The Yeast Motor Protein, Kar3p, Is Essential for Meiosis I

The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.     

Identification of Human Astrovirus Genome-Linked Protein (VPg) Essential for Virus Infectivity

Viral genome-linked proteins (VPgs) have been identified in several single-stranded positive-sense RNA virus families. The presence of such protein in the family Astroviridae has not been fully elucidated, although a putative VPg coding region in open reading frame 1a (ORF1a) of astrovirus with high amino acid sequence similarity to the VPg coding region of Caliciviridae has been previously identified. In this work we present several experimental findings that show that human astrovirus (HAstV) RNA encodes a VPg essential for viral infectivity: (i) RNase treatment of RNA purified from astrovirus-infected cells results in a single protein of 13 to 15 kDa, compatible with the predicted astrovirus VPg size; (ii) the antibody used to detect this 13- to 15-kDa protein is specifically directed against a region that includes the putative VPg coding region; (iii) the 13- to 15-kDa protein detected has been partially sequenced and the sequence obtained is contained in the computationally predicted VPg; (iv) the protein resulting from this putative VPg coding region is a highly disordered protein, resembling the VPg of sobemo-, calici- and potyviruses; (v) proteolytic treatment of the genomic RNA leads to loss of infectivity; and (vi) mutagenesis of Tyr-693 included in the putative VPg protein is lethal for HAstV replication, which strongly supports its functional role in the covalent link with the viral RNA.     

The Flagellar Basal Body-Associated Protein FlgT Is Essential for a Novel Ring Structure in the Sodium-Driven Vibrio Motor

In Vibrio alginolyticus, the flagellar motor can rotate at a remarkably high speed, ca. three to four times faster than the Escherichia coli or Salmonella motor. Here, we found a Vibrio-specific protein, FlgT, in the purified flagellar basal body fraction. Defects of FlgT resulted in partial Fla⁻ and Mot⁻ phenotypes, suggesting that FlgT is involved in formation of the flagellar structure and generating flagellar rotation. Electron microscopic observation of the basal body of ΔflgT cells revealed a smaller LP ring structure compared to the wild type, and most of the T ring was lost. His₆-tagged FlgT could be coisolated with MotY, the T-ring component, suggesting that FlgT may interact with the T ring composed of MotX and MotY. From these lines of evidence, we conclude that FlgT associates with the basal body and is responsible to form an outer ring of the LP ring, named the H ring, which can be distinguished from the LP ring formed by FlgH and FlgI. Vibrio-specific structures, e.g., the T ring and H ring might contribute the more robust motor structure compared to that of E. coli and Salmonella.The bacterial flagellar motor is a rotary nanomotor, which converts the electrochemical potential difference of the coupling ion (H⁺ or Na⁺) into rotational energy. Escherichia coli and Salmonella spp. have H⁺-driven motors, and Vibrio alginolyticus has Na⁺-driven motors. The rotation speed of the Vibrio motor is remarkably fast, 1,100 Hz on average and up to 1,700 Hz maximum, which is more than four times faster than that of the E. coli motor (24, 27).The flagellum is coordinately and hierarchically constructed from more than 30 related proteins and is composed of rotor, stator, universal joint (hook), and helical filament (22, 43). The rotor part (also called the basal body) contains several rings and a drive shaft, which are named the L, P, MS, and C rings and the rod (1, 14). The L, P, MS, and C rings are thought to be located in positions corresponding to the outer membrane, peptidoglycan layer, cytoplasmic membrane, and cytoplasm, respectively (Fig. ​(Fig.1).1). Because the LP ring is thought to be a bushing for rotation of the rod, the LP ring seems not to rotate. Analyses of the basal body components of Salmonella were carried out in detail, thereby identifying all of the gene products that are responsible for the substructures. The L, P and MS rings are composed of FlgH, FlgI, and FliF, respectively, while the C ring is composed of three different proteins, FliG, FliM, and FliN, and the rod is composed of FlgB, FlgC, FlgF, and FlgG (14, 17, 18, 39, 44).Open in a separate windowFIG. 1.Model of the flagellar basal body in Vibrio. The H ring and the T ring are shown in dark gray. The LP ring and the other basal body parts are shown in light gray. The PomA/B complex is shown in the medium gray. OM, outer membrane; PG, peptidoglycan layer; IM, inner membrane.The stator part is responsible for torque generation. The torque generation unit of the stator is composed of MotA and MotB in E. coli or PomA and PomB in Vibrio spp. and is a hexamer of four A subunits and two B subunits. They assemble around the rotor and transfer the coupling ions (H⁺ in E. coli and Na⁺ in Vibrio) across the membrane due to the electrochemical potential (2, 4, 11, 15, 37, 38, 40, 41). MotX and MotY are species-specific (e.g., Vibrio and Shewanella spp.) stator proteins, and defects in these proteins result in a mot phenotype in which flagellar morphogenesis is normal but the flagella cannot rotate (21, 30, 31, 33, 36). Pseudomonas spp. have only MotY but not MotX; MotY is required for flagellar rotation (12). In Vibrio alginolyticus it has been shown that MotX and MotY are produced as precursor proteins with signal sequences and are translocated to the periplasmic space by a general secretion pathway (35). MotX and MotY form a ring structure called the T ring in addition to the LP ring (Fig. ​(Fig.1).1). The N-terminal domain of MotY has been suggested to directly associate with the basal body, probably the P ring and MotX (23, 42), and MotX has been suggested to interact with PomB (34). Based on these lines of evidence, the T ring was proposed to be involved in the incorporation and/or stabilization of the PomA/B complex into the motor and provide a connection between the rotor and PomA/B in Vibrio (42).When flagellar basal bodies were purified from various species, the basic structures were similar but the details were different. When we compared the structures from Vibrio cells and E. coli cells, the Vibrio LP rings were bigger than those of E. coli (42). We speculated that additional proteins were present in the Vibrio LP rings. In the present study, we recognized a novel ring structure on the basal body of V. alginolyticus, and it was composed of the product of a recently identified motility gene, flgT. It was reported in that in Vibrio cholerae FlgT is somehow involved in motility and flagellar formation (9, 29). Furthermore, V. cholerae strains with defects in FlgT develop outer membrane blebbing and release the flagellum into the medium, suggesting that FlgT is involved in anchoring the flagellar base on the cell surface (29). We found that FlgT is necessary to form an outer ring of the LP ring, named the H ring (for holding ring of the flagellar base on the cell surface). The H ring is thought to be involved in assembly of MotX and MotY to the basal body.     

Microtubule-associated protein tau. Identification of a novel peptide from bovine brain

The microtubule-associated protein tau isolated from bovine brain was cleaved with CNBr and the 3 largest peptides of approx. 21, 19 and 18 kDa were obtained. Dephosphorylation of the CNBr digest of tau with alkaline phosphatase changed the electrophoretic mobility of these peptides to 19, 18 and 17 kDa. Amino acid sequencing of the total CNBr digest of tau revealed at least 3 sequences, two of which were highly homologous to previously published mouse and human tau sequences derived from cDNAs. A third amino acid sequence of 17 residues with heterogeneity at position 11 showed no homology with the cDNA-derived tau sequences. These studies suggest that the amino acid sequences of mammalian tau predicted from their cDNAs might be incomplete.     

人微管结合蛋白4微管结合区的体外表达

目的：构建表达人微管结合蛋白4微管结合区（Microtubule-binding domain,MTB）的大肠杆菌工程菌。方法：将1.27Kb编码MTB的DNA片段按正确方向亚克隆到原核生物表达载体pET-3α。得到表达质粒JS3,并转化大肠杆菌BL21（DE3）。结果：IPTG诱导下,表达产物的SDS-PAGE分子量大约是40kD,并能被抗MAP4抗体特异地识别。此外,重组MTB蛋白能在体外结合微管。结论：人微管结合蛋白4微管结合区具有微管结合活性。     

Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.     

UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.     

Protein Synthesis as a Requirement for Wound Xylem Differentiation

An increasing rate of protein synthesis was observed during the first 2 days after the isolation of 2 mm thick internodal stem slices of Coleus on a sucrose-agar medium. This rise in the rate of protein synthesis preceeded the first visible signs of wound-vessel member differentiation in the cultured stem slices. Irradiation of tissue slices with 4000 R of x-rays at isolation reduced the numbers of wound-vessel members differentiated after 7 days in culture by 51 per cent, and this level of x-irradiation was observed to inhibit protein synthesis by the cultured stem slices. Treatment of the tissue slices with exogenous auxin (0.05 mg/1) after irradiation did not alter the degree of inhibition of xylem differentiation. Actinomycin D inhibited wound-vessel member differentiation, but it had no effect on the endogenous growth of the cultured stem slices. Similarly, auxin at 0.05 mg/1 was without effect on the endogenous growth rate of the stem slices. Actinomycin D treatment was highly effective in inhibiting xylem differentiation if it was supplied to the tissues within the first 48 hours after isolation; actinomycin D treatment had no significant effect on xylem differentiation when it was given after the first 2 days of culture. Chloramphenicol (10^?3M) inhibited both xylem differentiation and the endogenous growth of the cultured stem slices.     

Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

Background

Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins.

Method

In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification.

Results

Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC).

Conclusions

LIDC is more effective for the prediction of essential proteins than other recently developed methods.     

Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.     

Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper,a Microtubule-associated Protein,and Dynein

Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.     

Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.Members of the family Baculoviridae are large rod-shaped enveloped viruses containing a circular double-stranded DNA genome that varies in size from 80 to 180 kb (3). Baculoviruses are unique viruses that only replicate in invertebrates. In general, isolates of each baculovirus species exhibit a narrow host range. For example, Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) is known to infect only the spruce budworm (Choristoneura fumiferana), but Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in hosts derived from several families of Lepidoptera (14). The restriction of baculovirus replication in nonpermissive hosts has been studied, and a number of genes, expressed at different points in the virus replication cycle, have been identified as playing some role in this restriction (40). Most of these identified genes are associated with viral DNA replication and late gene expression.Nine AcMNPV genes (ie-1, ie-2, p143, dnapol, lef-1, lef-2, lef-3, pe38, and p35) are required for directing transient replication of plasmids in transfected cells, suggesting that these genes are involved in baculovirus DNA replication (19, 27, 46). Only two of these genes, p143 and dnapol, have been shown to be essential for AcMNPV DNA replication in vivo (26, 41). Another gene, lef-11, although not essential for replication in transient assays, is also essential for DNA replication in vivo (24), indicating that questions concerning DNA replication need to be studied within the context of the whole virus genome.LEF-3 is a single-stranded DNA-binding protein (SSB) that self-localizes to the nucleus (15, 45). LEF-3 is also responsible for transporting P143, a predicted DNA unwinding (helicase) protein, into the nucleus, where it is required for viral DNA replication (26, 29, 45). LEF-3 may also regulate the activity of a viral alkaline nuclease (AN) during viral DNA replication (32). We have previously mapped the region carrying the nuclear localization signal of LEF-3 to residues 26 to 32 within the N-terminal 56-amino-acid domain (1, 7). By fusing this domain in frame with P143 and testing the construct in transient plasmid replication assays, we showed that additional functions of LEF-3 are required during replication, in addition to interacting with P143 to transport it into the nucleus. In fact, we have demonstrated that there is a close interaction between LEF-3 and P143 (as well as the immediate-early 1 [IE-1] protein) on viral DNA in the nucleus (17), suggesting that direct interaction of LEF-3 and P143 is required during viral DNA replication. The LEF-3 domain necessary for directing P143 to the nucleus is included within the N-terminal 125 amino acids (7). Two conserved cysteine residues in this region (C82 and C106) are not essential for this function, so it is unknown which specific amino acids are involved in the LEF-3-P143 interaction (1).In this study, a lef-3 knockout genome was constructed by exploiting a baculovirus shuttle vector (bacmid) system. Bacmids (a baculovirus genome carrying independent origins for replication in either bacteria or insect cells) were originally developed to prepare recombinant baculoviruses in Escherichia coli prior to transfection into insect cells (28). The system takes advantage of the site-specific transposition properties of the Tn7 transposon to simplify and enhance the process of generating recombinant bacmid DNA. In our case, we used the AcMNPV-derived bacmid as a template for deletion of the AcMNPV lef-3 gene and then examined the effect of this deletion on viral protein synthesis, budded virus (BV) production, and viral DNA replication. We also examined the ability of LEF-3 from another Alphabaculovirus species member, CfMNPV, to substitute for AcMNPV in a recombinant bacmid.     

Pex19p, a Farnesylated Protein Essential for Peroxisome Biogenesis

We report the identification and molecular characterization of Pex19p, an oleic acid-inducible, farnesylated protein of 39.7 kDa that is essential for peroxisome biogenesis in Saccharomyces cerevisiae. Cells lacking Pex19p are characterized by the absence of morphologically detectable peroxisomes and mislocalization of peroxisomal matrix proteins to the cytosol. The human HK33 gene product was identified as the putative human ortholog of Pex19p. Evidence is provided that farnesylation of Pex19p takes place at the cysteine of the C-terminal CKQQ amino acid sequence. Farnesylation of Pex19p was shown to be essential for the proper function of the protein in peroxisome biogenesis. Pex19p was shown to interact with Pex3p in vivo, and this interaction required farnesylation of Pex19p.     

Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

Background

Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia.

Methods

In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis.

Results

Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia.

Conclusion/Significance

This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers.