brp and blh Are Required for Synthesis of the Retinal Cofactor of Bacteriorhodopsin in Halobacterium salinarum

Abstract

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the ⌬brp strain, bacteriorhodopsin levels are decreased ϳ4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, ␤-carotene, is increased ϳ3.8-fold, whereas retinal is decreased by ϳ3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the ⌬brp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of ␤-carotene increased ϳ5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of ␤-carotene to retinal.Rhodopsins are integral membrane proteins containing seven transmembrane ␣-helices and a covalently bound molecule of retinal. Two distinct rhodopsin families are known: the visual rhodopsins, which bind 11-cis retinal or related compounds and function as photoreceptors in vertebrates (1) and invertebrates (2), and the archaeal rhodopsins, which bind all-trans-retinal and function as light-driven ion pumps and phototaxis receptors in archaea (3). Archaeal rhodopsin orthologs have been found recently in bacteria (4) and fungi (5), suggesting that retinal-based pigments are of widespread significance. Despite their importance, the biogenesis of these molecules is not fully understood. In particular, relatively little is known about how retinal is assembled with the opsin apoprotein in vivo. Thus, a goal in elucidating rhodopsin biogenesis is to identify the cellular factors that mediate the biosynthesis or uptake of retinal, the transport of retinal in the cell, and the binding of retinal to the corresponding opsin.To this end, we have studied the biogenesis of bacteriorhodopsin (BR), 1 a light-driven proton pump in the archaeon Halobacterium salinarum. BR consists of the membrane protein bacterioopsin (BO) and all-trans-retinal. Under microaerobic conditions, BR is induced ϳ50-fold (6) and forms a twodimensional crystal known as the purple membrane. This system has served as a model for studying key steps in membrane protein biogenesis, including protein insertion into the membrane (7,8) and the assembly of protein-lipid complexes (9, 10). H. salinarum is genetically tractable, and the genome sequence of a closely related organism, Halo...

Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme Environment through Computational Analysis of Its Genome Sequence

“…Recent success in solving the structure of the large ribosomal subunit from a related halophile, Haloarcula marismortui (Ban et al 2000), genomic and genetic dissection of the archaeal regulon for purple membrane synthesis in Halobacterium NRC-1 (Baliga et al 2001), and reverse genetic analysis of carotenoid and retinal biosynthesis (Peck et al 2001) further solidify halophiles as viable and indispensable model systems in both prokaryotic and eukaryotic fields. Undoubtedly, on-going work using genetic arrays, gene knockouts, and further computational analysis will yield insights into life in extreme environments generally and adaptation to hypersaline environments specifically.…”

Retinal biosynthesis in Eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp. PCC 6803

“…Recent evidence indicates that in H. salinarum retinal is synthesized from b-carotene via an unknown mechanism. The halobacterial retinal biosynthesis requires two enzymes (Brp and Blh) exhibiting significant homology to each other but not to known carotenoid oxygenases (Peck et al, 2001). An example for a carotenoid derivative produced through two different pathways is represented by the phytohormone abscisic acid (ABA).…”

Parallel evolution of transcriptome architecture during genome reorganization

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