Fouquaert Elke, Hanton Sally L, Brandizzi Federica, Peumans Willy J, Van Damme Els J M
Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, B-9000 Gent, Belgium.
Plant Cell Physiol. 2007 Jul;48(7):1010-21. doi: 10.1093/pcp/pcm071. Epub 2007 Jun 13.
The Galanthus nivalis agglutinin (GNA) is synthesized as a preproprotein. To corroborate the role of the different targeting peptides in the topogenesis of GNA and related proteins, different constructs were made whereby both the complete original GNA gene and different truncated sequences were coupled to the enhanced green fluorescent protein (EGFP). In addition, a GNA ortholog from rice that lacks the signal peptide and C-terminal propeptide sequence was fused to EGFP. These fusion constructs were expressed in tobacco BY-2 cells and their localization analyzed by confocal fluorescence microscopy. We observed that the processed preproprotein of GNA was directed towards the vacuolar compartment, whereas both the truncated forms of GNA corresponding to the mature lectin polypeptide and the rice ortholog of GNA were located in the nucleus and the cytoplasm. It can be concluded, therefore, that removal of the C-terminal propeptide and the signal peptide is sufficient to change the subcellular targeting of a normally vacuolar protein to the nuclear/cytoplasmic compartment of the BY-2 cells. These findings support the proposed hypothesis that cytoplasmic/nuclear GNA-like proteins and their vacuolar homologs are evolutionarily related and that the classical GNA-related lectins might have evolved from cytoplasmic orthologs through an evolutionary event involving the insertion of a signal peptide and a C-terminal propeptide.
雪花莲凝集素（GNA）最初以前体蛋白原的形式合成。为了证实不同靶向肽在GNA及相关蛋白的拓扑结构形成中的作用，构建了不同的结构，将完整的原始GNA基因和不同的截短序列与增强型绿色荧光蛋白（EGFP）偶联。此外，将来自水稻的缺少信号肽和C端前肽序列的GNA直系同源物与EGFP融合。这些融合构建体在烟草BY-2细胞中表达，并通过共聚焦荧光显微镜分析其定位。我们观察到，加工后的GNA前体蛋白原被导向液泡区室，而对应于成熟凝集素多肽的GNA截短形式以及GNA的水稻直系同源物则位于细胞核和细胞质中。因此可以得出结论，去除C端前肽和信号肽足以将正常定位于液泡的蛋白的亚细胞靶向改变为BY-2细胞的核/细胞质区室。这些发现支持了所提出的假说，即细胞质/核内的类GNA蛋白及其液泡同源物在进化上相关，并且经典的GNA相关凝集素可能是通过涉及信号肽和C端前肽插入的进化事件从细胞质直系同源物进化而来的。
