FtsZ filament dynamics at steady state: subunit exchange with and without nucleotide hydrolysis.
We have measured three aspects of FtsZ filament dynamics at steady state: rates of GTP hydrolysis, subunit exchange between protofilaments, and disassembly induced by dilution or excess GDP. All three reactions were slowed with an increase in the potassium concentration from 100 to 500 mM, via replacement of potassium with rubidium, or with an increase in the magnesium concentration from 5 to 20 mM. Electron microscopy showed that the polymers assembled under the conditions of fastest assembly were predominantly short, one-stranded protofilaments, whereas under conditions of slower dynamics, the protofilaments tended to associate into long, thin bundles. We suggest that exchange of subunits between protofilaments at steady state involves two separate mechanisms: (1) fragmentation or dissociation of subunits from protofilament ends following GTP hydrolysis and (2) reversible association and dissociation of subunits from protofilament ends independent of hydrolysis. Exchange of nucleotides on these recycling subunits could give the appearance of exchange directly into the polymer. Several of our observations suggest that exchange of nucleotide can take place on these recycling subunits, but not directly into the FtsZ polymer. Annealing of protofilaments was demonstrated for the L68W mutant in EDTA buffer but not in Mg buffer, where rapid cycling of subunits may obscure the effect of annealing. We also reinvestigated the nucleotide composition of FtsZ polymers at steady state. We found that the GDP:GTP ratio was 50:50 for concentrations of GTP >100 microM, significantly higher than the 20:80 ratio previously reported at 20 microM GTP.
Chen
Yaodong
Y
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710-3709, USA.
Erickson
Harold P
HP
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019943:Amino Acid Substitution / Q000187:drug effects; D001426:Bacterial Proteins / Q000378:metabolism* / Q000648:ultrastructure; D002021:Buffers; D003598:Cytoskeletal Proteins / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D021122:Protein Subunits / Q000378:metabolism*; D012413:Rubidium / Q000494:pharmacology
D001426:Bacterial Proteins; D002021:Buffers; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D021122:Protein Subunits; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D008274:Magnesium; D012413:Rubidium; D011188:Potassium