Molecular Genetics and Genomics

, Volume 278, Issue 1, pp 105–123

In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1

  • Jennifer L. Fuentes
  • Kaustuv Datta
  • Susan M. Sullivan
  • Angela Walker
  • Janine R. Maddock
Original Paper

DOI: 10.1007/s00438-007-0233-1

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

Abstract

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.

Keywords

NOG1GTPase60SRibosome biogenesisObg/CgtA

Supplementary material

438_2007_233_MOESM1_ESM.doc (178 kb)
Supplemental tables (DOC 177 kb)
438_2007_233_MOESM2_ESM.ppt (15.1 mb)
Figure S1. Ribosome association of nog1GTP-binding domain mutants. Wild type cells expressing HIS6Nog1, HIS6Nog1G179A, HIS6Nog1S181N, or HIS6Nog1D220A were analyzed on 7-47% sucrose gradients. The absorbance (A254) of the sucrose gradients was monitored and fractions were collected, as indicated by vertical lines. (A) A representative polysome profile is shown. (B) Proteins in the fractions were TCA precipitated and analyzed by immunoblot with anti-Nog1 antibodies. The positions of the 40S, 60S, 80S and polysomes are indicated. The positions of the episomal HIS6Nog1 and chromosomal Nog1 are also indicated (PPT 15.1 Mb)

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Jennifer L. Fuentes
    • 1
  • Kaustuv Datta
    • 1
  • Susan M. Sullivan
    • 1
  • Angela Walker
    • 2
  • Janine R. Maddock
    • 1
  1. 1.Department of Molecular, Cellular and Developmental BiologyUniversity of MichiganAnn ArborUSA
  2. 2.Department of Biological ChemistryUniversity of MichiganAnn ArborUSA