Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

Iryna Berezniuk,Hang T Vu,Peter J Lyons,Juan J Sironi,Hui Xiao,Berta Burd,Mitsutoshi Setou,Ruth H Angeletti,Koji Ikegami,Lloyd D Fricker

doi:10.1074/jbc.m111.309138

Abstract

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.

Highlights

Several cellular functions for cytosolic carboxypeptidase 1 (CCP1) have been proposed
CCP1 mRNA Is the Most Abundant CCP mRNA in human embryonic kidney 293T cells (HEK293T) Cells—To study CCP1 function in a simple system, we searched for a cell line that expresses high levels of CCP1 mRNA and low levels of other CCP transcripts
The hyperglutamylation of ␤-tubulin detected in the pcd brain was reduced and almost reached the same level as WT by the deletion of TTLL1 (Fig. 8, A and B). These results suggest that TTLL1 is involved in hyperglutamylation of both ␤- and ␣-tubulin and that the deletion of TTLL1 is sufficient for counteracting the hyperglutamylation in pcd mouse brain

Summary

Introduction

Several cellular functions for cytosolic carboxypeptidase 1 (CCP1) have been proposed. The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). The hyperglutamylation of ␣- and ␤-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates ␣- and ␤-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from ␤- as well as ␣-tubulin in vitro and in vivo

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Conclusion