The molecular dissection of the chaperone–usher pathway

Secretion systems are specialized in transport of proteins, DNA or nutrients across the cell envelope of bacteria and enable them to communicate with their environment. The chaperone–usher (CU) pathway is used for assembly and secretion of a large family of long adhesive protein polymers, termed pili, and is widespread among Gram-negative pathogens [1]. Moreover, recent evidence has indicated that CU secretion systems are also involved in sporulation  and . In this review we focus on the structural biology of the paradigmatic type 1 and P pili CU systems encoded by uropathogenic Escherichia coli (UPEC), where recent progress has provided unprecedented insights into pilus assembly and secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.     

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.     

Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C‐terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β‐turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X‐ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre‐organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild‐type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide.     

Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.