Differences in the polar clustering of the high- and low-abundance chemoreceptors of
            <i>Escherichia coli</i>

Lybarger, Suzanne R.; Maddock, Janine R.

doi:10.1073/pnas.130195397

Cited by 47 publications

(53 citation statements)

References 30 publications

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“…It is likely that the pentapeptide motif is required for maintaining an appropriate level of receptor methylation by increasing the local concentration of CheR around the receptor cluster in the cell. This interpretation is consistent with the following facts : (i) CheR is much less abundant than the receptor molecules (Simms et al, 1987) ; (ii) the overproduction of CheR suppresses the defect of the mutations in the pentapeptide sequence (Okumura et al, 1998 ;Shiomi et al, 2000) ; (iii) the receptor molecules form a cluster with CheW and CheA at a cell pole (Maddock & Shapiro, 1993 ;Lybarger & Maddock, 2000) ; (iv) a receptor dimer with the CheRbinding sequence can help methylation of a neighbouring receptor dimer without the CheR-binding sequence (Le Moual & Koshland, 1997 ;Li et al, 1997) ; (v) GFP-CheR localized to cell poles in the presence, but not in the absence, of a chemoreceptor with the NWETF sequence (Shiomi et al, 2002). Okumura et al (1998) showed that cells expressing mutant Tcp (NWESLA) respond to, but do not adapt to the repellent glycerol.…”

Section: Discussionsupporting

confidence: 74%

Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase

2002

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In the chemotaxis of Escherichia coli, receptor methylation is the key process of adaptation. The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors. The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheRbinding ability. In this study, six independent intragenic suppressors of the mutation were isolated. They were divided into two classes. Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA. These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate. Some other CheAactivating mutations were also found to serve as class I suppressors. These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor. The class II suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif. This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar. This effect was reversed by introducing a mutation in the xWxxF motif. These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus.

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Section: Discussionsupporting

confidence: 74%

Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase

2002

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“…This could indicate that the polar accumulation is functionally relevant and that it supports proper function of the two sensor kinases in an unknown way. Clusters of chemotaxis sensors together with the sensor-related proteins CheA and CheW are well documented (Lybarger & Maddock, 2000). The clusters are believed to support stimulus integration and the sensitivity of the sensors (Sourjik, 2004;Thiem et al, 2007).…”

Section: Comparison Of Dcus Accumulation With That Of Other Localizedmentioning

confidence: 99%

“…The localization of these histidine kinases within the cell often relates to their site of function, and typically features a polar localization. The chemoreceptors of chemotaxis (Lybarger & Maddock, 2000) show also an asymmetric distribution within E. coli cells, although no asymmetric distribution is expected for chemotaxis regulation. A uniform distribution over the cell membrane is assumed for kinases controlling metabolic processes or perceiving stimuli that are evenly distributed over the cell.…”

Section: Introductionmentioning

confidence: 99%

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Polar accumulation of the metabolic sensory histidine kinases DcuS and CitA in Escherichia coli

et al. 2008

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Signal transduction in prokaryotes is frequently accomplished by two-component regulatory systems in which a histidine protein kinase is the sensory component. Many of these sensory kinases control metabolic processes that do not show an obvious requirement for inhomogeneous distribution within bacterial cells. Here, the sensory kinases DcuS and CitA, two histidine kinases of Escherichia coli, were investigated. Both are membrane-integral and involved in the regulation of carboxylate metabolism. The two-component sensors were fused with yellow fluorescent protein (YFP) and live images of immobilized cells were obtained by confocal laser fluorescence microscopy. The fluorescence of the fusion proteins was concentrated at the poles of the cells, indicating polar accumulation of the sensory kinases. For quantitative evaluation, line profiles of the imaged fluorescence intensities were generated; these revealed that the fluorescence intensity of the polar bright spots was 2.3-8.5 times higher than that of the cytoplasm. With respect to the cylindrical part of the membrane, the values were lower by about 40 %. The polar accumulation was comparable to that of methyl-accepting chemotaxis proteins (MCPs) and MCP-related proteins. The degree of DcuS-YFP localization was independent of the presence of MCP and the expression level of dcuS-yfp (or DcuS concentration). The presence of effector (fumarate or citrate, respectively) increased the polar accumulation by more than 20 %. Cell fractionation demonstrated that polar accumulation was not related to inclusion body formation. Therefore, sensory kinases DcuS and CitA, which regulate metabolic processes without obvious polar function, exhibit polar accumulation.

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“…Wild-type, sec7, and sec12 cells expressing HA-Atg8 under the control of the native ATG8 promoter were grown in rich medium at 24°C and then shifted to 37°C in either the same medium or in SD-N to induce autophagy for 2 h. Cells were then fixed with formaldehyde and glutaraldehyde, treated with sodium periodate, dehydrated with ethanol, and finally embedded with LR White resin (Ted Pella, Redding, CA) as described previously (Lybarger and Maddock, 2000). After resin polymerization, 70-to 80-nm sections were mounted on nickel grids, incubated first with anti-HA antibody (1:25 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and successively with 12-nm colloidal gold-Affinipure goat anti-mouse IgG (1:30 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA).…”

Section: Immunoelectron Microscopymentioning

confidence: 99%

Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly inSaccharomyces cerevisiae

Reggiori

Wang

Nair

et al. 2004

MBoC

129

163

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The Cvt pathway is a biosynthetic transport route for a distinct subset of resident yeast vacuolar hydrolases, whereas macroautophagy is a nonspecific degradative mechanism that allows cell survival during starvation. Yet, these two vacuolar trafficking pathways share a number of identical molecular components and are morphologically very similar. For example, one of the hallmarks of both pathways is the formation of double-membrane cytosolic vesicles that sequester cargo before vacuolar delivery. The origin of the vesicle membrane has been controversial and various lines of evidence have implicated essentially all compartments of the endomembrane system. Despite the analogies between the Cvt pathway and autophagy, earlier work has suggested that the origin of the engulfing vesicle membranes is different; the endoplasmic reticulum is proposed to be required only for autophagy. In contrast, in this study we demonstrate that the endoplasmic reticulum and/or Golgi complex, but not endosomal compartments, play an important role for both yeast transport routes. Along these lines, we demonstrate that Berkeley bodies, a structure generated from the Golgi complex in sec7 cells, are immunolabeled with Atg8, a structural component of autophagosomes. Finally, we also show that none of the yeast t-SNAREs are located at the preautophagosomal structure, the presumed site of double-membrane vesicle formation. Based on our results, we propose two models for Cvt vesicle biogenesis. INTRODUCTIONThe lysosome/vacuole is the major cellular center for degradation, recycling, and storage of biological constituents. Several well-characterized transport routes are implicated in protein delivery to this organelle during normal growth conditions. These pathways are conserved among eukaryotic organisms, but work in the yeast Saccharomyces cerevisiae has had a major impact on their characterization and in the identification of the molecular machinery (Conibear and Stevens, 1998). The route called the alkaline phosphatase pathway provides a direct transport connection between the Golgi complex and the vacuole Piper et al., 1997). A second itinerary from the Golgi complex to the vacuole passes through the late endosome and is known as the carboxypeptidase Y pathway (Piper et al., 1995;Babst et al., 1998;Conibear and Stevens, 1998). These two pathways are required for the biogenesis and maintenance of vacuoles, whereas a third route, endocytosis, has a catabolic function. Endocytosis mediates the downregulation of plasma membrane proteins by delivering them via the late endosome to the vacuole for degradation (Hicke, 1999;Katzmann et al., 2002). Before reaching the late endosome, these proteins pass through the early endosome where some components are recycled back to the cell surface (Hicke et al., 1997;Prescianotto-Baschong and Riezman, 1998;Lewis et al., 2000). The carboxypeptidase Y pathway and endocytosis converge at the endosome where another route, the multivesicular body pathway, delivers both resident enzymes and substrates to the vac...

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Differences in the polar clustering of the high- and low-abundance chemoreceptors of Escherichia coli

Cited by 47 publications

References 30 publications

Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase

Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase

Polar accumulation of the metabolic sensory histidine kinases DcuS and CitA in Escherichia coli

Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly inSaccharomyces cerevisiae

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