Fig. 6.

In vitro synthesis and folding of β-tubulin. Panel A, A coupled transcription/translation system utilizing rabbit reticulocyte lysate was used to synthesize radiolabeled β-tubulin from plasmid DNA. Following synthesis, reaction mixtures were incubated with (+) or without (−) purified bovine brain tubulin and fractionated by native gel electrophoresis to assay for the presence of complexes formed between the labeled β-tubulin and folding cofactors present in the reticulocyte lysate. Results from reactions containing wild-type β-tubulin cDNA (lanes 1 and 2) and mutant cDNAs encoding a 12 amino acid deletion characteristic of strain 6H2 (lanes 3 and 4), an L187R mutation from strain A5 (lanes 5 and 6), and a Y398C mutation from strain 5L1 (lanes 7 and 8) are shown. Complexes formed between β-tubulin and CCT (CCTβ), cofactor D (Fdβ), cofactor A (Faβ), and α-tubulin (αβ) are indicated. Panel B, Reconstituted in vitro folding assay. [³⁵S]methionine labeled wild-type (WT) and mutant Y398C HAβ1-tubulin were produced in E. coli, purified, denatured, and diluted into an in vitro assay containing some or all of the components required to reconstitute the tubulin heterodimer assembly pathway (CCT, ATP, GTP, folding cofactors, and native bovine brain tubulin). The reaction products were resolved by native gel electrophoresis and detected by autoradiography. Complexes formed when HAβ1-tubulin was incubated with CCT alone (lanes 1 and 4), CCT + Fd (lanes 2 and 5), or CCT + Fd + FC + Fe + purified tubulin (i.e., the complete cocktail) (lanes 3 and 6). Panel C, CCT cycles the HAβ1-tubulin. The reactions contained purified, denatured HAβ1-tubulin plus CCT alone (lanes 1 and 4), CCT + a 10X excess of Hsp60 + ATP (lanes 2 and 5), or CCT + Hsp60 + ATP + glucose + hexokinase (lanes 3 and 6). The complex between HAβ1-tubulin and Hsp60 (Hsp60β) is labeled to the left of panel B.