| In this thesis project, the biogenesis of two very different mitochondrial proteins was explored: Taffazin (Taz1p) and polynucleotide phosphorylase (PNPase). Both are membrane anchored and both reside in the intermembrane space, but the similarities end there. Taffazin shares homology with a family of acyltransferases, remodels the mitochondrial phospholipid cardiolipin, and has been implicated in a recessive, X-linked disease called Barth Syndrome. PNPase is a major exoribonuclease found in bacteria and some eukaryotic organelles such as the mitochondria. It is known to degrade, process and polyadenylate RNA, but its specific role in the mitochondrion remains unclear. The import pathway of both proteins was characterized using a combined biochemical and genetic approach in S. Cereviseae, where radiolabelled peptides were imported into purified mitochondria from strains that harbor mutations in specific translocons subunits. Biochemical techniques such as crosslinking with immunoprecipitation, blue native gel electrophoresis and differential digitonin solubilization were also used to determine which translocons were involved. This work resulted in the discovery of a new mitochondrial import pathway, in the case of Taz1p, where the peptide first passes through the TOM (Translocon of the Outer Membrane) complex, interacts with the Tim9p/Tim10p complex in the IMS (inter membrane space) and is then inserted into the trans side of the outer membrane. The PNPase project resulted in the assignment of a new role for the inner membrane protease Yme1 that involves the regulation of incoming precursor peptides. Both findings have provided important insights into the biological functions of both proteins as well as furthered our understanding of the import pathways of mitochondrial proteins. |
