RESUMO
FtsZ polymerizes in a ring-like structure at mid cell to initiate cell division in Escherichia coli. The ring is stabilized by a number of proteins among which the widely conserved ZapA protein. Using antibodies against ZapA, we found surprisingly that the cellular concentration of ZapA is approximately equal to that of FtsZ. This raised the question of how the cell can prevent their interaction and thereby the premature stabilization of FtsZ protofilaments in nondividing cells. Therefore, we studied the FtsZ-ZapA interaction at the physiological pH of 7.5 instead of pH 6.5 (the optimal pH for FtsZ polymerization), under conditions that stimulate protofilament formation (5 mM MgCl(2)) and under conditions that stimulate and stabilize protofilaments (10 mM MgCl(2)). Using pelleting, light scattering, and GTPase assays, it was found that stabilization and bundling of FtsZ polymers by ZapA was inversely correlated to the GTPase activity of FtsZ. As GTP hydrolysis is the rate-limiting factor for depolymerization of FtsZ, we propose that ZapA will only enhance the cooperativity of polymer association during the transition from helical filament to mid cell ring and will not stabilize the short single protofilaments in the cytoplasm. All thus far published in vitro data on the interaction between FtsZ and ZapA have been obtained with His-ZapA. We found that in our case the presence of a His tag fused to ZapA prevented the protein to complement a DeltazapA strain in vivo and that it affected the interaction between FtsZ and ZapA in vitro.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biopolímeros/química , Biopolímeros/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Tamanho Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/ultraestrutura , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Histidina/química , Histidina/genética , Concentração de Íons de Hidrogênio , Luz , Cloreto de Magnésio/química , Cloreto de Magnésio/metabolismo , Cloreto de Magnésio/farmacologia , Oligopeptídeos/química , Oligopeptídeos/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento de Radiação , UltracentrifugaçãoRESUMO
In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue [125I]TID-PC or the small hydrophobic probe [125I]TID-BE. The most prominent difference was the very specific labeling of a 70 kDa protein by [125I]TID-PC. Mass spectrometric analysis of a tryptic digest of the corresponding 2D-gel spot identified the protein as the GUT2 gene product, the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate carrier (P(i)C) in the inner membrane. A hemagglutinin-tagged version of Gut2p was shown to be membrane-bound. Carbonate extraction released the protein from the membrane, whereas a high concentration of NaCl did not, demonstrating that Gut2p is a peripheral membrane protein bound to the inner membrane via hydrophobic interactions. The significance of the observed interactions between Gut2p and PC is discussed.