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. The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43-GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant. This allele-specific phenotype appears to be due to a G-R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G(1), no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes.     

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. We have developed an approach using atomic force microscopy imaging in liquid media, which allows us to study part of the motor in native liquid environment. In this work, we are interested in the FliG proteins, identified as the key functional proteins of this nano-machine. We report 3D images of their assembly on surfaces, which could be representative of the so-called M-ring part of the nano-motor. These images have been acquired on both mica surfaces and on supported bilayer membranes mimetics of E. coli native membrane. The systematic analysis of the shape and the size of different recorded assemblies made us believe that the FliG organization we observed could lead to a new model for the structure and mechanism of the flagellar nano-motor.     

Effect of ultrasonic treatment on enzyme activity and bioactives of strawberry puree

The aim of this study was to evaluate the impact of ultrasound (US) at different frequencies (20, 370, and 583 kHz) and power levels (35 and 48 W) on the residual activity (RA) of peroxidase (POD) and polyphenol oxidase (PPO) in strawberry puree. Total anthocyanin content (TAC), total phenolic content (TPC), ferric ion reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) were also assessed. Results were compared with untreated, thermally treated at 40 °C (control) and pasteurized (90 °C) strawberry puree. POD and PPO RA were significantly (P < 0.05) reduced, whilst there was a significant (P < 0.05) increase in TAC (5%–19%) in all US-treated samples in comparison with the untreated samples and the controls. US at 20 kHz (35 W) increased significantly (P < 0.05) TPC (9%) and FRAP (6%) in strawberry puree, whereas the effect of 583 kHz (48 W) on these parameters was insignificant (P > 0.05). Pasteurization inactivated POD and PPO, however, decreased dramatically TAC (14%), TPC (17%) and FRAP (9.5%) in strawberry puree. These findings suggest that US is a promising novel non-thermal food technology that can be tailored to improve the quality of strawberry puree by inactivating enzymes responsible for food deterioration whilst maintaining the content of bioactive compounds.     

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. This called for an in-depth structure-function analysis of the Knr4 protein, which is reported in the present paper. Computational analysis supported by biochemical and biophysical data characterize Knr4 as a newly identified member of the growing family of intrinsically disordered proteins. Despite disordered regions that are located at the N- and C-termini and are probably responsible for fine regulatory function; this protein contains a structured central core (amino acid residues 80-340) that is able to restore wild-type phenotypes of knr4Delta mutant in stress conditions. However, this fragment was unable to complement synthetic lethality between knr4 mutations and deletions of genes encoding protein kinases of the PKC1-dependent pathway. For these crucial events to occur, the presence of the N-terminal part of Knr4 protein is indispensable. Moreover, we demonstrate that this protein is essential for cell viability in the absence of a functional Pkc1-Slt2 pathway, since the lethality caused by KNR4 deletion in such a genetic background could not be compensated by overexpression of any gene from yeast genomic libraries.