Plant and Cell Physiology, 2003, Vol. 44, No. 12 1341-1349
© 2003 Oxford University Press
Molecular and Biochemical Analyses of OsRab7, a Rice Rab7 Homolog
1 Division of Applied Life Science (BK21), Gyeongsang National University, Jinju, 660-701 Korea
2 Research Institute of Natural Science, Gyeongsang National University, Jinju, 660-701 Korea
3 Agricutural Plant Stress Research Center, Chonnam National University, Gwangju, 500-757 Korea
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 GTP
S 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.
4 Corresponding author: E-mail, jdbahk{at}nongae.gsnu.ac.kr; Fax, +82-55-752-7062.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Limpens, S. Ivanov, W. van Esse, G. Voets, E. Fedorova, and T. Bisseling Medicago N2-Fixing Symbiosomes Acquire the Endocytic Identity Marker Rab7 but Delay the Acquisition of Vacuolar Identity PLANT CELL, September 1, 2009; 21(9): 2811 - 2828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhu, X. Fu, Y. D. Koo, J.-K. Zhu, F. E. Jenney Jr., M. W. W. Adams, Y. Zhu, H. Shi, D.-J. Yun, P. M. Hasegawa, et al. An Enhancer Mutant of Arabidopsis salt overly sensitive 3 Mediates both Ion Homeostasis and the Oxidative Stress Response Mol. Cell. Biol., July 15, 2007; 27(14): 5214 - 5224. [Abstract] [Full Text] [PDF]
|
|