Investigation of Human Pix Protein Regulation during Cell Cycle Progression

Proteomic analyses of centrosomes from diverse species are helping to identify conserved components of these organelles. Amongst these studies, a group of novel proteins has been identified and postulated as candidate core centriolar components, dubbed Poc proteins, for proteome of centriole. Meanwhile, studies on the Xenopus germ plasm protein, Xpat, led to the identification of an interacting protein, named Pix for protein that interacts with Xpat. Later sequence comparison revealed that Poc1 and Pix are the same protein and will here be referred to as Pix. Pix proteins localize to centrosomes and spindle poles in human cells and the basal bodies of Chlamydomonas and Tetrahymena. It has been suggested that Pix proteins are required for centriole duplication and length control, as well as potentially ciliogenesis. Pix proteins have also been localized to mitochondria in human cells where they might act as molecular adaptors to anchor microtubules to mitochondria. Human cells encode two Pix proteins and the aim of this thesis was to investigate the cell cycle-dependent regulation of Pix1 and Pix2 in human cells. For this purpose, we first generated polyclonal rabbit antibodies that were specific for either Pix1 or Pix2. We then showed for the first time that both Pix isoforms localize to centrosomes throughout the cell cycle and independently from one another. Using the antibodies, we also confirmed that Pix1, but not obviously Pix2, is not an intrinsic mitochondrial protein but localizes to the surface of mitochondria. Through a proteomic approach, we identified two molecular chaperons that potentially interact with Pix proteins, HSP90 and TCP1, and demonstrated that Pix proteins can form dimers. In addition, we produced the first experimental evidence that human Pix proteins are alternatively spliced, suggesting that additional, undiscovered Pix isoforms might be expressed in human cells. Finally, we found that Pix1 is phosphorylated in a mitosis-specific manner, and that this is potentially regulated by Cdk1. Thus, we propose that Pix proteins have a specific and previously unidentified role in human mitotic cell division.