Phytoene synthase (PSY) converts two molecules of geranyl-geranyl diphosphate to phytoene, the key regulatory step in carotenogenesis. However, post-translational mechanisms that control PSY expression are scarcely understood. Carotenoid biosynthesis (mainly bacterioruberin) is a distinctive feature of haloarchaea thriving in hypersaline environments. Carotenogenesis is negatively regulated by the AAA+ LonB protease in the haloarchaeon Haloferax volcanii as it controls PSY degradation. We investigated the relevance of the C-terminal portion of HvPSY as a regulatory element for carotenoid biosynthesis. H. volcanii mutants were constructed to express full-length HvPSY protein (strain HVPSYwt) and truncated HvPSY lacking 10 (HVPSY10), 20 (HVPSY20) or 34 amino acids (HVPSY34) at the C-terminus. Cells of HVPSY20 and HVPSY34 showed hyperpigmentation (bacterioruberin content 3-fold higher than HVPSYwt) which correlated with increased PSY protein abundance (2-fold in HVPSY34) while they contained less psy transcript level compared with HVPSYwt. In vivo degradation assays showed that HvPSY34 was more stable than HvPSYwt. Collectively, these results show that the C-terminal region of HvPSY contains a ‘recognition determinant’ for proteolysis in H. volcanii. Preliminary evidence suggests that LonB is involved in the recognition mechanism. This study provides the first identification of a regulatory sequence in an archaeal PSY for the post-translational control of carotenogenesis.

中文翻译：

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八氢番茄红素合成酶的 C 端区域是控制盐生古菌 Haloferax volcanii 类胡萝卜素生物合成的关键因素

八氢番茄红素合酶 (PSY) 将两个香叶基-香叶基二磷酸分子转化为八氢番茄红素，这是胡萝卜素生成中的关键调节步骤。然而，人们对控制 PSY 表达的翻译后机制知之甚少。类胡萝卜素生物合成（主要是细菌红素）是在高盐环境中繁衍生息的盐古菌的一个显着特征。类胡萝卜素生成受 haloarchaeon Haloferax volcanii 中 AAA+ LonB 蛋白酶的负调控，因为它控制 PSY 降解。我们调查了 HvPSY 的 C 端部分作为类胡萝卜素生物合成调节元件的相关性。H. volcanii 突变体被构建为表达全长 HvPSY 蛋白（菌株 HVPSYwt）和在 C 末端缺少 10 (HVPSY10)、20 (HVPSY20) 或 34 个氨基酸 (HVPSY34) 的截短 HvPSY。HVPSY20 和 HVPSY34 的细胞显示出色素沉着过度（细菌红素含量比 HVPSYwt 高 3 倍），这与 PSY 蛋白丰度增加（HVPSY34 中的 2 倍）相关，而与 HVPSYwt 相比，它们含有较少的 psy 转录物水平。体内降解测定显示 HvPSY34 比 HvPSYwt 更稳定。总的来说，这些结果表明，HvPSY 的 C 端区域包含 H. volcanii 蛋白水解的“识别决定簇”。初步证据表明 LonB 参与识别机制。本研究首次鉴定了古细菌 PSY 中用于胡萝卜素生成的翻译后控制的调控序列。