-BAR and Red-BAR, that are candidate eisosome organizers. We propose a model for eisosome formation wherein (i) positively charged recognition patches first establish contact with target membrane regions and (ii) a (partial) unwinding of the coiled-coil conformation of the BAR domains then allows interactions between the hydrophobic faces of their amphipathic helices and the lipid phase of the inner membrane leaflet, generating the striated patterns. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00106-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33904285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 37
Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster. 黄曲霉的转录组分析揭示了vea依赖性次级代谢产物基因簇的调控,包括新的黄素簇。
Eukaryotic Cell Pub Date : 2015-10-01 Epub Date: 2015-07-24 DOI: 10.1128/EC.00092-15
J W Cary, Z Han, Y Yin, J M Lohmar, S Shantappa, P Y Harris-Coward, B Mack, K C Ehrlich, Q Wei, N Arroyo-Manzanares, V Uka, L Vanhaecke, D Bhatnagar, J Yu, W C Nierman, M A Johns, D Sorensen, H Shen, S De Saeger, J Diana Di Mavungu, A M Calvo
{"title":"Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster.","authors":"J W Cary,&nbsp;Z Han,&nbsp;Y Yin,&nbsp;J M Lohmar,&nbsp;S Shantappa,&nbsp;P Y Harris-Coward,&nbsp;B Mack,&nbsp;K C Ehrlich,&nbsp;Q Wei,&nbsp;N Arroyo-Manzanares,&nbsp;V Uka,&nbsp;L Vanhaecke,&nbsp;D Bhatnagar,&nbsp;J Yu,&nbsp;W C Nierman,&nbsp;M A Johns,&nbsp;D Sorensen,&nbsp;H Shen,&nbsp;S De Saeger,&nbsp;J Diana Di Mavungu,&nbsp;A M Calvo","doi":"10.1128/EC.00092-15","DOIUrl":"https://doi.org/10.1128/EC.00092-15","url":null,"abstract":"<p><p>The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00092-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33934702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 62
The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing. 八肽重复蛋白Raa8是叶绿体反式剪接所必需的。
Eukaryotic Cell Pub Date : 2015-10-01 Epub Date: 2015-07-24 DOI: 10.1128/EC.00096-15
Christina Marx, Christiane Wünsch, Ulrich Kück
{"title":"The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing.","authors":"Christina Marx,&nbsp;Christiane Wünsch,&nbsp;Ulrich Kück","doi":"10.1128/EC.00096-15","DOIUrl":"https://doi.org/10.1128/EC.00096-15","url":null,"abstract":"<p><p>The mRNA maturation of the tripartite chloroplast psaA gene from the green alga Chlamydomonas reinhardtii depends on various nucleus-encoded factors that participate in trans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the psaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the trans-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized trans-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential trans-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated trans-splicing mutants. Our assay revealed that the trans-splicing defect of mutant F19 was restored by a new factor we named RAA8; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first psaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring psaA mRNA trans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying trans-splicing factors. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00096-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33867576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 19
Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X)nL Motif-Containing Target Proteins. 酵母Bro1家族V结构域与含YP(X)nL基序靶蛋白相互作用的保守模式
Eukaryotic Cell Pub Date : 2015-10-01 Epub Date: 2015-07-06 DOI: 10.1128/EC.00091-15
Yoko Kimura, Mirai Tanigawa, Junko Kawawaki, Kenji Takagi, Tsunehiro Mizushima, Tatsuya Maeda, Keiji Tanaka
{"title":"Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X)nL Motif-Containing Target Proteins.","authors":"Yoko Kimura,&nbsp;Mirai Tanigawa,&nbsp;Junko Kawawaki,&nbsp;Kenji Takagi,&nbsp;Tsunehiro Mizushima,&nbsp;Tatsuya Maeda,&nbsp;Keiji Tanaka","doi":"10.1128/EC.00091-15","DOIUrl":"https://doi.org/10.1128/EC.00091-15","url":null,"abstract":"<p><p>Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity. </p>","PeriodicalId":11891,"journal":{"name":"Eukaryotic Cell","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1128/EC.00091-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34264092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 8
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