Figure 1.
Assembly pathway and location of point mutations in BtubA/B.
(a) The proposed assembly pathway in which BtubA and B first assemble into heterodimers, and the heterodimers then further assemble into protofilaments. (b) Two classes of protofilament interface mutants are indicated. One disrupts the interface between dimers, and the other within dimers. The four mutants tested experimentally are indicated. (c) BtubA and BtubB are shown separately in ribbon diagram, and the mutated amino acids are shown in green spacefill. GDP in BtubA is in yellow spacefill. The figures were created in PyMol (DeLano Scientific) from the PDB file 2BTQ [4].
Figure 2.
Between-dimer mutant BtubB-D249K fails to assemble and disassembles wild type polymers.
(a) Assembly of 5 µM each wild type BtubA plus BtubB is shown by the red curve. The time of GTP addition is indicated. The blue curve shows that wild type BtubA plus BtubB-D249K gave no assembly. (b) polymers were first assembled to steady state from 5 µM each wild type BtubA and BtubB. At the arrows BtubB-D249K was added to 2.5 µM or 10 µM, causing disassembly.
Figure 3.
Between-dimer mutant BtubA-V179K.
The red line shows assembly of wild type BtubA/B (5 µM each). The black line shows that BtubA-V179K plus wild type BtubB does not assemble. The blue line shows that addition of 5 µM BtubA-V179K to the 5 µM preformed BtubA/B protofilaments caused their disassembly.
Figure 4.
Within-dimer mutants fail to assemble but have no effect on pre-assembled wild type polymers.
(a) Within-dimer mutant BtubB-N100E did not assemble when mixed with wild type BtubA (black line). When added to pre-assembled wild type polymer (all subunits at 5 µM) it did not cause any disassembly (blue line). (b) Within-dimer mutant BtubA-E258K behaved the same as BtubB-N100 in both assays.
Table 1.
GTPase activity
Table 2.
Sedimentation equilibrium
Figure 5.
Between-dimer mutants disassemble wild type polymers by sequestering subunits into inactive heterodimers.
The equilibrium on the left shows wild type BtubA/B protofilaments exchanging with dimers. These dimers are also exchanging with a small pool of monomers, not shown. The bracket on the right shows what happens when the reaction is flooded with between-dimer BtubB subunits. These are capable of forming dimers, and because they are in excess of the monomer/dimer pool they will replace the wild type BtubB. This sequesters most of the wild type BtubA into inactive dimers. This depletion of wild type dimers to below the critical concentration results in depolymerization.