Ribosome biogenesis in eukaryotes is a complex process that requires the participation of several accessory proteins that are not part of the mature particle. Efl1 is a yeast GTPase required for the cytoplasmic maturation of the 60S ribosomal subunit. Together with Sdo1, the yeast ortholog of the protein mutated in the Shwachman-Diamond Syndrome (SBDS), Efl1 releases the anti-association factor Tif6 from the surface of the 60S subunit allowing the assembly of mature ribosomes. We characterized the structural content and folding stability of the Saccharomyces cerevisiae and human EFL1 GTPases, as well as their enzymatic properties alone and in the presence of Sdo1 and SBDS, respectively. The human and S. cerevisiae EFL1 GTPases are composed of a mixture of alpha-helices and beta-sheets. Despite being orthologs, the yeast protein elicited a non-two state thermal unfolding behavior while the human EFL1 was highly resistant to thermal denaturation. Steady-state kinetic analyses indicated slow GTP hydrolysis for both EFLI GTPases, with k(cat) values of 0.4 and 0.3 min(-1) and K-m for GTP of 110 and 180 mu M respectively. In the presence of the effector proteins, their k(cat) values remained unaltered while the K-m decreased twofold suggesting that Sdo1 and SBDS act as nucleotide exchange factors. (C) 2013 Elsevier Inc. All rights reserved.