The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids
The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids
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DOI：
10.1105/tpc.109.068288
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发表时间：
2009-09-01
期刊：
影响因子：
11.6
通讯作者：
Mitsui, Toshiaki
中科院分区：
文献类型：
--
作者：
Kitajima, Aya;Asatsuma, Satoru;Mitsui, Toshiaki
水稻分泌糖蛋白--水稻α-淀粉酶亚型I-1(AmyI-1)定位于质体内，参与水稻细胞器中淀粉粒的降解。此外，大量瞬时表达的AmyI-1与绿色荧光蛋白(AmyI-1-GFP)在洋葱(Allium Cepa)表皮细胞中与同时表达的荧光质体标记共定位。拟南芥ARF1和拟南芥SAR1的显性负性突变体和结构性活性突变体都抑制了AmyI-1的质体靶向性，它们阻止了内质网到高尔基体的运输。在表达反式高尔基体和叶绿体荧光标记的细胞中，当与AmyI-1共表达时，这些荧光标记经常共定位。高压冷冻/冷冻替代细胞的三维时间推移成像和电子显微镜显示，高尔基体衍生的膜小泡与货物接触，随后被吸收到质体中。一系列C端截短的AmyI-1-GFP融合蛋白在洋葱细胞系统中的瞬时表达表明，Trp-301到Gln-369区域是AmyI-1的质体靶向所必需的。此外，定位于AmyI-1蛋白表面和两侧的Trp-302和Gly-354的定点突变所获得的结果表明，多个表面区是叶绿体靶向所必需的。因此，高尔基体到叶绿体的运输似乎参与了糖蛋白到叶绿体的运输，而叶绿体的靶向似乎是以一种分类信号依赖的方式完成的。
The well-characterized secretory glycoprotein, rice (Oryza sativa) alpha-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner.