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首页> 美国卫生研究院文献>Plant Physiology >Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI- Requires ATP and Does Not Proceed When Large Subunits Are Synthesized at Temperatures greater than or equal to32degC.
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Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI- Requires ATP and Does Not Proceed When Large Subunits Are Synthesized at Temperatures greater than or equal to32degC.

机译：在大于或等于32℃的温度下合成大亚基时将体外合成的大亚基组装成核糖二磷酸羧化酶/加氧酶对CI-敏感需要ATP并且不进行。

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In higher plants, ribulose bisphosphate carboxylase/oxygenase (Rubisco) consists of eight large "L" subunits, synthesized in chloroplasts, and eight small "S" subunits, synthesized as precursors in the cytosol. Assembly of these into holoenzyme occurs in the chloroplast stroma after import and processing of the S subunits. A chloroplast chaperonin interacts with the L subunits, which dissociate from the chaperonin before they assemble into holoenzyme. Our laboratory has reported L subunit assembly into Rubisco in chloroplast extracts after protein synthesis in leaves, intact chloroplasts, and most recently in membrane-free chloroplast extracts. We report here that the incorporation of in vitro-synthesized L subunits into holoenzyme depends on the conditions of L subunit synthesis. Rubisco assembly did not occur after L subunit synthesis at 160 mM KCI. When L subunit synthesis occurred at approximately 70 mM KCI, assembly depended on the temperature at which L subunit synthesis took place. These phenomena were the result of postsynthetic events taking place during incubation for protein synthesis. We separated these events from protein synthesis by lowering the temperature during protein synthesis. Lower temperatures supported the synthesis of full-length Rubisco L subunits. The assembly of these completed L subunits into Rubisco required intervening incubation with ATP, before addition of S subunits. ATP treatment mobilized L subunits from a complex with the chloroplast chaperonin 60 oligomer. Addition of 130 mM KCI at the beginning of the intervening incubation with ATP blocked the incorporation of L subunits into Rubisco. The inhibitory effect of high KCI was due to CI- and came after association of newly synthesized L subunits with chaperonin 60, but before S subunit addition. It is interesting that L subunits synthesized at [greater than or equal to]32[deg]C failed to assemble into Rubisco under any conditions. These results agree with previous results obtained in this laboratory using newly synthesized L subunits made in intact chloroplasts. They also show that assembly of in vitro-synthesized L subunits into Rubisco requires ATP, that CI- inhibits Rubisco assembly, and that synthesis temperature affects subsequent assembly competence of L subunits.
机译：在高等植物中，核糖二磷酸羧化酶/加氧酶（Rubisco）由在叶绿体中合成的八个大“ L”亚基和在胞质溶胶中作为前体合成的八个小“ S”亚基组成。这些在S亚基的导入和加工后，在叶绿体基质中组装成全酶。叶绿体伴侣蛋白与L亚基相互作用，后者在结合成全酶之前从伴侣蛋白解离。我们的实验室报告了在叶片，完整叶绿体以及最近在无膜叶绿体提取物中蛋白质合成后，叶绿体提取物中的L亚基组装到Rubisco中。我们在这里报告，体外合成的L亚基掺入全酶取决于L亚基合成的条件。在160 mM KCI合成L亚基后，Rubisco组装未发生。当L亚基合成发生在大约70 mM KCI时，组装取决于L亚基合成发生的温度。这些现象是在蛋白质合成孵育过程中发生的后合成事件的结果。我们通过降低蛋白质合成过程中的温度来将这些事件与蛋白质合成分开。较低的温度支持全长Rubisco L亚基的合成。这些完整的L亚基组装成Rubisco之前，需要先与ATP进行温育，然后再添加S亚基。 ATP处理从具有叶绿体伴侣蛋白60寡聚物的复合物中动员了L亚基。在与ATP的中间孵育开始时添加130 mM KCI阻止了L亚基掺入Rubisco。高KCI的抑制作用是由于CI-引起的，并且是在新合成的L亚基与伴侣蛋白60缔合后，但在添加S亚基之前。有趣的是，在大于或等于32℃下合成的L个亚基在任何条件下都不能组装成Rubisco。这些结果与在实验室中使用完整的叶绿体中新合成的L亚基获得的先前结果一致。他们还表明，将体外合成的L亚基组装成Rubisco需要ATP，CI-抑制Rubisco组装，并且合成温度会影响L亚基的后续组装能力。

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