The Role of Non-Bilayer Forming Lipids in Chloroplast Protein Transport

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9604535 Bruce Technical This proposal addresses fundamental questions regarding the mechanism of protein translocation across membranes. Although the protein transport system being investigated is the targeting and translocation of a nuclear encoded protein into higher plant chlormplasts, the underlying mechanisms are potentially relevant to all protein transport systems. The primary focus is to investigate the role of the non-bilayer forming lipid, monogalactosyl-diacyglycerol (MGDG), in chloroplast protein transport. We propose that chloroplast precursors have evolved specific domains in their transit peptide that interact specifically with MGDG to alter the bilayer organization. Transit peptide-lipid interactions may be transient, temporally and spatially, yet may represent an essential first step in chloroplast protein transport, and perhaps in all transport processes. Our preliminary data indicates that the precursor to the small subunit of Rubisco (prSSU) contains a lipid interacting domain that is located primarily at the C-terminus of the transit peptide (SS-tp). Using liposomes whose composition can be controlled, we have shown by electron microscopy and fluorescence measurements that the ability of both purified prSSU and the full-length SS-tp to disrupt bilayers is totally dependent on the presence of the non-bilayer forming lipid, MGDG. The experiments outlined in this proposal are a direct extension of our in vitro analysis of the SS-tp/membrane interaction. The first objective is to refine the mapping of prSSU sequences that are responsible for lipid interaction/membrane destabilization. A complete set overlapping peptides will be synthesized that span SS-tp entirely and extend into the mature domain of SSU. In this way, the sequences of prSSU that are both necessary and sufficient for lipid interaction will be determined precisely and the potential involvement of sequences in the mature domain will be addressed. The second objective is to examine the chemical proper ties of MGDG that account for its role in transit peptide-mediated membrane destabilization. By utilizing liposomes containing alternative non-bilayer forming lipids such as phosphatidylethanolamine and cardiolipin, it will be possible to distinguish between the non-bilayer forming property of MGDG vs. other traits, such as hydrogen bonding activity of the qugar head group. The third objective is to alter the non-bilayer forming tendency of MGDG in vivo by utilizing Arabidopsis mutants that are deficient in one or more of their fatty acid desaturases fad. These fad mutants are ideal for our purpose since earlier in viro research clearly indicates that MGDG prefers to form non-bilayer structures only when its fatty acids are highly unsaturated. By utilizing various single, double, and triple fad mutants, we can produce chloroplasts that contain MGDG variants which are predicted to vary in their preferences to form non-bilayer structures. These mutants will ppovide the most intact system to directly evaluate the role of non-bilayer structures in chloroplast protein translocation. Non-technical Biological membranes consist of a bilayer of lipids in which are embedded proteins. In order to assemble intracellular structures it is necessary for proteins to be translocated through the membrane. Much research has been directed at determining the protein components of membranes that facilitate the movement and processing of proteins as they transit through a membrane. Dr. Bruce will look at this process from a different perspective and he will conduct research to examine what lipid components are essential to facilitate trafficking of chloroplast proteins. The chloroplast is a useful model system to conduct these studies because chloroplasts possess unique lipids not found in other intracellular organelles. Dr. Bruce will use mutants of lipid synthesis in order to provide experimental models to probe the process of protein translocation into the chloroplast. This work is important because it provides a basis of the fundamental process of the assembly of intracellular organelles. ***