IN-VITRO MUTATION ANALYSIS OF ARABIDOPSIS-THALIANA SMALL GTP-BINDING PROTEINS AND DETECTION OF GAP-LIKE ACTIVITIES IN PLANT-CELLS

被引:0
作者
ANAI, T
MATSUI, M
NOMURA, N
ISHIZAKI, R
UCHIMIYA, H
机构
[1] UNIV TOKYO, INST MOLEC & CELLULAR BIOSCI, BUNKYO KU, TOKYO 113, JAPAN
[2] HOKKAIDO UNIV, FAC SCI, DEPT BOT, KITA KU, SAPPORO 060, HOKKAIDO, JAPAN
[3] NIPPON MED COLL, INST GERONTOL, NAKAHARA KU, KAWASAKI 211, KANAGAWA, JAPAN
关键词
SMALL GTP-BINDING PROTEIN; GTPASE; YPT/RAB; SITE-DRECTED MUTAGENESIS; GAP; ARABIDOPSIS THALIANA;
D O I
暂无
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana. The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type. To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into nra cDNAs. Mutant proteins were expressed in E. coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro. The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding. A point mutation of Asn at position 72 for ARA-2 or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gin at position 126 for ARA-2, or 125 for ARA-4 to Leu suppressed GTP-hydrolysis activity. Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells.
引用
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页码:175 / 180
页数:6
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