Protein Synthesis in Euglena gracilis is Light-and Temperature-Dependent,Oscillating in a Circadian,Temperature-Compensated Manner

Abstract: Incorporation of radiolabelled amino acids into proteins of Euglena gracilis revealed that the amount of labelled protein depends on the conditions of illumination and temperature of cultivation. Protein synthesis was generally lower under dark conditions except at 37 °C. The largest amounts of labelled protein were measured at 21 °C and decreased at higher and lower temperatures. By separating the labelled proteins of the membraneous cell fraction from subcultures under a range of culture conditions, the synthesis of some specific proteins was found to be light- and/or temperature-dependent. On incubating cells taken at different times during a light/dark cycle and under constant conditions, a circadian rhythm of ³⁵S-methionine- as well as ³⁵S-cysteine-incorporation was detected. Thereby the cells incorporated ten-times less cysteine than methionine. Protein synthesis always peaked during the last quarter of the daily light phase, confirming the rhythmic rise in total protein. The length of the rhythm period, approximately 24 h, was nearly independent of the applied temperature in the range of 16 to 27 °C.     

A Mutation in Drosophila simulans that Lengthens the Circadian Period of Locomotor Activity

The length of the Thr-Gly repeat within the period gene of Drosophilids, coevolves with its immediate flanking region to maintain the temperature compensation of the fly circadian clock. In Drosophila simulans, balancing selection appears to maintain a polymorphism in this region, with three repeat lengths carrying 23, 24 or 25 Thr-Gly pairs, each in complete linkage disequilibrium with a distinctive flanking region amino acid moiety. We wondered whether separating a specific length repeat from its associated flanking haplotype might have functional implications for the circadian clock. We fortuitously discovered a population of flies collected in Kenya, in which a chimeric Thr-Gly haplotype was segregating that carried the (Thr-Gly)24 repeat, but the flanking region of a (Thr-Gly)23 allele. One of the five isofemale lines that carried this 'mutant' Thr-Gly sequence showed a dramatically long and temperature-sensitive free-running circadian period. This phenotype was mapped to the X chromosome, close to the D. simulans per gene, but there was also a significant effect of a modifying autosomal locus or loci. It seems remarkable that such a mutant phenotype should be discovered in a screen of chimeric Thr-Gly regions.     

Cooperative Interaction between Phosphorylation Sites on PERIOD Maintains Circadian Period in Drosophila

Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 “phospho-occupied” serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the per⁰¹ arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ∼30 hrs. Interestingly, the single S613A mutation extends the period by 2–3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period.     

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-Like Potato virus A in Nicotiana Species

The multifunctional nuclear inclusion protein a (NIa) of potyviruses (genus Potyvirus; Potyviridae) accumulates in the nucleus of virus-infected cells for unknown reasons. In this study, two regions in the viral genome-linked protein (VPg) domain of NIa in Potato virus A (PVA) were found to constitute nuclear and nucleolar localization signals (NLS) in plant cells (Nicotiana spp). Amino acid substitutions in both NLS I (residues 4 to 9) and NLS II (residues 41 to 50) prevented nuclear localization, whereas mutations in either single NLS did not. Mutations in either NLS, however, prevented nucleolar localization and prevented or diminished virus replication in protoplasts, accumulation in infected plant tissues, and/or systemic movement in plants. One NLS mutant was partially complemented by the wild-type VPg expressed in transgenic plants. Furthermore, NLS I controlled NIa accumulation in Cajal bodies. The VPg domain interacted with fibrillarin, a nucleolar protein, and depletion of fibrillarin reduced PVA accumulation. Overexpression of VPg in leaf tissues interfered with cosuppression of gene expression (i.e., RNA silencing), whereas NLS I and NLS II mutants, which exhibited reduced nuclear and nucleolar localization, showed no such activity. These results demonstrate that some of the most essential viral functions required for completion of the infection cycle are tightly linked to regulation of the NIa nuclear and nucleolar localization.     

Analysis of Nuclear Localization Signals Using a Green Fluorescent Protein-Fusion Protein Library

We describe here an efficient method for identifying intracellular localization signals in proteins with stereospecific intracellular localizations in culture cells. The method involves rapid fluorescence screening of cells transfected with a cDNA library in which cDNAs are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP). We analyzed nuclear localization and nuclear localization signals (NLSs) in a model application of this method. As a result, we identified classical NLSs in 75% of nuclear localized proteins. We identified some novel NLS candidates among the classical NLS-negative sequences whose nuclear localization was also identified in another cell line and with other molecular tag sequences. This method will be useful for identifying intracellular localization signals and for more detailed analysis of intracellular architecture.     

Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.     

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.     

The T-loop Extension of the Tomato Protein Kinase AvrPto-dependent Pto-interacting Protein 3 (Adi3) Directs Nuclear Localization for Suppression of Plant Cell Death

In tomato (Solanum lycopersicum), resistance to Pseudomonas syringae pv. tomato is elicited by the interaction of the host Pto kinase with the pathogen effector protein AvrPto, which leads to various immune responses including localized cell death termed the hypersensitive response. The AGC kinase Adi3 functions to suppress host cell death and interacts with Pto only in the presence of AvrPto. The cell death suppression (CDS) activity of Adi3 requires phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (Pdk1) and loss of Adi3 function is associated with the hypersensitive response cell death initiated by the Pto/AvrPto interaction. Here we studied the relationship between Adi3 cellular localization and its CDS activity. Adi3 is a nuclear-localized protein, and this localization is dictated by a nuclear localization signal found in the Adi3 T-loop extension, an ∼80 amino acid insertion into the T-loop, or activation loop, which is phosphorylated for kinase activation. Nuclear localization of Adi3 is required for its CDS activity and loss of nuclear localization causes elimination of Adi3 CDS activity and induction of cell death. This nuclear localization of Adi3 is dependent on Ser-539 phosphorylation by Pdk1 and non-nuclear Adi3 is found in punctate structures throughout the cell. Our data support a model in which Pdk1 phosphorylation of Adi3 directs nuclear localization for CDS and that disruption of Adi3 nuclear localization may be a mechanism for induction of cell death such as that during the Pto/AvrPto interaction.     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

The Influence of Huntingtin Protein Size on Nuclear Localization and Cellular Toxicity

Huntington disease is an autosomal dominant neurodegenerative disorder caused by the pathological expansion of a polyglutamine tract. In this study we directly assess the influence of protein size on the formation and subcellular localization of huntingtin aggregates. We have created numerous deletion constructs expressing successively smaller fragments of huntingtin and show that these smaller proteins containing 128 glutamines form both intranuclear and perinuclear aggregates. In contrast, larger NH₂-terminal fragments of huntingtin proteins with 128 glutamines form exclusively perinuclear aggregates. These aggregates can form in the absence of endogenous huntingtin. Furthermore, expression of mutant huntingtin results in increased susceptibility to apoptotic stress that is greater with decreasing protein length and increasing polyglutamine size. As both intranuclear and perinuclear aggregates are clearly associated with increased cellular toxicity, this supports an important role for toxic polyglutamine-containing fragments forming aggregates and playing a key role in the pathogenesis of Huntington disease.     

Identification and Characterization of Xlr5c as a Novel Nuclear Localization Protein in Mouse Germ Cells

Background

Spermatogenesis is the complex process by which diploid stem cells generate haploid germ cells in gamete production. Members of the Xlr (X-chromosome linked, lymphocyte regulated) superfamily play essential roles in spermatogenesis. The expression, localization and role in spermatogenesis of one such member, Xlr5c, has not been reported previously.

Methodology/Principal Findings

Xlr5c mRNA and protein levels in murine testes and other tissues were investigated using RT-PCR and Western blotting. Xlr5c was abundantly transcribed in mouse testes, particularly during the early stages of spermatogenesis and throughout prophase I in the nuclei of spermatocytes. Xlr5c was specifically localized at synaptonemal complexes(SCs) region in preleptotene and pachytene spermatocytes, as was the homologous Xlr protein Sycp3.

Conclusions/Significance

These results suggest that Xlr5c was abundantly transcribed in germ cells, localized at SCs region, where it may play a potential role during the early stages of spermatogenesis. Identification and characterization of this novel testis protein may offer a new perspective for understanding of the molecular mechanisms involved in germ cell differentiation.     

Protein Connectivity and Protein Complexity Promotes Human Gene Duplicability in a Mutually Exclusive Manner

It has previously been reported that protein complexity (i.e. number of subunits in a protein complex) is negatively correlated to gene duplicability in yeast as well as in humans. However, unlike in yeast, protein connectivity in a protein–protein interaction network has a positive correlation with gene duplicability in human genes. In the present study, we have analyzed 1732 human and 1269 yeast proteins that are present both in a protein–protein interaction network as well as in a protein complex network. In the human case, we observed that both protein connectivity and protein complexity complement each other in a mutually exclusive manner over gene duplicability in a positive direction. Analysis of human haploinsufficient proteins and large protein complexes (complex size >10) shows that when protein connectivity does not have any direct association with gene duplicability, there exists a positive correlation between gene duplicability and protein complexity. The same trend, however, is not found in case of yeast, where both protein connectivity and protein complexity independently guide gene duplicability in the negative direction. We conclude that the higher rate of duplication of human genes may be attributed to organismal complexity either by increasing connectivity in the protein–protein interaction network or by increasing protein complexity.     

Tom20 Mediates Localization of mRNAs to Mitochondria in a Translation-Dependent Manner

mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20Δ puf3Δ double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20Δ strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.mRNA localization to distinct cellular compartments is important for the efficiency and specificity of the translation process. Synthesis of proteins at their sites of action may decrease the likelihood of ectopic protein expression and facilitate assembly of large multiprotein complexes. Two general modes for mRNA localization are known. The first, which is common for endoplasmic reticulum (ER)-associated mRNAs, necessitates translation of a short region of the protein (the signal peptide). The signal is recognized by the signal recognition particle as it emerges from the ribosome exit tunnel, and the complex that includes the mRNA, ribosome, and signal recognition particle is targeted to the ER (18). As an outcome of this process, mRNAs that encode proteins destined for the ER and the secretory pathway are associated with this compartment (7). The second mode for mRNA localization occurs prior to translation and in many cases prevents initiation of protein synthesis. Sequences or structural elements of the mRNA are bound by RNA-binding proteins, and these interact with transport factors, which direct the mRNA to its destination (5, 35, 42). Genome-wide studies indicate that localization by either mode is a broad phenomenon that encompasses many mRNAs and various cellular destinations (6, 21, 32, 38). Interestingly, we along with others have recently shown that noncoding regions may also be involved in localization of ER-associated mRNAs (1, 26, 38), demonstrating that these two modes are not mutually exclusive.Most of the mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Various in vitro and in vivo assays led to the widely accepted notion that import may occur posttranslationally, i.e., after the protein is fully synthesized in the cytosol (33). However, mounting evidence also supports a cotranslational import of proteins into the mitochondria. Specifically, polysomes were shown to be associated with the mitochondrial surface, and these translated a distinct set of proteins (12, 19, 20). Moreover, isolated mitochondria are associated with many different mRNAs that encode mitochondrial proteins (28, 46). Elements from both the coding region (the mitochondrial targeting signal [MTS]) and the 3′ untranslated region (UTR) were shown to be important for targeting of some of these mRNAs (4, 29). One model for localization suggests association of the nascent peptide chain (specifically, the N-terminal MTS) with receptors on the mitochondria, coupled to cotranslational insertion of the protein (24). As an outcome of this cotranslational mechanism, polysomal mRNAs become associated with the mitochondria, analogously to what is observed in the ER. However, experimental support for this hypothesis is currently lacking.Recently, Saint-Georges et al. (41) have shown a role for Puf3p in localization of many mRNAs to the mitochondria of Saccharomyces cerevisiae. Puf3p is associated with the mitochondria outer membrane (11), and its role is mediated through interaction with UTRs. This may suggest a translation-independent mode of action. Intriguingly, however, most Puf3 targets appeared to be mislocalized also after treatment with the translation inhibitor cycloheximide (CHX), suggesting that an active translation process is required for their asymmetric localization (41). Moreover, a large number of mRNAs that are not Puf3 targets appeared to be affected from treatments with the translation inhibitors puromycin and cycloheximide (41), further supporting the existence of an additional, translation-dependent mode of mRNA targeting to the mitochondria.The translocase of the mitochondrial outer membrane (TOM complex) is a multiprotein machinery which mediates the import of the vast majority of proteins into the mitochondria (36, 39). Its core protein (Tom40) forms a β-barrel structure and serves as the main component of the import pore. Tom20 is a peripheral component of the TOM complex that functions as a primary receptor for mitochondrial precursor proteins (15). It was hypothesized that protein receptors interact with the incoming polypeptide while it is translated, and this leads to a local increase in mRNA concentration (24). An open question is whether the TOM complex, through Tom20, interacts with polypeptides while they are translated and thereby leads to higher local concentrations of mRNAs near the mitochondria. To address this issue, we analyzed the effects of TOM20 deletion on mRNA association with mitochondrial fractions and the role of the MTS on mRNA localization. We also tested the interactions between Tom20 and Puf3. We found that Tom20 is involved in mitochondrial association of many mRNAs by a process that requires the MTS. Tom20 deletion affects the localization of Puf3p, and a strain with deletions of both Tom20 and Puf3 exhibits a growth defect under conditions that require mitochondrial optimal function.     

多肽TAT与核定位信号介导的蛋白质入核递送

增强型绿色荧光蛋白与蛋白质转导结构域TAT、SV40大T抗原的核定位信号以融合蛋白的形式在大肠杆菌中表达 ,纯化后转导A431细胞 ,大部分细胞核内都可以观察到绿色荧光 ,说明TAT NLS可以有效介导蛋白质的入核递送。这种蛋白质递送系统可望用于转录治疗等研究领域。     

Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel's Kinetics in a Temperature-Dependent Manner

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I _h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I _h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I _h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.