Regulation of motor proteins by tubulin carboxy-termini tails

The ubiquitous cytoskeletal polymer, microtubules (MTs) is a heterogeneous mixture of multiple a/b-tubulin isotypes with posttranslational modifications (PTMs). Based on conserved tubulin isotype clades and their abundance in specific cellular locations, ‘Tubulin-code’ was proposed three decades ago. The ‘Tubulin-code’ hypothesizes that distinct tubulin isotypes or PTMs offer unique interactions with MT-associated proteins for specific cellular function. So far, lack of systems to obtain homogenous tubulin population has hindered testing this hypothesis directly. Here we focused on engineering tubulin carboxy-termini tails (CTTs), most divergent region among tubulin isotypes and hotspots of PTMs. Using recombinant yeast expression system a comprehensive series of (>30) unique CTT engineered recombinant tubulins were purified. The engineered MTs were used as tracks to study three motile motor proteins (kinesin-1, kinesin-2 and dynein) and depolymerising kinesin (kinesin-13). Single molecule TIRF microscopy of these motor proteins revealed that each motor protein recognizes unique molecular signatures present in the CTTs for their optimal function. In addition we demonstrate motor preference towards different tubulin isotypes and their cross-regulation by PTMs. Identifying how different motor proteins perceive unique CTT molecular signatures strongly supports the ‘tubulin-code’ hypothesis.