Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi

In the model organism Escherichia coli, the coupling protein CheW, which bridges the chemoreceptors and histidine kinase CheA, is essential for chemotaxis. Unlike the situation in E. coli, Borrelia burgdorferi, the causative agent of Lyme disease, has three cheW homologues (cheW₁, cheW₂ and cheW₃). Here, a comprehensive approach is utilized to investigate the roles of the three cheWs in chemotaxis of B. burgdorferi. First, genetic studies indicated that both the cheW₁ and cheW₃ genes are essential for chemotaxis, as the mutants had altered swimming behaviours and were non‐chemotactic. Second, immunofluorescence and cryo‐electron tomography studies suggested that both CheW₁ and CheW₃ are involved in the assembly of chemoreceptor arrays at the cell poles. In contrast to cheW₁ and cheW₃, cheW₂ is dispensable for chemotaxis and assembly of the chemoreceptor arrays. Finally, immunoprecipitation studies demonstrated that the three CheWs interact with different CheAs: CheW₁ and CheW₃ interact with CheA₂ whereas CheW₂ binds to CheA₁. Collectively, our results indicate that CheW₁ and CheW₃ are incorporated into one chemosensory pathway that is essential for B. burgdorferi chemotaxis. Although many bacteria have more than one homologue of CheW, to our knowledge, this report provides the first experimental evidence that two CheW proteins coexist in one chemosensory pathway and that both are essential for chemotaxis.     

Three heat shock proteins are essential for rotifer thermotolerance

Heat shock proteins (HSPs) are important molecules in the stress response of organisms from prokaryotes to mammals, and thus may be useful biomarkers for environmental stress. Here we characterize the functional roles of genes belonging to four distinct families of HSPs (hsp40, hsp60, hsp70, and hsp90) in the monogonont rotifer Brachionus manjavacas. Because B. manjavacas inhabits ponds of varying thermal regimes, including ephemeral ponds that may experience temperature fluctuations, HSP-mediated thermotolerance likely is important to its survival and adaptation. Using interference RNA (RNAi), we provide the first conclusive evidence that HSPs are required for rotifer survival following heat stress. Effective RNAi-mediated suppression of all hsp genes except hsp90 was verified via quantitative PCR. Hsp40, hsp60, and hsp70 are required for rotifer thermotolerance (P < 0.05); however, our data do not indicate hsp90 is essential. Quantitative PCR further revealed immediate up-regulation of hsp40 mRNA following heat stress. Additionally, we demonstrated expression of hsp40 mRNA in multiple tissues using fluorescent in situ hybridization. Our characterization of mRNA expression and functional roles for four distinct hsp genes provides a baseline for molecular-level comparisons of the stress response of rotifers with other taxonomic groups, and the technique for in-depth studies of the role of specific genes in rotifer stress responses. Considering the potential for ambient temperatures to impact species survival, competitive interactions, and body size of individuals, thermotolerance may be an important influence on zooplankton community structure.     

Bcl-2 family proteins are essential for platelet survival

Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.     

Two trypanosome-specific proteins are essential factors for 5S rRNA abundance and ribosomal assembly in Trypanosoma brucei

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-amino-acid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a higher-molecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.     

Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture

Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases that catalyze the synthesis of glucan and the nonglucosyltransferase glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest that these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties were quantified by the biofilm quantification software comstat. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.     

RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis

When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli.     

Stem-forming regions that are essential for the amyloidogenesis of prion proteins

Prion diseases represent fatal neurodegenerative disorders caused by the aggregation of prion proteins. With regard to the formation of the amyloidogenic cross-β-structure, the initial mechanism in the conversion to a β-structure is critically important. To explore the core regions forming a stem of the amyloid, we designed and prepared a series of peptides comprised of two native sequences linked by a turn-inducing dipeptide moiety and examined their ability to produce amyloids. A sequence alignment of the peptides bearing the ability to form amyloid structures revealed that paired strands consisting of VNITI (residues 180-184) and VTTTT (residues 189-193) are the core regions responsible for initiating the formation of cross-β-structures and for further ordered aggregation. In addition, most of the causative mutations responsible for inherited prion diseases were found to be located in these stem-forming regions on helix H2 and their counterpart on helix H3. Moreover, the volume effect of the nonstem domain, which contains ~200 residues, was deduced to be a determinant of the nature of the association such as oligomerization, because the stem-forming domain is only a small part of a prion protein. Taken together, we conclude that the mechanism underlying the initial stage of amyloidogenesis is the exposure of a newly formed intramolecular β-sheet to a solvent through the partial transition of a native structure from an α-helix to a β-structure. Our results also demonstrate that prion diseases caused by major prion proteins except the prions of some fungi such as yeast are inherent only in mammals, as evidenced by a comparison of the corresponding sequences to the stem-forming regions among different animals.     

Arabidopsis stromal 70-kDa heat shock proteins are essential for chloroplast development

70 kDa heat shock proteins (Hsp70s) act as molecular chaperones involved in essential cellular processes such as protein folding and protein transport across membranes. They also play a role in the cell’s response to a wide range of stress conditions. The Arabidopsis family of Hsp70s homologues includes two highly conserved proteins, cpHsc70-1 and cpHsc70-2 which are both imported into chloroplasts (Su and Li in Plant Physiol 146:1231–1241, 2008). Here, we demonstrate that YFP-fusion proteins of both cpHsc70-1 and cpHsc70-2 are predominantly stromal, though low levels were detected in the thylakoid membrane. Both genes are ubiquitously expressed at high levels in both seedlings and adult plants. We further show that both cpHsc70-1 and cpHsc70-2 harbour ATPase activity which is essential for Hsp70 chaperone activity. A previously described T-DNA insertion line for cpHsc70-1 (ΔcpHsc70-1) has variegated cotyledons, malformed leaves, growth retardation, impaired root growth and sensitivity to heat shock treatment. In addition, under stress conditions, this mutant also exhibits unusual sepals, and malformed flowers and sucrose concentrations as low as 1% significantly impair growth. cpHsc70-1/cpHsc70-2 double-mutants are lethal. However, we demonstrate through co-suppression and artificial microRNA (amiRNA) approaches that transgenic plants with severely reduced levels of both genes have a white and stunted phenotype. Interestingly, chloroplasts in these plants have an unusual morphology and contain few or no thylakoid membranes. Our data show that cpHsc70-1 and cpHsc70-2 are essential ATPases, have overlapping roles and are required for normal plastid structure.     

Two histidine residues are essential for catalysis by lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) is a novel membrane bound enzyme that catalyzes the formation of retinyl esters from vitamin A and lecithin. The enzyme is both essential for vision and for the general mobilization of vitamin A. The sequence of LRAT defines it as a novel enzyme unrelated to any other protein of known function. LRAT possesses a catalytically essential active site cysteine residue. The enzyme also contains six histidine residues. It is shown here that two of these residues (H57 and H163) are essential for catalysis. A mechanistic hypothesis is presented to account for these observations.     

Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.     

Palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity

Coronavirus spike (S) proteins are palmitoylated at several cysteine residues clustered near their transmembrane-spanning domains. This is achieved by cellular palmitoyl acyltransferases (PATs), which can modify newly synthesized S proteins before they are assembled into virion envelopes at the intermediate compartment of the exocytic pathway. To address the importance of these fatty acylations to coronavirus infection, we exposed infected cells to 2-bromopalmitate (2-BP), a specific PAT inhibitor. 2-BP profoundly reduced the specific infectivities of murine coronaviruses at very low, nontoxic doses that were inert to alphavirus and rhabdovirus infections. 2-BP effected only two- to fivefold reductions in S palmitoylation, yet this correlated with reduced S complexing with virion membrane (M) proteins and consequent exclusion of S from virions. At defined 2-BP doses, underpalmitoylated S proteins instead trafficked to infected cell surfaces and elicited cell-cell membrane fusions, suggesting that the acyl chain adducts are more critical to virion assembly than to S-induced syncytial developments. These studies involving pharmacologic inhibition of S protein palmitoylation were complemented with molecular genetic analyses in which cysteine acylation substrates were mutated. Notably, some mutations (C1347F and C1348S) did not interfere with S incorporation into virions, indicating that only a subset of the cysteine-rich region provides the essential S-assembly functions. However, the C1347F/C1348S mutant viruses exhibited relatively low specific infectivities, similar to virions secreted from 2-BP-treated cultures. Our collective results indicate that the palmitate adducts on coronavirus S proteins are necessary in assembly and also in positioning the assembled envelope proteins for maximal infectivity.     

Two distinct domains in pro-region of Nodal-related 3 are essential for BMP inhibition

The transforming growth factor-beta (TGF-beta) superfamily member, Xenopus nodal-related 3 (Xnr3), induces neural tissues through inhibition of bone morphogenetic proteins (BMPs). We recently identified an inhibitory mechanism in which the pro-region of Xenopus tropicalis nodal-related 3 (Xtnr3) physically interacts with BMP ligands. Here, we show that disulfide-linked heterodimerization does not contribute to BMP inhibition by Xtnr3 and that the Xtnr3 mature region, overexpression of which can induce the same phenotype as full-length Xtnr3, does not inhibit BMP signaling. Furthermore, we find that the BMP-inhibitory domains of Xtnr3 are separately located in the N- and C-terminal regions of the pro-region. These results indicate the pro-region of Nodal-related 3 is both necessary and sufficient for its BMP inhibition.     

Cellular retinoic acid-binding proteins are essential for hindbrain patterning and signal robustness in zebrafish

The vitamin A derivative retinoic acid (RA) is a morphogen that patterns the anterior-posterior axis of the vertebrate hindbrain. Cellular retinoic acid-binding proteins (Crabps) transport RA within cells to both its nuclear receptors (RARs) and degrading enzymes (Cyp26s). However, mice lacking Crabps are viable, suggesting that Crabp functions are redundant with those of other fatty acid-binding proteins. Here we show that Crabps in zebrafish are essential for posterior patterning of the hindbrain and that they provide a key feedback mechanism that makes signaling robust as they are able to compensate for changes in RA production. Of the four zebrafish Crabps, Crabp2a is uniquely RA inducible and depletion or overexpression of Crabp2a makes embryos hypersensitive to exogenous RA. Computational models confirm that Crabp2a improves robustness within a narrow concentration range that optimizes a 'robustness index', integrating spatial information along the RA morphogen gradient. Exploration of signaling parameters in our models suggests that the ability of Crabp2a to transport RA to Cyp26 enzymes for degradation is a major factor in promoting robustness. These results demonstrate a previously unrecognized requirement for Crabps in RA signaling and hindbrain development, as well as a novel mechanism for stabilizing morphogen gradients despite genetic or environmental fluctuations in morphogen availability.     

Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin

Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein–TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.     

QQT proteins colocalize with microtubules and are essential for early embryo development in Arabidopsis

During Arabidopsis embryogenesis, the control of division between daughter cells is critical for pattern formation. Two embryo-defective (emb) mutant lines named quatre-quart (qqt) were characterized by forward and reverse genetics. The terminal arrest of qqt1 and qqt2 embryos was at the octant stage, just prior to the round of periclinal divisions that establishes the dermatogen stage . Homozygous embryos of a weaker allele of qqt1 were able to divide further, resulting in aberrant periclinal divisions. These phenotypic analyses support an essential role of the QQT proteins in the correct formation of the tangential divisions. That an important proportion of qqt1 embryos were arrested prior to the octant stage indicated a more general role in cell division. The analysis of QQT1 and QQT2 genes revealed that they belong to a small subgroup of the large family encoding ATP/GTP binding proteins, and are widely conserved among plants, vertebrates and Archaea. We showed that QQT1 and QQT2 proteins interact with each other in a yeast two-hybrid system, and that QQT1 and QQT2 tagged by distinct fluorescent probes colocalize with microtubules during mitosis, in agreement with their potential role in cell division and their mutant phenotype. We propose that QQT1 and QQT2 proteins participate in the organization of microtubules during cell division, and that this function is essential for the correct development of the early embryo.     

Two newly identified sites in the ubiquitin-like protein Atg8 are essential for autophagy

Atg8, a member of a novel ubiquitin-like protein family, is an essential component of the autophagic machinery in yeast. This protein undergoes reversible conjugation to phosphatidylethanolamine through a multistep process in which cleavage of Atg8 by a specific protease is followed by ubiquitin-like conjugation processes. Here, we identify two essential sites in Atg8, one of them involving residues Phe 77 and Phe 79 and the other, located on the opposite surface of Atg8, residues Tyr 49 and Leu 50. We show that these two sites are associated with different functions of Atg8: Phe 77 and Phe 79 seem to be part of the recognition site for Atg4, a cystein protease that acts also as a deubiquitination enzyme, whereas Tyr 49 and Leu 50 act downstream of the lipidation step. These two newly identified distinct sites that are essential for Atg8 activity provide an explanation for the many protein-protein interactions of this low-molecular-weight protein.     

CAPS-1 and CAPS-2 are essential synaptic vesicle priming proteins

Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca(2+) levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca(2+) triggered release of transmitters.