Crystallographic studies of the structured core domain of Knr4 from Saccharomyces cerevisiae.

The potentially structured core domain of the intrinsically disordered protein Knr4 from Saccharomyces cerevisiae, comprising residues 80-340, was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Selenomethionine-containing (SeMet) protein was also purified and crystallized. Crystals of both proteins belonged to space group P6522, with unit-cell parameters a = b = 112.

Finding undetected protein associations in cell signaling by belief propagation.

External information propagates in the cell mainly through signaling cascades and transcriptional activation, allowing it to react to a wide spectrum of environmental changes. High-throughput experiments identify numerous molecular components of such cascades that may, however, interact through unknown partners. Some of them may be detected using data coming from the integration of a protein-protein interaction network and mRNA expression profiles.

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth.

The Saccharomyces cerevisiae protein Knr4 is composed of a globular central core flanked by two natively disordered regions. Although the central part of the protein holds most of its biological function, the N-terminal domain (amino acids 1-80) is essential in the absence of a functional CWI pathway. We show that this specific protein domain is required for the proper cellular localization of Knr4 at sites of polarized growth during vegetative growth and sexual differentiation (bud tip and 'shmoo' tip).

Functional dissection of an intrinsically disordered protein: understanding the roles of different domains of Knr4 protein in protein-protein interactions.

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N-terminal and large natively unfolded C-terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4.

Contribution to the elucidation of the structure of the bacterial flagellum nano-motor through AFM imaging of the M-Ring.

The flagellar nano-motor of bacteria is one of the most interesting and amazing natural nano-machine. Despite its discovery 30 years ago, some details of its structure and mechanisms are not yet elucidated. Several studies have revealed some important aspects of its structure and numerous data are available today; however, the inner mechanisms of the nano-motor have not been yet resolved, partially due to the lack of information about the 3D assembly, shape and interactions of the different parts in experimental environment as close as possible as the native cellular conditions.

Structure-function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1-SLT2 pathway in Saccharomyces cerevisiae.

The coordination between cell wall synthesis and cell growth in the yeast Saccharomyces cerevisiae implicates the PKC1-dependent MAP kinase pathway. KNR4, encoding a 505 amino acid long protein, participates in this coordination, since it displays synthetic lethality with all the members of the PKC1 pathway and shows physical interaction with Slt2/Mpk1. The recent finding that KNR4 interacts genetically or physically with more than 100 partners implicated in different cellular processes raised the question of how these interactions may occur and their physiological significance.

The 'interactome' of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae.

The integrity of the Saccharomyces cerevisiae cell wall requires a functional Pkc1-Slt2 MAP kinase pathway that contributes to transient growth arrest, enabling coordination of cell division with cell wall remodelling. How this coordination takes place is still an open question. Recently, we brought evidence that Knr4 protein, whose absence leads to several cell wall defects, may play a role in this function.

Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells.

Of the 13 two-component signal transduction systems (TCS) identified in Streptococcus pneumoniae, two, ComDE and CiaRH, are known to affect competence for natural genetic transformation. ComD and ComE act together with the comC-encoded competence-stimulating peptide (CSP) and with ComAB, the CSP-dedicated exporter, to co-ordinate activation of genes required for differentiation to competence. Several lines of evidence suggest that the CiaRH TCS and competence regulation are interconnected, including the observation that inactivation of the CiaR response regulator derepresses competence.

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis.

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery.

The interaction of Slt2 MAP kinase with Knr4 is necessary for signalling through the cell wall integrity pathway in Saccharomyces cerevisiae.

In budding yeast, PKC1 plays an essential role in cell integrity and proliferation through a linear MAP (Mitogen Activated Protein) kinase phosphorylation cascade, which ends up with the activation of the Slt2-MAP kinase by dual phosphorylation on two conserved threonine and tyrosine residues. In this phosphorylated form, Slt2p kinase activates by phosphorylation at least two known downstream targets: Rlm1p, which is implicated in the expression of cell wall-related genes, and SBF, required for transcription activation of cell cycle-regulated genes at the G1 to S transition. In this paper, we demonstrate by two-hybrid, in vitro immunoprecipitation and tandem affinity purification (TAP) methods that Knr4p physically interacts with Slt2p.

KNR4 is a member of the PKC1 signalling pathway and genetically interacts with BCK2, a gene involved in cell cycle progression in Saccharomyces cerevisiae.

In budding yeast, PKC1 plays an essential role in cell wall integrity and cell proliferation through a bifurcated PKC1/mitogen-activated protein (MAP) kinase pathway. The evidence that KNR4 is a member of the PKC1 pathway and genetically interacts with BCK2, a gene involved together with Cln3-Cdc28 in the G1 to S transition phase of the cell cycle, was as follows. Both KNR4 and BCK2 were isolated as a dosage suppressor of a calcofluor white hypersensitive ( cwh43) mutant.

Involvement of GFA1, which encodes glutamine-fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae.

Cell-wall damage caused by mutations of cell-wall-related genes triggers a compensatory mechanism which eventually results in hyperaccumulation of chitin reaching 20% of the cell-wall dry mass. We show that activation of chitin synthesis is accompanied by a rise, from 1.3-fold to 3.

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway.

The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a growth defect and increased release of beta-1,6-glucan and beta-glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster.

Interaction of Knr4 protein, a protein involved in cell wall synthesis, with tyrosine tRNA synthetase encoded by TYS1 in Saccharomyces cerevisiae.

The Knr4 protein, known to be involved in the regulation of cell wall assembly in Saccharomyces cerevisiae, strongly interacts with the tyrosine tRNA synthetase protein encoded by TYS1 as demonstrated by the genetic two-hybrid system and a biochemical pull-down experiment using GST--Tys1p fusion. Data reported here raise the possibility that this physical interaction between these proteins is required for dityrosine formation during the sporulation process. In addition, it is shown that the efficiency of spores formation was drastically reduced in diploid cells homozygous for the disruption of KNR4 or for a temperature-sensitive mutation of TYS1, although this effect could be independent of their protein interaction.

KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes.

The KNR4 gene, originally isolated by complementation of a K9 killer-toxin-resistant mutant displaying reduced levels of both 1,3-beta-glucan and 1,3-beta-glucan synthase activity, was recloned from a YCp50 genomic library as a suppressor of Saccharomyces cerevisiae calcofluor-white-hypersensitive (cwh) mutants. In these mutants, which were characterized by increased chitin levels, the suppressor effect of KNR4 resulted, for some of them, in a lowering of polymer content to close to wild-type level, with no effect on the contents of beta-glucan and mannan. In all cases, this effect was accompanied by a strong reduction in mRNA levels corresponding to CHS1, CHS2 and CHS3, encoding chitin synthases, without affecting expression of FKS1 and RHO1, two genes encoding the catalytic subunit and a regulatory component of 1,3-beta-glucan synthase, respectively.