Screening for Chloroplast Inner Envelope-thylakoid Membrane Contact Site Proteins

Most life on Earth is dependent upon the capture of solar energy and subsequent fixation of carbon. In members of the Archaeplastida lineage these biological processes take place in semi-autonomous organelles called chloroplasts. Chloroplasts are bounded by a double membrane envelope and contain the photosynthetic membrane system, thylakoid membranes, where light capture takes place. While a great deal is known about how thylakoid lipids are synthesized and protein complexes therein are properly maintained, very little is known regarding the processes by which lipids are transported to thylakoids which lack de novo glycerolipid synthesis. We have worked to identify candidate proteins which could be involved in chloroplast lipid trafficking, particularly at membrane contact sites. Chapter 1 will provide an overview of photosynthetic lipid synthesis and trafficking to the chloroplast envelopes. This includes the biosynthesis of glycerolipids and the movement of lipids between the various membranes where they are made. Chapter 2 is an investigation of select candidate proteins which were selected as potential components of chloroplast lipid trafficking. This chapter is a combination two orthologous approaches; one which was based on homology and targeting prediction, the second which is based on isolation of membrane contact site enriched membrane fractions. Four candidates were identified by overlap with chlorophyll autofluorescence, two were identified by fractionation, and one protein was selected for cloning and further study based its likely role in chloroplast membrane contact sites. Advisor: Rebecca L. Roston