Abstract
Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.
Highlights
Microtubules (MTs) are dynamic polymers that modulate fundamental cellular processes through dynamic αβ-tubulin polymerization and depolymerization at their ends, and serve as polarized tracks for molecular motor proteins (Akhmanova and Steinmetz, 2008)
Our studies reveal a new role for tubulin cofactors TBCD, TBCE, and Arl2, which together assemble a GTP-hydrolyzing tubulin chaperone critical for the biogenesis, maintenance, and degradation of soluble αβ-tubulin, defects in which have a profound effect on MT dynamics in vivo
We found that TBCA, TBCB, and TBCC are each soluble when expressed on their own in Escherichia coli, while TBCD, TBCE, and Arl2 are insoluble on their own
Summary
Microtubules (MTs) are dynamic polymers that modulate fundamental cellular processes through dynamic αβ-tubulin polymerization and depolymerization at their ends, and serve as polarized tracks for molecular motor proteins (Akhmanova and Steinmetz, 2008). Polarity and dynamic instability are fundamental features of the MT polymer, originating from the head-to-tail polymerization of αβ-tubulin heterodimers (Nogales et al, 1999; Alushin et al, 2014). Intracellular MT dynamics critically relies on a tightly controlled pool of soluble αβ-tubulin dimers in the cytoplasm. Despite their importance, the mechanisms for biogenesis, maintenance, and degradation of soluble αβ-tubulin dimers remain poorly understood (Tian and Cowan, 2013)
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