Cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules through kinetochores and the spindle assembly checkpoint (SAC) is satisfied. We show that the amino-terminal localization module of the SAC protein kinase MPS1 (monopolar spindle 1) directly interacts with the HEC1 (highly expressed in cancer 1) calponin homology domain in the NDC80 (nuclear division cycle 80) kinetochore complex in vitro, in a phosphorylation-dependent manner. Microtubule polymers disrupted this interaction. In cells, MPS1 binding to kinetochores or to ectopic NDC80 complexes was prevented by end-on microtubule attachment, independent of known kinetochore protein-removal mechanisms. Competition for kinetochore binding between SAC proteins and microtubules provides a direct and perhaps evolutionarily conserved way to detect a properly organized spindle ready for cell division.

Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of ⍺-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of ⍺-tubulin, and purified vasohibins removed the C-terminal tyrosine of ⍺-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle.

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin–small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo–electron microscopy structures established MATCAP’s cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.Microtubules are key components of the eukaryotic cytoskeleton, involved in cell division, morphogenesis, motility and intracellular transport. Post-translational modifications of tubulin heterodimers, the so-called 'tubulin code' , includes the enzymatic removal and ligation of the C-terminal tyrosine 1 . Although the tubulin tyrosine ligase that reverts α-tubulin to the translated form has been described 2 and structurally characterized 3,4 , the carboxypeptidase that removes it has remained elusive for four decades. Recently, we 5 and others 6 described a complex between SVBP, a 66-residue peptide, and vasohibins (VASH1 or VASH2), as the long-sought-for tubulin carboxypeptidases. Vasohibins increase detyrosination of α-tubulin in cells and in vitro 5 , especially in the presence of SVBP, and both proteins have been implicated in neuronal function, a role that may be associated with their role in tubulin detyrosination 6 .A chaperone-like function was possible because SVBP enhances the levels of detyrosinated α-tubulin, and concomitantly affects the cellular abundance and solubility of vasohibins. VASH1 might have a transglutaminase-like protease fold, with a non-canonical Cys-His-Ser catalytic triad 7 ; however, low similarity to existing structures precludes establishment of a reliable structural model. To study the folding of VASH1, to understand how SVBP affects vasohibins and to examine how VASH1 recognizes and cleaves the α-tubulin C-terminal tyrosine, we co-expressed a VASH1-SVBP complex in insect cells, and then purified and crystallized it. The crystal structure was determined by sulfur single-wavelength anomalous dispersion (S-SAD) phasing, notably averaging 16 data sets of 360° sweeps using a PRIGo multi-axis goniometer 8 . The structure was refined to 2.1 Å resolution to an R free of 21.4% (see Methods and Supplementary Table 1 for crystallographic details).Only residues 60-304 of VASH1 (1-315 expressed) and 26-52 of SVBP (1-66) were visible in the electron density maps and modeled