Development of a method to study inter-organelle lipid transport in vivo: mammalian betaine lipid transport (mBLT)

Thesis

Studying lipid transport in vivo has remained a major challenge. This is because transport occurs through vesicular trafficking as well as through lipid transfer proteins, many of which exhibit redundancies and function in a context-dependent manner. By confining the stepwise synthesis of betaine lipids across two membranes, betaine lipid transport (BLT) provides a biorthogonal strategy to study lipid transport in vivo with spatial and temporal control. Betaine lipids synthesis proceeds via two successive enzymatic reactions: BtaA converts diacylglycerol (DAG) to DGHS, which is then converted to DGTS by BtaB. In BLT, BtaA and BtaB are artificially targeted to any two membranes, such that the accumulation of DGTS serves as a readout for directional lipid transport; in combination with metabolic labelling, temporal changes in transport can be assessed. This study strongly demonstrates the feasibility of adapting BLT for use in mammalian cells, following its initial development in yeast. Due to the complexity of the mammalian lipidome versus that of yeast, single quadrupole mass spectrometry did not provide adequate resolution for the robust detection of betaine lipids. Tandem mass spectrometry was therefore required to confirm ectopic betaine lipid synthesis and demonstrate that co-expression of BtaA and BtaB could report on basal lipid transport. However, low lipid abundance ultimately limited an assessment of changes in lipid transport during mitophagy, which provides an ideal context for obtaining proof-of-concept for mBLT. To guide further development, this study outlines the key biological and technical constraints which must be addressed to enable robust quantification of lipid transport using mBLT.

Authors: Laurent, C

• DOI: 10.5287/ora-qxa675a7r
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English