Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA.

Sari Kassem,Zoltan Villanyi,Martine A Collart

doi:10.1093/nar/gkx447

Abstract

Acetylation of histones regulates gene expression in eukaryotes. In the yeast S.cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase (HAT) complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4-Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4-Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. The Ccr4-Not complex is present at this site to promote the co-translational assembly of these subunits and this is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4-Not-dependent co-translational SAGA assembly and functions as a chaperone.

Highlights

Total protein extracts from these aliquots were separated on SDS-PAGE and transferred to membranes that were revealed with PAP antibodies. (B) 20 g of total extracts (TE) from wild type (WT) and not5Δ expressing SAGA subunits fused to a C-terminal Tap-tag (TAP) or MYC-tag as indicated were separated on SDS-PAGE and transferred to membranes that were revealed with PAP or MYC antibodies
Since we determined that the subunits of SAGA were stable and normally expressed in not5Δ, yet SAGA activity is compromised in these mutant cells, we investigated the integrity of the SAGA complex on native gels in a systematic way
Compatible with this observation, immunofluorescence microscopy of Gcn5 shows that this protein, which is normally nuclear in WT, was accumulated in punctate in the cytoplasm in not5Δ, which implicates that this phenotype is an in vivo assembly defect rather than an in vitro disassembly related phenotype

Summary

Introduction

The outcome of this study is a modular SAGA with a total of 4 small modules: the first module was named SPT and was put in the center It combined the Scaffold Tra, the two TBP binding Spts (3 and 8), and the already known structural subunits of the complex (Spt, Ada, and Spt). Additional studies include an atomic model of CNOT9, the human orthologue of Caf (Garces et al, 2007), which proposed a tendency of this subunit to dimerize and to bind nucleic acids Another set of studies has tackled via crystallography the interactions of individual subunits of the Ccr4-Not complex with domains of Not, the scaffold protein of the complex.

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