Abstract
SummaryMicrotubule (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.
Highlights
In SEC on Superose 6, some g-TuSCAlp6-MBP:Mzt1 was not recovered from the column, most of the g-TuSCAlp6-MBP: Mzt1 that did elute from the column eluted at a position expected for g-tubulin small complex (g-TuSC), and a much smaller proportion eluted in the void volume (Figure 1E)
Our results indicate that S. pombe Mzt1 maintains Alp6, the homolog of human GCP3, in an ‘‘interaction-competent’’ state that prevents large-scale aggregation of g-TuSCs and allows Mto1/2[bonsai] to bind to g-TuSC without disrupting g-TuSC integrity
Two of our key findings concerning the role of S. pombe Mzt1 in MT nucleation differ significantly from recent in vitro work involving the equivalent proteins (‘‘Ca’’ proteins) in the dimorphic yeast C. albicans [10]
Summary
In SEC on Superose 6, some g-TuSCAlp6-MBP:Mzt1 was not recovered from the column, most of the g-TuSCAlp6-MBP: Mzt1 that did elute from the column eluted at a position expected for g-TuSCs, and a much smaller proportion eluted in the void volume (Figure 1E). We generated the suggest that MGM may have an overall structure similar to MGM holocomplex via coexpression of Mto1/2[bonsai] with g-TuRC; further work may be required to optimize morphological g-TuSCAlp6-MBP:Mzt1 (Figure S2B).
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