The signal recognition particle receptor alpha subunit of the hyperthermophilic archaeon Acidianus ambivalens exhibits an intrinsic GTP-hydrolyzing activity.
Two adjacent genes of the acidophilic and hyperthermophilic crenarchaeon Acidianus ambivalens were cloned and sequenced. The 1.6 kb genomic nucleotide sequence under investigation consists of the 1.12 kb SRa gene encoding the putative signal recognition particle receptor alpha subunit (SR alpha, 42.2 kDa) and the 186 basepair secE gene coding for the putative secretory component secE subunit (6800 Da). The SR alpha protein is structured by three distinct regions: the N-terminal hydrophilic H-region, the following X-region and the C-terminal GTP-binding domain. A polyclonal anti-E. coli lacZ/A. ambivalens SR alpha antiserum detects a 51 kDa cell protein (p51) on immunoblots. Proteolysis of the recombinant SR alpha protein by Proteinase K produces a 31.6 kDa protease-resistant protein fragment comprising X-region and G-domain. The protein binds tightly to the GTP-agarose affinity matrix in a temperature-dependent manner. It hydrolyzes GTP readily at higher temperatures only in the presence of Mg2+. Point mutations (T326N) and (D329A) in the G-4 element of A. ambivalens SR alpha G-domain diminish the GTPase activity significantly. In contrast, the deletion mutant protein SR alpha (delta1-92) lacking the hydrophilic H-region displays a higher GTP-hydrolyzing activity when compared to the unmodified recombinant protein. Addition of GDP greatly inhibits GTP hydrolysis in mutant and unmodified A. ambivalens SR alpha.
Moll
R
R
Institut für Biochemie, Medizinische Universität zu Lübeck, Germany.moll@physik.mu-luebeck.de
Schmidtke
S
S
Petersen
A
A
Schäfer
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry; D008969:Molecular Sequence Data; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics; D018271:Signal Recognition Particle / Q000737:chemistry*; D016943:Sulfolobaceae / Q000737:chemistry* / Q000235:genetics
D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D018271:Signal Recognition Particle; C036318:signal peptide receptor; D006160:Guanosine Triphosphate; D006867:Hydrolases