In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1
- First Online:
- Cite this article as:
- Fuentes, J.L., Datta, K., Sullivan, S.M. et al. Mol Genet Genomics (2007) 278: 105. doi:10.1007/s00438-007-0233-1
- 266 Downloads
The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347–456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function.