Various widely known GTPases are associated with diverse crucial cellular processes. However, the functional targets of the universally conserved homologous GTPases Drg1 and Drg2, constituting the DRG subfamily in eukaryotes, remain completely unknown despite their pleiotropic cell growth effects. Contrary to expectations of functional redundancy between Drg1 and Drg2 due to their high homology, the different binding proteins Dfrp1 and Dfrp2, respectively, have been previously identified. Here, we report the first systematic characterization of all these proteins in mammals by analyses in physiological conditions. Our findings are: (11) At least one of the components of the Drg1/Dfrp1 and the Drg2/Dfrp2 complexes is specifically and drastically stabilized by each unique complex formation: and (2) the Drg1/Dfrp1 complex cosediments with polysome, while neither Drg2 nor Dfrp2 is found in ribosomal fractions at all. These results suggest that the Drg1/Dfrp1 complex independently modulates a protein synthesis mechanism different from the Drg2/Dfrp2 complex in mammalian cells. (C) 2009 Elsevier Inc. All rights reserved.