In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1 |
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Authors: | Jennifer L. Fuentes Kaustuv Datta Susan M. Sullivan Angela Walker Janine R. Maddock |
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Affiliation: | (1) Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109, USA;(2) Department of Biological Chemistry, University of Michigan, 300 North Ingalls Building, Ann Arbor, MI 48109, USA |
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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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | NOG1 GTPase 60S Ribosome biogenesis Obg/CgtA |
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