Integrity of the yeast mitochondrial genome,but not its distribution and inheritance,relies on mitochondrial fission and fusion

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin–dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.Mitochondrial DNA (mtDNA) is essential for respiratory growth of all eukaryotic cells, and all multicellular organisms depend on mtDNA for their development. Not surprisingly, given the fundamental importance of mtDNA, mutations within mtDNA have been identified as the cause for a plethora of human diseases (1). mtDNA in Saccharomyces cerevisiae encodes for seven essential subunits of the respiratory chain, one protein and two RNA subunits of the mitochondrial ribosome, 24 tRNAs, and the RNA subunit of RNase P (2). Every cell contains 50–100 copies of mtDNA that are organized into nucleoprotein complexes termed nucleoids, each containing 1–10 copies of mtDNA (3, 4). Nucleoids are distributed throughout the mitochondrial network, which is likely important for equivalently supplying spatially separated mitochondrial segments with mitochondrially encoded proteins.How the distribution of mtDNA throughout the mitochondrial network is established and maintained is not fully understood. Previous work from our laboratory and others has shown that the movement of nucleoids within the mitochondrial network is limited, suggesting that the mechanisms of nucleoid distribution are tightly interlinked with the dynamics of mitochondria themselves (5, 6). Mitochondria undergo constant fusion and fission events that are mediated by dedicated machineries, with the central components Fzo1 and Dnm1 required for fusion and fission, respectively (7). Recently, we have provided support for a role of mitochondrial fission in mtDNA distribution. We have shown that mtDNA localizes to sites of Dnm1-dependent mitochondrial fission and that it is segregated after scission to both of the newly generated mitochondrial tips (8). Localizing mtDNA to the newly formed tips would then allow transport of mitochondrial tips and mtDNA to distal parts in the cell, where fusion with the mitochondrial network may drive mtDNA distribution. Such a mechanism would be particularly important during inheritance of mtDNA to daughter buds during cell division, where mtDNA needs to be transported over a relatively large distance. In S. cerevisiae, mitochondria are inherited in a myosin- and F-actin–dependent process, in which a mitochondrial tubule invades the budding daughter cell and is subsequently anchored at the distal membrane (9). An active mtDNA partition and inheritance apparatus has been postulated (6); however, the spatiotemporal relationship between the inheritance of mitochondria and the inheritance of mtDNA has not been examined.If mitochondrial fusion and fission were essential for the distribution and inheritance of mtDNA, their loss would impair the process. Indeed, fusion-defective cells lose mtDNA (10, 11), most likely due to excessive fragmentation. By contrast, however, fission-defective cells, as well as cells defective in fusion and fission, remain capable of respiratory growth, indicating that a functional mitochondrial genome must be maintained (10, 12). These observations suggest that fission-independent mechanisms must exist that facilitate mtDNA inheritance.In this work, we investigated the role of mitochondrial fusion and fission in mtDNA distribution and inheritance. Through the development of a noninvasive method to quantify the spatial organization of mtDNA within mitochondrial tubules, we found that cells deficient in fusion and fission maintain a WT distribution of mtDNA. Live-cell imaging showed that this distribution is facilitated by the de novo generation of tubules from the sides of existing tubules, a process coupled to the spatial positioning of mtDNA to the newly formed tip. Unexpectedly, although dispensable for maintaining mtDNA distribution and inheritance, fusion and fission were required to maintain the integrity of the mitochondrial genome.     

Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

AIM: To investigate the biological function of F protein by yeast two-hybrid system. METHODS: We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH 109 was mated with yeast Y187 (a type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. RESULTS: Thirty-six colonies were selected and sequenced. Among them, 11 colonies were zymogen granule protein, 5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor I, 1 colony was vitronectin, and 2 colonies were new genes with unknown function. CONCLUSION: The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins.     

禽流感病毒NP基因的酵母双杂交饵载体表达及自激活检测

目的构建H5N1亚型禽流感病毒NP基因的酵母双杂交诱饵载体,验证其在酵母中的表达并检测其自激活作用。方法以PCR法从pGEMT/H5NP扩增NP基因编码区序列,将其定向克隆到PGBKT7载体,经测序鉴定后,PEG/Li-Ac法转化酵母菌株AH109,用表型筛选法及颜色筛选法检测其自激活作用同时Westernblot验证诱饵蛋白的表达。结果获得NP编码区基因,并成功构建酵母双杂交系统中的诱饵载体pGBKT7-NP,对宿主细胞酵母菌株AH109无毒性和自激活作用,并能在酵母细胞中稳定表达。结论诱饵载体pGBKT7-NP可用于GAL4酵母双杂交系统钓取与禽流感病毒核蛋白相互作用的蛋白。     

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

DNA double-strand breaks (DSBs) are potentially lethal lesions that threaten genomic integrity and cell viability. DSBs can occur spontaneously as a result of faulty DNA metabolism or by exposure to genotoxins. In eukaryotes, these DSBs have “dirty ends” that lack ligatable 3ʹ-hydroxyl/5ʹ-phosphate groups and are often firmly attached to proteins such as the Ku70-80 heterodimer and topoisomerases (1, 2). During meiosis, the topoisomerase-like Spo11 protein generates DSBs and remains covalently attached to the 5ʹ DNA ends (3). To enable further processing, these DSB ends must be converted to “clean” ends with 3ʹ-hydroxyl/5ʹ-phosphate groups properly exposed. This step is achieved by endonucleolytic cleavage, or clipping, by Mre11 (4–7).In mammals, the MRE11, RAD50, and NBS1 complex (Mre11-Rad50-Nbs1 [MRN]), together with CtIP, is involved in the clipping reaction. MRE11 is the nuclease subunit that has both endonuclease and 3′-to-5′ exonuclease activities, but only the former is essential for clipping (4, 8–12). RAD50, a member of the Structural Maintenance of Chromosomes protein family, binds to MRE11 to form an (MRE11)₂-(RAD50)₂ ring structure (MR complex) (13–15). NBS1 binds to the MR complex via MRE11 to form the MRN complex (16). Homologs of CtIP include Ctp1 in Schizosaccharomyces pombe and Sae2 in Saccharomyces cerevisiae (17–19). Upon phosphorylation, these proteins physically interact with their cognate MRN complex via the N-terminal forkhead-associated domain of NBS1, leading to activation of the MRE11 endonucleolytic clipping activity (20–24). However, the mechanistic details underlying this activation have not yet been determined.Through biochemical reconstitution using fission yeast proteins, we made three key findings regarding how Ctp1 activates MRN. First, MRN activation is mediated by Ctp1 phosphorylation, which promotes the direct association of Ctp1 with the Nbs1 subunit of MRN. Second, the highly conserved extreme C terminus of Ctp1 retains the ability to promote the endonuclease activity of MRN. Strikingly, a synthetic polypeptide comprising the 15 amino acids from the extreme C terminus of Ctp1 was sufficient for the full activation of MRN. Third, we verified the evolutionary significance of these findings by demonstrating that the conserved C-terminal polypeptide of CtIP can also stimulate the endonuclease activity of human MRN. Together, our results strongly suggest that the Ctp1-promoted MRN activation mechanism consists of at least two fundamentally separable elements: phosphorylation-induced Ctp1-MRN association and activation of MRN by the C-terminal peptide of Ctp1. Thus, recruitment of the Ctp1 C terminus to MRN is likely pivotal in this activation mechanism.     

Low Toxicity of Deoxynivalenol-3-Glucoside in Microbial Cells

Host plants excrete a glucosylation enzyme onto the plant surface that changes mycotoxins derived from fungal secondary metabolites to glucosylated products. Deoxynivalenol-3-glucoside (DON3G) is synthesized by grain uridine diphosphate-glucosyltransferase, and is found worldwide, although information on its toxicity is lacking. Here, we conducted growth tests and DNA microarray analysis to elucidate the characteristics of DON3G. The Saccharomyces cerevisiae PDR5 mutant strain exposed to DON3G demonstrated similar growth to the dimethyl sulfoxide control, and DNA microarray analysis revealed limited differences. Only 10 genes were extracted, and the expression profile of stress response genes was similar to that of DON, in contrast to metabolism genes like SER3, which encodes 3-phosphoglycerate dehydrogenase. Growth tests with Chlamydomonas reinhardtii also showed a similar growth rate to the control sample. These results suggest that DON3G has extremely low toxicity to these cells, and the glucosylation of mycotoxins is a useful protective mechanism not only for host plants, but also for other species.     

Candida albicans chronic colonisation in cystic fibrosis may be associated with inhaled antibiotics

Candida albicans is increasingly recognised as a coloniser of the respiratory tract in cystic fibrosis (CF) patients. Yet, the potential role, if any, of the micro‐organism in the progress of the disease remains unclear. In this study, we investigated the association between inhaled antibiotics and C. albicans chronic colonisation in patients with CF. A cohort of 121 CF patients born from 1988 to 1996 was, respectively, studied. The medical records of each patient were reviewed from the first time they attended the CF Centre until the occurrence of C. albicans chronic colonisation or their last visit for the year 2010. Chronic colonisation was defined as the presence of C. albicans in more than 50% of cultures in a given year. A number of possible confounders were included in the multivariate logistic regression analysis to identify an independent association between inhaled antibiotics and C. albicans chronic colonisation. Fifty‐four (44.6%) of the 121 patients enrolled in the study developed chronic colonisation by the micro‐organism. Multivariate logistic regression analysis determined the independent effect of inhaled antibiotic treatment on the odds of chronic colonisation (OR 1.112, 95% CI [1.007–1.229], P = 0.036). Candida albicans chronic colonisation may be associated with the duration of inhaled antibiotic treatment.     

From the Cover: A disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates adiponectin multimerization

Impairments in adiponectin multimerization lead to defects in adiponectin secretion and function and are associated with diabetes, yet the underlying mechanisms remain largely unknown. We have identified an adiponectin-interacting protein, previously named GST-kappa, by yeast 2-hybrid screening. The adiponectin-interacting protein contains 2 thioredoxin domains and has very little sequence similarity to other GST isoforms. However, this protein shares high sequence and secondary structure homology to bacterial disulfide-bond A oxidoreductase (DsbA) and is thus renamed DsbA-like protein (DsbA-L). DsbA-L is highly expressed in adipose tissue, and its expression level is negatively correlated with obesity in mice and humans. DsbA-L expression in 3T3-L1 adipocytes is stimulated by the insulin sensitizer rosiglitazone and inhibited by the inflammatory cytokine TNFα. Overexpression of DsbA-L promoted adiponectin multimerization while suppressing DsbA-L expression by RNAi markedly and selectively reduced adiponectin levels and secretion in 3T3-L1 adipocytes. Our results identify DsbA-L as a key regulator for adiponectin biosynthesis and uncover a potential new target for developing therapeutic drugs for the treatment of insulin resistance and its associated metabolic disorders.     

高尿酸血症动物模型的实验研究

[目的]制备稳定持续、简便易行的高尿酸血症动物模型。[方法]采用单纯氧嗪酸钾、氧嗪酸钾+次黄嘌呤、腺嘌呤+乙胺丁醇、单纯酵母膏、酵母膏+腺嘌呤、酵母膏+腺嘌呤+氧嗪酸钾不同药物与不同浓度进行实验,检测模型动物血清尿酸值。[结果]单纯氧嗪酸钾、氧嗪酸钾+次黄嘌呤模型组尿酸值升高,与空白组比较有差异,但模型不稳定,重复性差,个体差异较大。腺嘌呤+乙胺丁醇、单纯酵母膏、酵母膏+腺嘌呤模型组尿酸值与空白组比较无差异。酵母膏+腺嘌呤+氧嗪酸钾模型组尿酸值升高,与空白组比较有显著性差异(P0.01),且模型稳定,个体差异小。[结论]采用酵母膏+腺嘌呤+氧嗪酸钾复制高尿酸血症动物模型最佳。     

Anti-Candida activity of the water-soluble lectin from Moringa oleifera seeds (WSMoL)

This work reports the effects of the water-soluble lectin from Moringa oleifera seeds (WSMoL) on growth and survival of Candida species. In addition, cellular alterations linked to the antifungal effect were investigated. The minimal inhibitory (MIC) and fungicidal (MFC) concentrations were determined and 24-h growth curves in absence and presence of lectin were established. Flow cytometry was used to evaluate the induction of apoptosis/necrosis, alterations in mitochondrial membrane potential (ΔΨm), and occurrence of lysosomal damage. WSMoL inhibited the growth of C. albicans, C. glabrata, C. krusei and C. parapsilosis with MIC of 20 μg/mL. The lowest MFC (20 μg/mL) was detected for C. glabrata and the highest (80 μg/mL) for C. albicans and C. parapsilosis. The inhibitory effect started from the ninth to nineteenth hour of incubation depending on the fungal species. Incubation with the lectin at the MIC for 24 h increased the number of cells undergoing apoptosis and necrosis. Hyperpolarization of the mitochondrial membrane was detected after 12-h treatment, followed by reduction of ΔΨm or depolarization after 24 h. No lysosomal damage was detected in treated cells. In conclusion, WSMoL is a fungistatic and fungicide agent against Candida with differential effects depending on the species.