Date of this Version
Ishimwe, J. 2023. Exploring Membrane Contact Sites Components Enabling Biogenesis of Photosynthetic Membrane. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
Plant chloroplasts capture light energy in the thylakoid membrane, a system of proteins and pigments embedded in a lipid bilayer. Lipids needed for thylakoid formations are biosynthesized in the chloroplast envelope membranes and must be trafficked to the thylakoid by unknown means. One likely mechanism of moving lipids to/from the thylakoid involves its close association with the inner envelope membrane (IEM) where the energetic barrier of lipid movement is decreased. The aim of this project is to identify chloroplast proteins located at thylakoid/IEM contact sites (MCS) from Genes of Interest and then use a technique called split enzyme-catalyzed proximity labeling (Split-PL). One-half of an engineered split-biotin ligase will be targeted to the IEM, one half to the thylakoid. Where the two membranes come into close contact the enzyme can reconstitute and promiscuously biotinylated proteins within a small radius. Biotinylated proteins can then be isolated using affinity purification and identified by mass spectrometry. Candidate proteins of interest identified using split-PL will then be tagged with a fluorescent protein and chloroplast localization will be tested using confocal laser scanning microscopy.