REQUIREMENTS FOR NUCLEAR-LOCALIZATION AND SUPRAMOLECULAR ASSEMBLY OF A BACULOVIRUS POLYHEDRIN PROTEIN
REQUIREMENTS FOR NUCLEAR-LOCALIZATION AND SUPRAMOLECULAR ASSEMBLY OF A BACULOVIRUS POLYHEDRIN PROTEIN
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DOI：
10.1016/0042-6822(91)90551-l
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发表时间：
1991-12-01
期刊：
影响因子：
3.7
通讯作者：
GARCIA, A
中科院分区：
文献类型：
--
作者：
JARVIS, DL;BOHLMEYER, DA;GARCIA, A
这项研究明确了杆状病毒多角体蛋白在鳞翅目昆虫细胞中的核定位、稳定的核结合和超分子组装的要求。将多角体蛋白片段与两种不同的非核报告蛋白进行基因融合，并分析融合蛋白在感染昆虫细胞中的分布。间接免疫荧光分析表明，30-57位氨基酸之间的结构域可介导多角体蛋白的核定位。然而，生化分级实验表明，这个结构域不足以使多角体蛋白与细胞核以洗涤剂稳定的方式结合。这需要一个稍大的结构域，介于30到110个氨基酸之间。差示干涉显微镜显示，多角体的超分子组装成核闭塞样颗粒需要氨基酸19和110之间的结构域。最有可能获得最小核定位信号的候选序列是KRKK序列，它位于氨基酸32和35之间。因此，采用寡核苷酸定向突变的方法将该序列改变为NGNN，并对突变蛋白的胞内分布进行了分析。结果表明，突变蛋白主要定位于感染细胞的细胞质中，在那里它组装成大的立方体闭塞样颗粒。因此，KRKK序列对于多角体的核定位是必要的，但它的超分子组装成闭塞样颗粒并不需要核定位。
This study defines the requirements for the nuclear localization, stable nuclear association, and supramolecular assembly of a baculovirus polyhedrin protein in lepidopteran insect cells. Fragments of the polyhedrin protein were genetically fused to two different nonnuclear reporter proteins and the intracellular distribution of the fusion proteins was analyzed in infected insect cells. Analysis by indirect immunofluorescence showed that the domain between amino acids 30 and 57 could mediate nuclear localization of polyhedrin. However, biochemical fractionation experiments showed that this domain was not sufficient for a detergent-stable association of polyhedrin with the nucleus. This required a slightly larger domain, between amino acids 30 and 110. Differential interference-contrast microscopy showed that the supramolecular assembly of polyhedrin into nuclear occlusion-like particles required the domain between amino acids 19 and 110. The most likely candidate for a minimal nuclear localization signal was the sequence KRKK, located between amino acids 32 and 35. Therefore, oligonucleotide-directed mutagenesis was used to change this sequence to NGNN and the intracellular distribution of the mutant protein was analyzed. The results showed that the mutant protein was predominantly localized in the cytoplasm of infected cells, where it assembled into large, cubic, occlusion-like particles. Thus, the KRKK sequence is necessary for the nuclear localization of polyhedrin, but nuclear localization is not required for its supramolecular assembly into occlusion-like particles.