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Abstract Microtubule network remodeling is an essential process for cell development, maintenance, cell division, and motility. Microtubule‐severing enzymes are key players in the remodeling of the microtubule network; however, there are still open questions about their fundamental biochemical and biophysical mechanisms. Here, we explored the ability of the microtubule‐severing enzyme katanin to depolymerize stabilized microtubules. Interestingly, we found that the tubulin C‐terminal tail (CTT), which is required for severing, is not required for katanin‐catalyzed depolymerization. We also found that the depolymerization of microtubules lacking the CTT does not require ATP or katanin's ATPase activity, although the ATP turnover enhanced depolymerization. We also observed that the depolymerization rate depended on the katanin concentration and was best described by a hyperbolic function. Finally, we demonstrate that katanin can bind to filaments that lack the CTT, contrary to previous reports. The results of our work indicate that microtubule depolymerization likely involves a mechanism in which binding, but not enzymatic activity, is required for tubulin dimer removal from the filament ends.
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A Tale of 12 Tails: Katanin Severing Activity Affected by Carboxy-Terminal Tail Sequences
In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule “tubulin code,” which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament.
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- Award ID(s):
- 1817948
- PAR ID:
- 10428201
- Date Published:
- Journal Name:
- Biomolecules
- Volume:
- 13
- Issue:
- 4
- ISSN:
- 2218-273X
- Page Range / eLocation ID:
- 620
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/biom13040620
