A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

Abstract

Edited by Velia M. FowlerMicrotubule dynamics involves the polymerization and depolymerization of tubulin dimers and is an essential and highly regulated process required for cell viability, architecture, and division. The regulation of the microtubule network also depends on the maintenance of a pool of ␣␤-tubulin heterodimers. These dimers are the end result of complex folding and assembly events, requiring the TCP1 Ring Complex (TriC or CCT) chaperonin and five tubulin-specific chaperones, tubulin binding cofactors A-E (TBCA-TBCE). However, models of the actions of these chaperones are incomplete or inconsistent. We previously purified TBCD from bovine tissues and showed that it tightly binds the small GTPase ARL2 but appears to be inactive. Here, in an effort to identify the functional form of TBCD and using non-denaturing gels and immunoblotting, we analyzed lysates from a number of mouse tissues and cell lines to identify the quaternary state(s) of TBCD and ARL2. We found that both proteins co-migrated in native gels in a complex of ϳ200 kDa that also contained ␤-tubulin. Using human embryonic kidney cells enabled the purification of the TBCD⅐ARL2⅐␤-tubulin trimer found in cell and tissue lysates as well as two other novel TBCD complexes. Characterization of ARL2 point mutants that disrupt binding to TBCD suggested that the ARL2-TBCD interaction is critical for proper maintenance of microtubule densities in cells. We conclude that the TBCD⅐ARL2⅐␤-tubulin trimer represents a functional complex whose activity is fundamental to microtubule dynamics.Microtubules are highly dynamic polymers that are best known for their roles as the central cytoskeletal structure in cells and in mitotic spindles during cell division. They are also the tracks on which organelles traffic, particularly important in polarized cells that generate great distances between parts of the cell. Additionally, they are the core of sensory and motile cilia or flagella. The formation and destruction of microtubules and microtubule bundles are orchestrated by a large number of proteins that include the microtubule-associated proteins. Microtubules are polymers of the ␣␤-tubulin dimer, with several genes encoding each ␣-or ␤-tubulin subunit (e.g. see Lewis et al. (1)), resulting in some diversity in composition. Tubulins can also be modified by posttranslational modifications, including acetylation, tyrosination, and phosphorylation, which can alter the dynamics of the polymerization and depolymerization reactions. Because of the essential role of microtubules in cell division, they have also been the target of many antitumor therapies, e.g. the taxanes and Vinca alkaloids (2). However, despite their importance to cells and in the clinic and decades of research, we still lack a complete molecular-level understanding of the biosynthesis and regulation of the formation of ␣␤-tubulin.Tubulins are typically the most abundant proteins in mammalian cells, but the generation of the ␣␤-tubulin dimer requires a complex series of biosynthetic steps to ...

Nucleotide Binding to ARL2 in the TBCD ∙ ARL2 ∙ β-Tubulin Complex Drives Conformational Changes in β-Tubulin

“…It has been inferred that perhaps β-tubulin is similarly liganded, though direct evidence for this subunit is lacking. This foundational work provided the first model for the steps in the tubulin-folding pathway, but aspects of it are challenged by more recent studies [11, 12]. A detailed model for tubulin folding that withstands rigorous testing would include the roles played by each of the required components, allow the generation of key biochemical reagents for multiple studies, and generate novel opportunities for therapeutic development.…”

“…This is in marked contrast to the other four cofactors, A-C and E [13]. We recently described the use of human embryonic kidney (HEK293T/17) cells to overexpress and purify human or bovine TBCD and showed it to be soluble and stable [11]. That study identified a number of novel complexes containing TBCD and strong physical connection to the regulatory GTPase, ARL2.…”

“…Also, a spate of recent reports describe point mutations in TBCD found in patients with links to early-onset encephalopathy [28–31] and intractable seizures [32]. A subset of these mutations were specifically shown to disrupt formation and stability of the TBCD•ARL2•β-tubulin complex [29], which we have recently purified and characterized [11]. …”

“…In contrast, ARL2 was first purified from bovine tissues as a dimer of TBCD•ARL2 [33]. We recently showed that in most cells and tissues a species of ~200 kDa is the predominant form of ARL2 and TBCD and that it represents the TBCD•ARL2•β-tubulin trimer [11]. The TBCD•ARL2 dimer purified from bovine brain was inactive in the tubulin folding assay and displayed the unique property of binding GDP but not GTP [33].…”

“…The TBCD•ARL2 dimer purified from bovine brain was inactive in the tubulin folding assay and displayed the unique property of binding GDP but not GTP [33]. To allow more detailed functional and structural studies, we developed an expression system in mammalian cells capable of generating milligram amounts of TBCD complexes [11]. This study found that only when co-expressed with ARL2 does TBCD purify as a monodisperse species, a 1:1:1 complex of TBCD•ARL2•β-tubulin.…”