
TTLL8 is a monoglycylase that adds glycines to multiple sites on both α- and β-tubulin, while TTLL10 is a polyglycylase that can only elongate pre-existing monoglycine branches. TTLL10 binding to microtubules is stimulated by monoglycylation but inhibited by polyglycylation in a chain length-dependent manner, suggesting an autonomous mechanism for polyglycine chain length control.


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Microtubules in cells have complex and developmentally stereotyped posttranslational modifications that support diverse processes such as cell division, ciliary growth and axonal specification. Glycylation, the addition of glycines, singly (monoglycylation) or in chains (polyglycylation), is primarily found on axonemal microtubules where it functions in cilia maintenance and motility. It is catalyzed by three enzymes in the tubulin tyrosine ligase- like family, TTLL3, 8 and 10. We show that TTLL8 monoglycylates both α- and β-tubulin, unlike TTLL3 which prefers β-tubulin. Microscopy and mass spectrometry show that TTLL10 requires monoglycylation for high affinity microtubule binding and elongates polyglycine chains only from pre-existing glycine branches. Surprisingly, tubulin polyglycylation inhibits TTLL10 recruitment to microtubules proportional with the number of posttranslationally added glycines, suggesting an autonomous mechanism for polyglycine chain length control. In contrast, tubulin glutamylation, which developmentally precedes polyglycylation in cilia, increases TTLL10 recruitment to microtubules, suggesting a mechanism for sequential deposition of tubulin modifications on axonemes. Our work sheds light on how the tubulin code is written by establishing the substrate preference and regulation of TTLL glycylases, and provides a minimal system for generating differentially glycylated microtubules for in vitro analyses of the tubulin code.

### Competing Interest Statement

The authors have declared no competing interest.

The TTLL10 polyglycylase is stimulated by tubulin glutamylation and inhibited by polyglycylation

the-ttll10-polyglycylase-requires-monoglycylation-for-microtubule-binding-and-is-inhibited-by-polyglycylation


The TTLL10 Polyglycylase Requires Monoglycylation for Microtubule Binding and is Inhibited by Polyglycylation
