n nnnThe well-characterized secretory glycoprotein, rice (Oryza sativa)-amylase isoform I-1 (AmyI-1), was localized within the plastidsand proved to be involved in the degradation of starch granulesin the organelles of rice cells. In addition, a large portionof transiently expressed AmyI-1 fused to green fluorescent protein(AmyI-1-GFP) colocalized with a simultaneously expressed fluorescentplastid marker in onion (Allium cepa) epidermal cells. The plastidtargeting of AmyI-1 was inhibited by both dominant-negativeand constitutively active mutants of Arabidopsis thaliana ARF1and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgitraffic. In cells expressing fluorescent trans-Golgi and plastidmarkers, these fluorescent markers frequently colocalized whencoexpressed with AmyI-1. Three-dimensional time-lapse imagingand electron microscopy of high-pressure frozen/freeze-substitutedcells demonstrated that contact of the Golgi-derived membranevesicles with cargo and subsequent absorption into plastidsoccur within the cells. The transient expression of a seriesof C-terminal-truncated AmyI-1-GFP fusion proteins in the onioncell system showed that the region from Trp-301 to Gln-369 isnecessary for plastid targeting of AmyI-1. Furthermore, theresults 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 plastidtargeting. Thus, Golgi-to-plastid traffic appears to be involvedin the transport of glycoproteins to plastids and plastid targetingseems to be accomplished in a sorting signal–dependentmanner.展开▼
机译:ABSTRACTn FONT> TH> TR> TABLE> n
n TOP n <字体颜色= 464c53>抽象 FONT> n 介绍 n 结果 n 讨论 n 方法 n 参考文献 n FONT> TH> TR> TABLE> n nnn特征明确的分泌糖蛋白水稻( Oryza sativa I>) SUP> -淀粉酶同工型I-1(AmyI-1)位于质体中 < / SUP>,并证明它参与了稻细胞细胞器中淀粉颗粒 SUP>的降解。此外,与绿色荧光蛋白 SUP>(AmyI-1-GFP)融合的大部分瞬时表达的AmyI-1与同时表达的荧光 SUP共定位>洋葱(葱属 I>)表皮细胞中的质体标记。 拟南芥 I> ARF1 SUP>的显性负性 SUP>和组成型活性突变体均抑制AmyI-1的质体 SUP>。和拟南芥 I> SAR1,它们阻止了内质网向高尔基体的流动[SUP> SUP>。在表达荧光 trans I>-高尔基体和质体 SUP>标记的细胞中,当 SUP>与AmyI-1共表达时,这些荧光标记经常共定位。高压冷冻/冷冻取代的 SUP>细胞的三维延时成像 SUP>和电子显微镜观察表明,高尔基体细胞膜 SUP>的接触带有货物的囊泡随后被吸收进入质体 SUP>。一系列 SUP> C末端截短的AmyI-1-GFP融合蛋白在洋葱 SUP>细胞系统中的瞬时表达表明,从Trp-301到Gln-369的区域 SUP>是AmyI-1质体靶向所必需的。此外,通过位于AmyI-1蛋白表面和相对侧的Trp-302和Gly-354的定点突变获得的 SUP>结果, SUP> > SUP>建议质体 SUP>定位需要多个表面区域。因此,高尔基体到质体的运输似乎参与了糖蛋白向质体的运输,而质体靶向 SUP>似乎是通过依赖于分类信号的 < / SUP>方式。 SUP>
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Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Saitama 351-0198, Japan|Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan;
Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan;