He Q, Schlich T, Paulsen H, Vermaas W
Department of Plant Biology and Center for the Study of Early Events in Photosynthesis, Arizona State University, Box 871601, Tempe, AZ 85287-1601, USA.
Eur J Biochem. 1999 Jul;263(2):561-70. doi: 10.1046/j.1432-1327.1999.00526.x.
A chimeric lhcb gene, coding for Lhcb, a higher plant chlorophyll a/b-binding light-harvesting complex of photosystem II (LHCII), was constructed using the Synechocystis sp. PCC 6803 psbA3 promoter and a modified lhcb gene from pea. This construct drives synthesis of full-length, mature Lhcb under the control of the strong psbA3 promoter that usually drives expression of the D1 protein of photosystem II. This chimeric gene was transformed into a photosystem I-less/chlL(-) Synechocystis sp. PCC 6803 strain that is unable to synthesize chlorophyll in darkness. In the resulting strain, a high level of lhcb transcript was detected and transcript accumulation was enhanced by addition of exogenous Zn-chlorophyllide b. The chimeric lhcb gene was translated to produce full-length Lhcb as demonstrated by pulse-labeling: a new radioactively labeled band of a size corresponding to full-length Lhcb was visible on autoradiograms. Using Triton X-114 phase fractionation, this labeled protein band was found to partition to the phase containing integral membrane proteins, indicating that the pulse-labeled Lhcb is readily integrated into the membrane. However, Lhcb was rapidly degraded and did not accumulate in thylakoid membranes to levels that were detectable other than by pulse labeling. Upon immunological detection with LHCII antibodies, a small protein (approximately 8 kDa) was found specifically in the lhcb-containing mutant. We interpret this protein to be a degradation product of the full-length Lhcb. This fragment was stabilized by supplementing cells with xanthophylls, which incorporated into thylakoid membranes only in the mutant carrying lhcb. The lutein/chlorophyll ratio of thylakoids of this mutant was about 1 : 10. These results indicate that in this cyanobacterial system Lhcb is synthesized, integrated into the membrane, and then degraded to a approximately 8 kDa fragment that is stabilized by pigment binding and does not require the presence of chlorophyll b.
利用集胞藻PCC 6803的psbA3启动子和来自豌豆的一个经过修饰的lhcb基因，构建了一个嵌合的lhcb基因，该基因编码Lhcb，即高等植物光系统II（LHCII）的叶绿素a/b结合捕光复合体。此构建体在通常驱动光系统II的D1蛋白表达的强psbA3启动子的控制下驱动全长成熟Lhcb的合成。将这个嵌合基因转化到一个无光系统I/ chlL(-)的集胞藻PCC 6803菌株中，该菌株在黑暗中无法合成叶绿素。在所得菌株中，检测到高水平的lhcb转录本，并且通过添加外源锌叶绿酸b可增强转录本积累。脉冲标记表明，嵌合的lhcb基因被翻译产生全长Lhcb：在放射自显影片上可见一条大小与全长Lhcb相对应的新的放射性标记条带。使用Triton X-114相分离法，发现这条标记蛋白条带分配到含有整合膜蛋白的相中，这表明脉冲标记的Lhcb很容易整合到膜中。然而，Lhcb迅速降解，并且没有在类囊体膜中积累到除脉冲标记外可检测到的水平。用LHCII抗体进行免疫检测时，在含有lhcb的突变体中特异性地发现了一种小蛋白（约8 kDa）。我们将这种蛋白解释为全长Lhcb的降解产物。通过用叶黄素补充细胞使该片段稳定，叶黄素仅在携带lhcb的突变体中掺入类囊体膜。该突变体类囊体的叶黄素/叶绿素比率约为1:10。这些结果表明，在这个蓝细菌系统中，Lhcb被合成、整合到膜中，然后降解为一个约8 kDa的片段，该片段通过色素结合而稳定，并且不需要叶绿素b的存在。
