The Substrate and Regulator of Acetyltransferase San

Lysine acetylation is one of the most common protein modifications in eukaryotes and plays critical roles in numerous cellular events. San is an acetyltransferase required for proper chromosome segregation in Drosophila and human, but little is known about its substrates or upstream regulators. To identify San substrates, I performed affinity purification and demonstrated that tubulin is a San substrate. Tubulins are the building blocks of microtubules, which display dynamic instability. By regulating the rate of microtubule assembly and disassembly, cells organize the microtubule cytoskeleton to accommodate their specific functions. Posttranslational modifications of tubulin have been implicated in regulating microtubule functions. San acetylates beta-tubulin on lysine-252, which only occurs on free tubulin heterodimers. The acetylation-mimicking mutants are incorporated into the microtubule cytoskeleton in HeLa cells without causing any obvious microtubule defect. However, after cold-induced catastrophe, microtubule regrowth is accelerated in San-siRNA cells while the incorporation of the acetylation-mimicking mutant tubulins is severely impeded. The lysine-252 of beta-tubulin localizes at the intradimer interface and interacts with the phosphate group of the alpha-tubulin-bound GTP. Based on these findings, I propose that San regulates tubulin polymerization by acetylating beta-tubulin lysine-252, which neutralizes the positive charge and makes the tubulin heterodimer adopt a conformation that disfavors tubulin polymerization. In addition, I present evidence indicating that the enzymatic activity of San is inhibited by a protein in an ATP-dependent but hydrolysis-independent manner. Purification and identification of this San inhibitor is still under way, and preliminary data from gel filtration chromatography suggest that the inhibitor may be a multi-subunit protein complex.