Involvement of Hat1p (Kat1p) Catalytic Activity and Subcellular Localization in Telomeric Silencing

Erica L Mersfelder,Mark R Parthun

doi:10.1074/jbc.m802564200

Abstract

Previous studies have shown that loss of the type B histone acetyltransferase Hat1p leads to defects in telomeric silencing in Saccharomyces cerevisiae. We used this phenotype to explore a number of functional characteristics of this enzyme. To determine whether the enzymatic activity of Hat1p is necessary for its role in telomeric silencing, a structurally conserved glutamic acid residue (Glu-255) that has been proposed to be the enzymes catalytic base was mutated. Surprisingly neither this residue nor any other acidic residues near the enzymes active site were essential for enzymatic activity. This suggests that Hat1p differs from most histone acetyltransferases in that it does not use an acidic amino acid as a catalytic base. The effects of these Hat1p mutants on enzymatic activity correlated with their effects on telomeric silencing indicating that the ability of Hat1p to acetylate substrates is important for its in vivo function. Despite its presumed role in the acetylation of newly synthesized histones in the cytoplasm, Hat1p was found to be a predominantly nuclear protein. This subcellular localization of Hat1p is important for its in vivo function because a construct that prevents its accumulation in the nucleus caused defects in telomeric silencing similar to those seen with a deletion mutant. Therefore, the presence of catalytically active Hat1p in the cytoplasm is not sufficient to support normal telomeric silencing. Hence both enzymatic activity and nuclear localization are necessary characteristics of Hat1p function in telomeric silencing.

Highlights

IntroductionConsistent with its designation as a type B histone acetyltransferase, Hat1p was originally isolated from yeast cytoplasmic extracts [3]
Mutational Analysis of the Hat1p Active Site—To determine whether the histone acetyltransferase activity of Hat1p is essential for its function in telomeric silencing, we sought to generate point mutants that would inactivate the enzymatic activity of Hat1p
The mutational analysis of Hat1p is greatly facilitated by the presence of a crystal structure for this enzyme [34]. This crystal structure was obtained in the presence of acetyl coenzyme A (Ac-CoA), which provides important information on the location of the active site of the enzyme

Summary

Introduction

Consistent with its designation as a type B histone acetyltransferase, Hat1p was originally isolated from yeast cytoplasmic extracts [3]. Relative to most other histone acetyltransferases, which exist in large, multisubunit complexes, Hat1p is found in comparatively simple complexes in yeast cells [12]. When isolated from the cytoplasm, yeast Hat1p is found associated with Hat2p [3]. Hif1p is an H3/H4-specific histone chaperone that has chromatin assembly activity in vitro [15]. It is not known whether a complex comparable to the yeast NuB4 complex exists in other eukaryotes

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