PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in the ciliate Tetrahymena thermophila

Proteins in order to perform their biological functions, have to be properly folded. To sustain the conformational integrity of proteins, cells maintain an integrated network of several hundred proteins including molecular chaperones. Chaperonin CCT is one of the proteins involved in the quality control system in the cell and is primarily responsible for folding of cytoskeletal proteins – actin and tubulin. Recent studies have implicated the evolutionary conserved phosducin-like protein-2 (Phlp2) in regulation of CCT, however the exact molecular function of Phlp2 is unclear. Here we investigate the significance of Phlp2 in a ciliated unicellular model, Tetrahymena thermophila. Cells lacking Phlp2p became larger and died within 96h. Overexpressed Phlp2-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2 had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2. Growing GFP-Phlp2-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2-overexpressing cells. Thus, in Tetrahymena, Phlp2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.

Date of publication
25 October 2013