Spb1p Is a Yeast Nucleolar Protein Associated with Nop1p and Nop58p That Is Able To BindS-Adenosyl-l-Methionine In Vitro

Abstract

We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis. A cold-sensitive allele for that gene (referred to here asspb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here asspb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae. The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content. In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated. Spb1p is a 96.5-kDa protein that is localized to the nucleolus. Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p. Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-l-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases. We show here that Spb1p is able to bind [³H]AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.

ACKNOWLEDGMENTS

We are indebted to A. Sachs who initiated this work and who gave us generously the cloned SPB1 gene (pAS16), as well as many other reagents. We thank J. Aris who kindly provided several antibodies, including large amounts of anti-Nop1p and anti-Nop5/58p, and B. Trumpower for the anti-Qsr1p antibody. We thank J. de la Cruz and P. Linder for sharing materials and results prior to publication and for stimulating discussion. We are grateful to F. Martin for his help with the chromatography experiments. We thank members of our laboratory for fruitful discussions and J. Morrissey for careful reading of the manuscript.

This work was supported by the Centre National de la Recherche Scientifique and by grants from the Ligue contre le Cancer, from the Fondation pour la Recherche Médicale, and from the Philippe Foundation. L.P. had a fellowship from the MENESR. D.K. was supported by a grant from the Swiss National Science Foundation to P. Linder.