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BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess the GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.

Several data analyses are conducted to explain the contribution of the domains of the translational GTPase BipA in determining the biological properties of the protein. The GTPase activities are shown to be highly related to the ribosome binding of the protein.

BipA is a multi-domain prokaryotic GTPase that associates with the ribosome. Although the exact cellular function of this protein remains elusive, BipA homologs have been implicated in a number of cellular processes including expression of virulence genes, low temperature growth, flagella mediated motility, and other cellular processes associated with stress response. We are utilizing biochemical and biophysical techniques to study the Salmonella typhimurium homolog of BipA in order to better understand its cellular function. Using sucrose density gradients and pelleting assays we have shown that BipA interacts with the ribosome in vivo, and that the GTP bound state of the protein associates with the ribosome in vitro. We have also determined that association with 70S ribosomes significantly stimulates the GTPase activity of the protein. The C-terminal region of the protein has been found to mediate the stimulatory effect of the ribosome on BipA hydrolysis. As is the case with other multidomain switch proteins, intramolecular interactions play a role in regulating the GTPase activities of BipA. We have also employed competition assays with antibiotics to identify the general location on the ribosome occupied by BipA. Together, these studies provide us with some initial details as to the nature of the BipA-ribosome interaction. [ABSTRACT FROM AUTHOR]

Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion>95 correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response.