MZT1 regulates microtubule nucleation by linking γTuRC assembly to adapter-mediated targeting and activation

Abstract

Regulation of the γ-tubulin ring complex (γTuRC) through targeting and activation restricts nucleation of microtubules to microtubuleorganizing centers (MTOCs), aiding in the assembly of ordered microtubule arrays. However, the mechanistic basis of this important regulation remains poorly understood. Here, we show that, in human cells, γTuRC integrity, determined by the presence of γ-tubulin complex proteins (GCPs; also known as TUBGCPs) 2-6, is a prerequisite for interaction with the targeting factor NEDD1, impacting on essentially all γ-tubulin-dependent functions. Recognition of γTuRC integrity is mediated by MZT1, which binds not only to the GCP3 subunit as previously shown, but cooperatively also to other GCPs through a conserved hydrophobic motif present in the N-termini of GCP2, GCP3, GCP5 and GCP6. MZT1 knockdown causes severe cellular defects under conditions that leave γTuRC intact, suggesting that the essential function of MZT1 is not in γTuRC assembly. Instead, MZT1 specifically binds fully assembled γTuRC to enable interaction with NEDD1 for targeting, and with the CM1 domain of CDK5RAP2 for stimulating nucleation activity. Thus, MZT1 is a 'priming factor' for γTuRC that allows spatial regulation of nucleation.

A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers

“…It is therefore remarkable that CnnT, with its simple composition of the CM1-containing γ-TuRC recruitment/binding domain fused to a mitochondrial targeting domain, is capable of organizing robust MTOCs at the mitochondrial surface without involvement of the key PCM proteins. The small protein MOZART (MZT) [35, 36] is a direct partner of CM1 [27] that also associates with GCP3 [37] and other GCP proteins [38], and cooperates with GCP6 [39]. GCP3, in particular, may directly control conformational changes of the γ-TuRC and thus regulate its MT nucleation activity [40, 41], and MZT1 appears to be a key link [27, 38].…”

“…NEDD1 appears to be a γ-TuRC targeting factor [42, 43], associates with γ-TuRCs independently of CM1 in solution, and appears to anchor the γ-TuRC rather than activate it [28]. MZT1 is associated with both NEDD1-containing and CM1-containing γ-TuRCs [38]. …”

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma‐tubulin complex

“…Furthermore, recent studies have shown that MOZART1 together with Spc110 would act as oligomerization chaperones to cooperatively promote oligomerization of γTuSCs into well‐organized and active microtubule nucleation template . Altogether, these data indicate that MOZART1 may contribute to the recruitment of γTuRCs to microtubule‐organizing centers (MTOCs), and to the activation of microtubule nucleation . Consistently, defects in MOZART1 lead to abnormalities in mitotic spindle assembly and in cell division and development …”

“…Various proteins such as pericentrin, NEDD1/GCP‐WD, and CDK5RAP2 have been implicated in the regulation of γTuRCs . In addition, recent studies revealed that MOZART1 (human mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1) protein may equally be involved in this regulation …”

“…) . These proteins are predicted to have an α‐helical fold and would interact with a 10‐amino‐acid hydrophobic sequence motif present in the N‐terminal extension of GCP3, GCP5, GCP6, and potentially of GCP2 proteins . The binding of MOZART1 to a subset of GCPs may support the assembly of multiple γTuSCs into γTuRCs, and a cooperative binding of other proteins to the GCPs, such as proteins carrying CM1‐motifs, and NEDD1 .…”