Identification of Tubulin Deglutamylase among Caenorhabditis elegans and Mammalian Cytosolic Carboxypeptidases (CCPs)

Yoshishige Kimura,Nobuya Kurabe,Koji Ikegami,Koji Tsutsumi,Yoshiyuki Konishi,Oktay Ismail Kaplan,Hirofumi Kunitomo,Yuichi Iino,Oliver E Blacque,Mitsutoshi Setou

doi:10.1074/jbc.c110.128280

Abstract

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.

Highlights

The microtubule (MT)2 cytoskeleton plays critical roles in multiple cellular processes such as chromosome segregation, intracellular transport, cell morphogenesis, and polarity
We identified a cytosolic carboxypeptidase gene, ccpp-6, as a candidate tubulin deglutamylase gene, whose functional properties are opposite to those of the tubulin polyglutamylase gene, ttll-4
C. elegans Sensory Ciliary MTs Are Strongly Polyglutamylated, Requiring ttll-4—We determined the distribution of polyglutamylation signals in C. elegans sensory cilia using antibodies that recognize polyglutamylation

Summary

Introduction

The microtubule (MT)2 cytoskeleton plays critical roles in multiple cellular processes such as chromosome segregation, intracellular transport, cell morphogenesis, and polarity. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. We identified mammalian CCP5 as a functional homolog of nematode CCPP-6 and confirmed its deglutamylase activity in cultured cells and in vitro.

Results

Conclusion