Di Sansebastiano G P, Paris N, Marc-Martin S, Neuhaus J M
University of Neuchâtel, Biochemistry Laboratory, Switzerland.
Plant J. 1998 Aug;15(4):449-57. doi: 10.1046/j.1365-313x.1998.00210.x.
The green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C-terminal endoplasmic reticulum (ER)-retention motif of mGFP5-ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18-24 h) depending on the cell type. Most chloroplast-rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast-poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non-acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.
在原生质体瞬时转化后，维多利亚多管水母的绿色荧光蛋白（GFP）可在活的植物细胞中被检测到。GFP的表达可用于监测混合细胞群体中的蛋白质运输，还可用于研究不同细胞类型中细胞器的不同功能和重要性。我们开发了一种液泡形式的GFP，它是通过用烟草几丁质酶A的液泡靶向肽取代mGFP5-ER的C末端内质网（ER）保留基序而获得的。液泡GFP如预期那样被运输并积累在液泡中。然而，长时间孵育（18 - 24小时）后，根据细胞类型我们发现了两种GFP积累模式。大多数富含叶绿体的原生质体有一个荧光大中央液泡。相反，大多数叶绿体含量低的原生质体将GFP积累在一个较小的液泡中，而不在大中央液泡中积累，在同一细胞的光学显微镜下可以看到这种情况。这种差异积累反映了最近通过几种液泡标记物的免疫定位所描述的两种不同液泡区室的存在。我们能够将GFP特异性靶向的液泡区室表征为非酸性的，因为它不积累中性红，而酸性液泡不积累GFP。
