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Title: Pct1 regulates phosphatidylcholine synthesis in response to changes in surface curvature elastic stress sensed on the inner nuclear membrane
Author: Wei, Yu-Chen
ISNI:       0000 0004 7652 6930
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Cell and organelle membranes consist of a complex mixture of phospholipids that determine their size, shape, and function. Among the distinct types of phospholipids found in membranes of living organisms, phosphatidylcholine (PC) is the most abundant. The rate-limiting step of the predominant pathway for PC synthesis in eukaryotic cells is catalysed by the enzyme, CTP: phosphocholine cytidylyltransferase α (CCTα or PCYT1A). CCTα has a critical role in lipid metabolism and also has direct clinical relevance as mutations in CCTα result in an interesting spectrum of human diseases, such as lipodystrophy with fatty liver, growth plate dysplasia and cone-rod related dystrophy. Numerous biochemical and structural studies on purified CCTα have revealed its membrane-bound activation and suggested that it acts as a lipid compositional sensor, yet the in vivo mechanism of how CCTα senses and regulates PC levels in membranes remains unclear. Here I show that in budding yeast Saccharomyces cerevisiae, Pct1, the yeast homolog of CCTα, is intranuclear and translocates to the nuclear membrane in response to changes in membrane properties and the need for membrane PC synthesis. By aligning imaging with lipidomic analysis and data-driven modelling, Pct1 membrane association is demonstrated to correlate with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous Pct1. These data suggest an ancient mechanism by which CCTα senses lipid packing defects and regulates phospholipid homeostasis from the nucleus. Additionally, I identified the importance of mammalian CCTα in early adipogenesis and investigated the enzymatic function of PCYT1A mutants in fibroblasts from lipodystrophic patients. The allele Val142Met is evaluated to be the main cause of loss-of-function in the compound heterozygous mutations by using yeast survival assay. These results collectively provide preliminary evidence for the pathogenicity of PCYT1A mutations in adipose tissue. From yeast to humans, this study uncovers the critical role of Pct1/CCTα in maintaining the internal membrane environment.
Supervisor: Savage, David Sponsor: Cambridge Trust ; Taiwan Ministry of Education
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: CCT ; Pct1 ; Kennedy Pathway ; PCYT1A ; lipidomics ; phosphatidylcholine ; stored curvature elastic stress