Targeting, assembly and regulation of the pyruvate dehydrogenase complex

Ward, Jason David (1998) Targeting, assembly and regulation of the pyruvate dehydrogenase complex. PhD thesis, University of Glasgow.

Abstract

The majority of polypeptides in the mitochondrion are nuclear encoded and post-translationally transported to the mitochondrion from the cytosol. These include the polypeptides which make up the pyruvate dehydrogenase complex (PDC), the largest multienzyme complex of the 2-oxoacid dehydrogenase complex family. This transportation is initiated by an N-terminal mitochondrial targeting signal. However, whereas standard mitochondrial matrix targeting signals are approx. 15-30 amino acids long in mammals, the N-terminal presequence of the PDC-E2 component is 73 amino acids in length. Such long presequences usually carry additional information, such as a second targeting signal; however the presequence of PDC-E2 appears to have no such additional function. Furthermore, mammalian PDC-E2 has the capacity to spontaneously self assemble into 60-meric dodecahedral cores, which form the superstructure for the complex itself. An integral part of mitochondrial targeting is prevention of such self assembly to allow translocation of the polypeptide from the cytosol to the matrix inside the mitochondrion. This research details studies undertaken to examine the roles of the extended presequence of PDC-E2. A novel in vivo model was developed to monitor the natural behaviour of mitochondrial presequences with a reporter gene, utilising the reporter to measure both targeting and assembly. Sections of the PDC-E2 presequence were tested with this model system to discover their role within the translocation and folding of PDC-E2. The regulation of PDC by PDC kinase was also investigated. PDC activity is strictly controlled by a variety of effector molecules, through the inactivation of PDC-E1 by PDC kinase. The kinase, by exerting control over the first, irreversible and rate limiting step, can block the entire reaction sequence. Interestingly, phosphorylation of PDC-E1 can occur at 3 different sites, but previous research has demonstrated that not all sites must be phosphorylated to inactivate the complex. To understand the behaviour of the kinase, and the circumstances under which the 3 phosphorylations take place, electrospray mass spectroscopy was employed to measure the number of phosphorylations on PDC-E1 made by PDC kinase, over time, under different circumstances.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Professor Lindsay.
• Keywords: Molecular biology.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Date of Award: 1998
• Depositing User: Enlighten Team
• Unique ID: glathesis:1998-71835
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 18 Oct 2022 07:11
• Thesis DOI: 10.5525/gla.thesis.71835
• URI: https://theses.gla.ac.uk/id/eprint/71835