Generation of Differentially Modified Microtubules Using In Vitro Enzymatic Approaches

• Published in: Methods in Enzymology Vol. 540; pp. 149 - 166
• Main Authors: Vemu, Annapurna, Garnham, Christopher P., Lee, Duck-Yeon, Roll-Mecak, Antonina
• Format: Book Chapter Journal Article
• Language: English
• Published: United States Elsevier Science & Technology 2014
• Subjects: Acetylation; Acetyltransferases - genetics; Acetyltransferases - isolation & purification; Acetyltransferases - metabolism; Animals; Cell Line; Cloning, Molecular - methods; Glutamic Acid - analysis; Glutamic Acid - metabolism; Humans; Microtubule; Microtubules - chemistry; Microtubules - metabolism; Models, Molecular; Motors; Peptide Synthases - genetics; Peptide Synthases - isolation & purification; Peptide Synthases - metabolism; Protein Processing, Post-Translational; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Tubulin - chemistry; Tubulin - metabolism; Tubulin acetylation; Tubulin acetyltransferase (α-TAT); Tubulin glutamylation; Tubulin post-translational modifications; Tubulin tyrosination; Tubulin tyrosine ligase (TTL); Tubulin tyrosine ligase-like (TTLL); Tyrosine - analysis; Tyrosine - metabolism; Tubulin tyrosine ligase (TTL); Tubulin glutamylation; Motors; Microtubule; Tubulin tyrosination; Tubulin acetyltransferase (α-TAT); Tubulin post-translational modifications; Tubulin tyrosine ligase-like (TTLL); Tubulin acetylation
• ISBN: 9780123979247; 0123979242
• ISSN: 0076-6879; 1557-7988
• DOI: 10.1016/B978-0-12-397924-7.00009-1

Tubulin, the building block of microtubules, is subject to chemically diverse and evolutionarily conserved post-translational modifications that mark microtubules for specific functions in the cell. Here we describe in vitro methods for generating homogenous acetylated, glutamylated, or tyrosinated tubulin and microtubules using recombinantly expressed and purified modification enzymes. The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin post-translational modifications on the dynamics and mechanical properties of microtubules as well as the behavior of motors and microtubule-associated proteins.