Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1

Summary Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of the heterotetrameric γ-tubulin small complex (γ-TuSC) associate to form a ring-like structure (in metazoans, γ-tubulin ring complex; γ-TuRC) [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ]. Additional conserved regulators of the γ-TuC include the small protein Mzt1 (MOZART1 in human; GIP1/1B and GIP2/1A in plants) [ 8 , 9 , 10 , 11 , 12 , 13 ] and proteins containing a Centrosomin Motif 1 (CM1) domain [ 10 , 14 , 15 , 16 , 17 , 18 , 19 ]. Many insights into γ-TuC regulators have come from in vivo analysis in fission yeast Schizosaccharomyces pombe . The S. pombe CM1 protein Mto1 recruits the γ-TuC to microtubule-organizing centers (MTOCs) [ 14 , 20 , 21 , 22 ], and analysis of Mto1[bonsai], a truncated version of Mto1 that cannot localize to MTOCs, has shown that Mto1 also has a role in γ-TuC activation [ 23 ]. S. pombe Mzt1 interacts with γ-TuSC and is essential for γ-TuC function and localization to MTOCs [ 11 , 12 ]. However, the mechanisms by which Mzt1 functions remain unclear. Here we describe reconstitution of MT nucleation using purified recombinant Mto1[bonsai], the Mto1 partner protein Mto2, γ-TuSC, and Mzt1. Multiple copies of the six proteins involved coassemble to form a 34-40S ring-like “MGM” holocomplex that is a potent MT nucleator in vitro . Using purified MGM and subcomplexes, we investigate the role of Mzt1 in MT nucleation. Our results suggest that Mzt1 is critical to stabilize Alp6, the S. pombe homolog of human γ-TuSC protein GCP3, in an “interaction-competent” form within the γ-TuSC. This is essential for MGM to become a functional nucleator.