Targeting of EGFP chimeras within chloroplasts
Targeting of EGFP chimeras within chloroplasts
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DOI：
10.1007/s00438-003-0846-y
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发表时间：
2003-06-01
影响因子：
3.1
通讯作者：
Klösgen, RB
中科院分区：
文献类型：
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作者：
Marques, JP;Dudeck, I;Klösgen, RB
我们测试了 EGFP（绿色荧光蛋白 (GFP) 的衍生物）作为过客蛋白的潜力，用于分析叶绿体中跨类囊体膜的蛋白质运输过程。与此类研究中常用的大多数融合蛋白相反，EGFP 不是植物来源的，因此可以假设其行为类似于“中性”过客蛋白，不受任何内部植物调节电路的影响。我们的体外转运实验清楚地表明，EGFP 是一种合适的过客蛋白，如果与适当的转运肽融合，可以正确靶向基质或类囊体腔。然而，EGFP 穿过类囊体膜的运输显示出明显的路径偏好。虽然该蛋白质被 DeltapH/TAT 途径有效靶向，但无论是用分离的类囊体还是用完整的叶绿体，几乎检测不到 Sec 途径的转运。该途径的特异性表明 EGFP 在导入叶绿体基质后立即折叠，从而防止 Sec 易位酶进一步跨类囊体膜易位。获得的数据为开发使用 EGFP 作为过客蛋白的运输研究分子工具提供了良好的基础。此外，表达相应EGFP嵌合体的植物系有望允许对叶绿体生物发生中涉及的运输和分选机制进行体内研究。
We have tested the potential of EGFP, a derivative of the green fluorescent protein (GFP), as a passenger protein for the analysis of protein transport processes across the thylakoid membranes in chloroplasts. In contrast to the majority of fusion proteins commonly used in such studies, EGFP is not of plant origin and can therefore be assumed to behave like a 'neutral' passenger protein that is unaffected by any internal plant regulatory circuits. Our in vitro transport experiments clearly demonstrate that EGFP is a suitable passenger protein that can be correctly targeted either to the stroma or to the thylakoid lumen if fused to the appropriate transit peptide. The transport of EGFP across the thylakoid membrane shows, however, a clear pathway preference. While the protein is efficiently targeted by the DeltapH/TAT pathway, transport by the Sec pathway is barely detectable, either with isolated thylakoids or with intact chloroplasts. This pathway specificity suggests that EGFP is folded immediately after import into the chloroplast stroma, thus preventing further translocation across the thylakoid membrane by the Sec translocase. The data obtained provide a good basis for the development of molecular tools for transport studies using EGFP as a passenger protein. Furthermore, plant lines expressing corresponding EGFP chimeras are expected to allow in vivo studies on the transport and sorting mechanisms involved in the biogenesis of the chloroplast.