The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain
The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain
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DOI：
10.1105/tpc.000620
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发表时间：
2002-05-01
期刊：
影响因子：
11.6
通讯作者：
Paris, N
中科院分区：
文献类型：
--
作者：
Brandizzi, F;Frangne, N;Paris, N
tono质体被认为是没有特异性靶向信号的膜结合蛋白的默认目的地。为了研究这种靶向的性质，我们创建了I型融合蛋白，其中包含绿色荧光蛋白，然后是人溶酶体蛋白LAMP1的跨膜结构域。我们改变跨膜结构域的长度从23到20或17个氨基酸在疏水区域内的缺失。由此产生的嵌合体TM23、TM20和TM17在烟草中短暂或稳定地表达。免疫电镜证实质膜内明显积聚TM23。相比之下，TM17明显保留在内质网中，TM20积聚在小的移动结构中。通过与主要定位于高尔基体的标记物terd2融合到黄色荧光蛋白的共表达，研究了tm20标记的区室的性质。两种荧光蛋白的严格共定位表明TM20在高尔基体中积累。为了进一步测试I型膜蛋白的默认目的地，我们将绿色荧光蛋白融合到植物液泡分选受体BP-80的19个氨基酸跨膜结构域。由此产生的嵌合体也在高尔基体中积累，而不是在原生BP-80定位的后高尔基区室中积累。此外，当BP-80的跨膜结构域延长至22个氨基酸时，报告蛋白逃离高尔基体并在质膜中积累。因此，tono质体显然不是植物中I型膜蛋白的首选默认目的地。此外，嵌合体聚集的靶膜不是唯一的，至少部分取决于膜跨越域的长度。
The tonoplast was proposed as a default destination of membrane-bound proteins without specific targeting signals. To investigate the nature of this targeting, we created type I fusion proteins with green fluorescent protein followed by the transmembrane domain of the human lysosomal protein LAMP1. We varied the length of the transmembrane domain from 23 to either 20 or 17 amino acids by deletion within the hydrophobic domain. The resulting chimeras, called TM23, TM20, and TM17, were expressed either transiently or stably in tobacco. TM23 clearly accumulated in the plasmalemma, as confirmed by immunoelectron microscopy. In contrast, TM17 clearly was retained in the endoplasmic reticulum, and TM20 accumulated in small mobile structures. The nature of the TM20-labeled compartments was investigated by coexpression with a marker localized mainly in the Golgi apparatus, AtERD2, fused to a yellow fluorescent protein. The strict colocalization of both fluorescent proteins indicated that TM20 accumulated in the Golgi apparatus. To further test the default destination of type I membrane proteins, green fluorescent protein was fused to the 19-amino acid transmembrane domain of the plant vacuolar sorting receptor BP-80. The resulting chimera also accumulated in the Golgi instead of in post-Golgi compartments, where native BP-80 localized. Additionally, when the transmembrane domain of BP-80 was lengthened to 22 amino acids, the reporter escaped the Golgi and accumulated in the plasma membrane. Thus, the tonoplast apparently is not a favored default destination for type I membrane proteins in plants. Moreover, the target membrane where the chimera concentrates is not unique and depends at least in part on the length of the membrane-spanning domain.