| The Obg subfamily of GTPases is a novel family of GTP-binding proteins that are universally conserved and predicted to play a role in translation. Interestingly, all Obg proteins studied to date are involved in some aspect of ribosome function. In Chapters 2 and 3 of this study I demonstrate that the Saccharomyces cerevisiae GTPase, Mtg2p (Yhr168wp), is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to respiratory incompetent cells. In addition, cells expressing a temperature sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lower levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. Consistent with a role for Mtg2p in mitochondrial ribosome biogenesis, I show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.; The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, in Chapter 4 we generated mutations in the Caulobacter crescentus obg gene, (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. I purified CgtACS173N (analogous to RasS17N) and CgtACP168V (analogous to RasG12V) protein and measured their guanine nucleotide affinities, exchange rates and half life of hydrolysis. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Further, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. |
