Microtubule nucleation is spatiotemporally regulated in cells by several molecules, including the template γ-tubulin and the polymerase XMAP215. Recently, XMAP215 and the γ-tubulin ring complex were reported to function synergistically, and this synergy was hypothesized to be due to direct binding between XMAP215 and γ-tubulin. Here, we address this hypothesis by 1) probing domain requirements for XMAP215 to promote microtubule nucleation and 2) testing whether XMAP215 functions synergistically with γ-tubulin in the absence of the other ring complex proteins. We confirm that γ-tubulin and XMAP215 are classically defined nucleators that reduce the nucleation lag seen in bulk tubulin assembly. Then, using deletion constructs, we show that XMAP215's ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and γ-tubulin promote αβ-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct, and the synergy reported between XMAP215 and the γ-tubulin ring complex is not due to γ-tubulin alone. ### Competing Interest Statement The authors have declared no competing interest.
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