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

Abstract

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligaselike (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. The microtubule (MT)2 cytoskeleton plays critical roles in multiple cellular processes such as chromosome segregation, intracellular transport, cell morphogenesis, and polarity. Tubulin, which is the major protein subunit of MT fibers, exhibits polymorphic protein variation and undergoes a variety of unique post-translational modifications at its C-terminal tail, such as detyrosination/tyrosination (1, 2), polyglycylation (3), and polyglutamylation (4). These modifications regulate tubulin and MT function. Polyglutamylation enzymes add multiple glutamate moieties to specific glutamate residues within substrate proteins. Physiological roles of tubulin polyglutamylation for MT-related processes were reported recently (5, 6) and reviewed by Ikegami and Setou (7).Polyglutamylase enzymes have recently been identified as belonging to the tubulin tyrosine ligase-like (TTLL) protein family (8 -10) (supplemental Fig. 1). In contrast, the enzyme(s) underlying deglutamylation have not been discovered yet, although such proteins are suggested to be present in mouse brain neurons and in cultured cells (11,12).In this study, we employed the genetically tractable nematode, Caenorhabditis elegans, to identify genes that regulate tubulin polyglutamylation/deglutamylation. Specifically, we investigated tubulin post-translational modification in C. elegans sensory cilia, which are MT-based organelles that extend from the distal dendrite tips of 60 sensory neurons found in sensory organs named amphid (head) and phasmid (tail) sensilla (see Fig. 1A). In this system, we identified a cytosolic carboxypeptidase (ccpp) gene, ccpp-6, as a candidate tubulin deglutamylase gene, whose functional properties are opposite ...

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