Journal Article

Molecular and Biochemical Analyses of <i>OsRab7</i>, a Rice <i>Rab7</i> Homolog

Min Yeop Nahm, Sam Woong Kim, Daejin Yun, Sang Yeol Lee, Moo Je Cho and Jeong Dong Bahk

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 44, issue 12, pages 1341-1349
Published in print December 2003 | ISSN: 0032-0781
Published online December 2003 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcg163
Molecular and Biochemical Analyses of OsRab7, a Rice Rab7 Homolog

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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 GTPγS 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.

Keywords: Keywords: Dominant negative mutant — Environmental stimuli — Oryza sativa Rab7 — Tryptic proteolysis — Vesicular transport.; Abbreviations: CHAPS, 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate; DTT, dithiothreitol; GAP, GTPase activating protein; GDI, GDP dissociation inhibitor; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; OsRab7, Oryza sativa Rab7; REP, Rab escort protein; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone.

Journal Article.  5663 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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