Biogenesis, assembly, and regulation of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum

Many integral membrane proteins require specific organic cofactors that are essential for function. These cofactors must be transported from their site of biosynthesis or uptake in the cell and assembled with membrane apoproteins within the lipid bilayer. When cofactors are synthesized in the cell, cofactor and apoprotein synthesis is coordinately regulated. The focus of this thesis is the identification of proteins that are potentially involved in cofactor assembly or coordinate regulation of cofactor and apoprotein synthesis in integral membrane protein-cofactor complexes.;The archaeon Halobacterium salinarum produces bacteriorhodopsin (BR), an integral membrane protein with a bound retinal cofactor. The relative simplicity of the BR structure and the experimental tractability of H. salinarum make it an excellent model system to study cofactor assembly to apoproteins and regulation of cofactor biosynthesis. I developed an efficient method for gene-replacement in H. salinarum and used this method to make deletions in genes involved with metabolism of the retinal cofactor.;Deletion of the brp and blh genes resulted in a loss of BR and retinal synthesis, and increased accumulation of the immediate retinal precursor, beta-carotene. These data suggest that brp and blh encode proteins involved in the conversion of beta-carotene to retinal or transport of retinal to the apoprotein bacterioopsin (BO). Deletion of the H. salinarum crtY gene resulted in a loss of BR, retinal, and beta-carotene and increased accumulation of lycopene. Heterologous expression of crtY in E. coli was sufficient to convert lycopene to beta-carotene. These results indicated that crtY encodes a lycopene cyclase required for the synthesis of the retinal cofactor of (BR),;In summary, I have employed a deletion approach to identify three genes involved in the synthesis of the retinal cofactor of BR. Brp and Blh may be involved with the transport or assembly of retinal to BO. CrtY is likely the lycopene cyclase in H. salinarum, and this identification will allow the testing of the regulatory model that BO stimulates lycopene cyclase activity. The proteins identified and techniques developed in this thesis may provide insight into cofactor assembly with integral membrane proteins and coordinate regulation of cofactor and apoprotein synthesis.