Cellular Localization and Biochemical Characterization of a Chimeric Fluorescent Protein Fusion of Arabidopsis Cellulose Synthase-Like A2 Inserted into Golgi Membrane
Cellular Localization and Biochemical Characterization of a Chimeric Fluorescent Protein Fusion of Arabidopsis Cellulose Synthase-Like A2 Inserted into Golgi Membrane
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DOI：
10.1155/2014/792420
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发表时间：
2014-01-01
影响因子：
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通讯作者：
Piro, Gabriella
中科院分区：
文献类型：
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作者：
De Caroli, Monica;Lenucci, Marcello S.;Piro, Gabriella
纤维素合成酶(CSL)基因被认为编码合成细胞壁基质多糖的酶。CSLA亚家族可能参与了β-甘露聚糖的生物合成。本文报道了拟南芥CSLA家族成员AtCslA2的细胞定位和酶活性的研究。我们发现，在烟草原生质体中瞬时表达的荧光蛋白融合蛋白AtCslA2-GFP在内质网中合成，并在高尔基体中积累。该嵌合体被插入高尔基体膜中，通过转化原生质体获得的膜制剂进行了以GDP-d-[U-C-14]甘露糖为底物的C-14-甘露聚糖的体外合成。与野生型相比，转化原生质体中的酶比活力提高了约38%。用蛋白酶K、生化数据和TM结构域预测的初步测试表明，AtCslA2的催化部位面向高尔基体腔。
Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily of CslA is putatively involved in the biosynthesis of beta-mannans. Here we report a study on the cellular localization and the enzyme activity of an Arabidopsis CslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out the in vitro synthesis of a C-14-mannan starting from GDP-d-[U-C-14] mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.