SummaryGrowth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/ LpoB and their PBP docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.
SummaryThe murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylasetranspeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.
We describe a method to label gamma-aminobutyric acid (GABA)A receptors on the surface of living hippocampal neurons in primary culture, and we compare the distribution of receptors with that of active synapses. To visualize GABAA receptors, the affinity-purified antibody beta3(1-13), recognizing the extracellular N-termini of the GABAA receptor beta2- and beta3-subunits, was used in combination with fluorescent secondary antibodies. The beta2- and beta3-subunits belong to the predominant GABAA receptor subunits in the hippocampus. As expected for aggregates of GABAA receptors in the somato-dendritic plasma membrane, a patchy staining pattern similar to that seen by labelling neurons after fixation was obtained. An antiserum recognizing an intracellular epitope of GABAA receptor beta3-subunits did not label the receptors in living neurons. Whole-cell recordings of GABA-evoked Cl - currents were not affected after decorating GABAA receptors with antibody beta3(1-13). Combining the staining of GABAA receptors with the labelling of active presynaptic terminals with the fluorescent dyes FM1-43 or FM4-64, consistently resulted in the detection of GABAA receptor clusters that were not located at active synapses. These amounted to approximately 50% of all labelled GABAA receptor clusters. GABAA receptor clusters that were not associated with active presynaptic terminals partially colocalized with the synaptic vesicle marker protein sv2, while another fraction had no presynaptic counterpart at all. These findings suggest the presence of presynaptically silent GABAergic synapses in cultured hippocampal neurons. They also indicate that for the maintenance of GABAA receptor aggregates, the release of GABA from an opposing active terminal is not essential.
Abstract:In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABAA receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABAA receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABAA receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the al-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with alsubunit-containing GABAA receptors. Results of a biochemical characterization of these GABAA receptor-tubulin complex-associated proteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.
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