Nahm Min Yeop, Kim Sam Woong, Yun Daejin, Lee Sang Yeol, Cho Moo Je, Bahk Jeong Dong
Division of Applied Life Science (BK21), Gyeongsang National University, Jinju, 660-701 Korea.
Plant Cell Physiol. 2003 Dec;44(12):1341-9. doi: 10.1093/pcp/pcg163.
Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.
Rab7是一种小GTP结合蛋白，在哺乳动物细胞早期到晚期内体/溶酶体囊泡运输中起重要作用。我们通过消减筛选法从冷处理的水稻cDNA文库中分离出一个Rab7 cDNA克隆，即OsRab7。该cDNA编码一个206个氨基酸的多肽，计算分子量约为23 kDa。其预测的氨基酸序列与其他Rab7蛋白的序列具有显著的高度同一性。His标签的OsRab7以特异性和化学计量的方式与放射性标记的GTPγS结合。将Rab7野生型（WT）蛋白的生化和结构特性与Q67L和T22N突变体的特性进行了比较。去污剂3-([3-胆酰胺丙基]二甲基铵)-1-丙烷磺酸盐（CHAPS）增加了Rab7WT的鸟嘌呤核苷酸结合和水解活性。与未用CHAPS处理的WT蛋白相比，OsRab7Q67L突变体的GTP酶活性低得多，而T22N突变体根本没有GTP结合活性。OsRab7Q67L突变体在鸟嘌呤核苷酸结合方面具有组成型活性，而T22N突变体（显性负性）则没有鸟嘌呤核苷酸结合活性。当与GTP结合时，Rab7WT和Q67L突变体受到胰蛋白酶消化的保护。然而，Rab7T22N突变体的切割模式不受GTP添加的影响。Northern和Western印迹分析表明，OsRab7分布在水稻的各种组织中。此外，水稻Rab7基因的表达受到冷、NaCl、脱水和ABA等各种环境刺激的差异调节。此外，通过使用GFP融合的OsRab7和FM4-64的双标记实验，在拟南芥原生质体中研究了OsRab7的亚细胞定位。GFP-OsRab7定位于液泡膜，表明OsRab7参与植物细胞中向液泡的囊泡运输。
