In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1.
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.
Fuentes
Jennifer L
JL
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109, USA.
Datta
Kaustuv
K
Sullivan
Susan M
SM
Walker
Angela
A
Maddock
Janine R
JR
D000595:Amino Acid Sequence; D000818:Animals; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012335:RNA, Ribosomal / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
C475027:NOG1 protein, S cerevisiae; D009687:Nuclear Proteins; D009711:Nucleotides; D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins