BtubA-BtubB heterodimer is an essential intermediate in protofilament assembly.
BACKGROUND
BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to alpha-tubulin and BtubA to beta-tubulin. A 1:1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament.


RESULTS
We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1:1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity.


CONCLUSIONS
An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity.
Sontag
Christopher A
CA
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America.
Sage
Harvey
H
Erickson
Harold P
HP
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002693:Chlamydiaceae / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003599:Cytoskeleton / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008027:Light; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D017354:Point Mutation; D011108:Polymers / Q000737:chemistry; D012542:Scattering, Radiation; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011108:Polymers; D014404:Tubulin; D006160:Guanosine Triphosphate