Fig. 2. dRheb is a highly conserved GTPase. Sequence alignment of S. pombe, D. melanogaster and two human Rheb proteins by Clustal (A) indicates that dRheb is a highly conserved Ras-like GTPase. The G boxes (G1-G5) and the CaaX box are indicated by lines above the sequences; asterisks and dots indicate identical and similar residues, respectively (A). GTP binding of dRheb was examined by incubating His-tagged dRheb with [-³⁵S]GTP in binding buffer in the presence of 1 mM MgCl₂; radioactivity bound to dRheb was assessed as described in Materials and Methods (B). To examine nucleotide specificity, radioactivity in [-³⁵S]GTP bound to His-tagged dRheb in the presence of 20-fold excess unlabeled ATP, CTP, GTP, UTP or GDP was compared with [-³⁵S]GTP bound in the absence of nucleotide (set to 100%), as described in Materials and Methods (C). GTPase activity of dRheb was examined as described in Materials and Methods: His-tagged dRheb was preloaded with [-³²P]GTP in 1 mM MgCl₂ and hydrolysis initiated by increasing MgCl₂ concentration to 10 mM; bound radioactivity was determined by nitrocellulose filter assay (D).