Cherry J R, Hondred D, Walker J M, Keller J M, Hershey H P, Vierstra R D
Department of Horticulture, University of Wisconsin-Madison 53706.
Plant Cell. 1993 May;5(5):565-75. doi: 10.1105/tpc.5.5.565.
A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.
一系列七个燕麦光敏色素A的羧基末端缺失突变体在转基因烟草中稳定表达，以定位参与生色团附着、光谱完整性、红光（Pr）吸收形式和远红光（Pfr）吸收形式之间的光可逆性、二聚化和生物活性的光敏色素结构域。体内生色团附着所需的氨基酸定位于氨基末端的398个残基，因为这么小的突变蛋白具有共价结合的生色团。从羧基末端到第653位残基的缺失突变体在光谱上与全长色素蛋白没有区别。相比之下，进一步截短至第399位残基会产生一种色素蛋白，其Pfr吸收光谱漂白，Pr和Pfr吸收最大值向较短波长移动，并且Pfr到Pr的光转化效率降低。因此，399至652位残基之间的序列是光谱完整性所必需的，但对于生色团附着不是必需的。919至1093位残基之间的序列似乎是二聚化所必需的。包含该区域的羧基末端突变体在体外非变性条件下表现为二聚体，而没有该区域的截短体表现为单体。即使截短的光敏色素表达水平比全长色素蛋白有效水平高6至15倍，表达高水平缺失35个羧基末端氨基酸的缺失突变体的植物中，没有一个表现出表达生物活性光敏色素A的植物所特有的光夸大表型。总体而言，这些数据表明，光敏色素蛋白包含几个结构/功能所需的可分离羧基末端结构域，并确定了羧基末端35个残基内的一个结构域，该结构域对体内光感受器的生物活性至关重要。
