The GTPase Der is universally conserved in bacteria and is structurally unique as it consists of two GTP-binding domains in tandem (G-domain 1 and G-domain 2) whereas all the other GTPases posses a single GTPase domain. In order to assess the function of Der we have fractionated whole cell lysates containing over expressed Der. This analysis indicated that Der was present in sucrose gradient fractions containing membrane proteins. The interaction with the membrane fraction was specific for Der, since the related GTPase, Era, did not form the membrane complex. In addition, three independent criteria suggested a high affinity interaction; (1) the interaction can be detected under partially denaturing conditions using a gel electrophoresis co-migration assay, (2) the interaction survived 16 h sucrose gradient centrifugation, and (3) the complex could be efficiently reconstituted from purified components. Microscopic examination of cells containing over expressed Der showed that the cell wall structure was disrupted at both cell poles. This phenotype required Der domain three since domain deletion mutations showed no affect on cell wall structure. Surprisingly point mutations that ablate nucleotide binding of either GTP binding domain result in a defect in cell wall structure at only a single cell pole. The data reported here were considered together with results presented previously to suggest that Der may engage in a functional cyclic interaction between ribosomes and the membrane in Escherichia coli.
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