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Biochemical characterization of rab proteins from Bombyx mori
Author(s) -
Uno Tomohide,
Moriwaki Tsubasa,
Nakamura Masahiko,
Matsubara Mamoru,
Yamagata Hiroshi,
Kanamaru Kengo,
Takagi Michihiro
Publication year - 2009
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.20273
Subject(s) - gtp' , rab , gtpase , biochemistry , biology , complementary dna , bombyx mori , gtp binding protein regulators , microbiology and biotechnology , enzyme , g protein , signal transduction , gene
The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT‐PCR to isolate cDNA clones of insect‐specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori . These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [ 3 H]‐GDP and [ 35 S]‐GTPγS with dissociation constants of 0.087 × 10 −6 M and 1.02 × 10 −6 M, respectively, whereas those of BRabN2 were 0.546 × 10 −6 M and 1.02 × 10 −6 M, respectively. Binding of [ 35 S]‐GTPγS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP‐hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [ 35 S]‐GTPγS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [ 35 S]‐GTPγS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti‐BRabN1 antibody revealed a single band in the testis of B. mori . These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP‐bound state to the GDP‐bound state by intrinsic GTP hydrolysis activity, and return to the GTP‐bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley‐Liss, Inc.