Abstract
Cathepsin L, a lysosomal protein in mouse embryonic stem cells has been shown to clip the histone H3 N- terminus, an activity associated with gene activity during mouse cell development. Glutamate dehydrogenase (GDH) was also identified as histone H3 specific protease in chicken liver, which has been connected to gene expression during aging. In baker’s yeast, Saccharomyces cerevisiae, clipping the histone H3 N-terminus has been associated with gene activation in stationary phase but the protease responsible for the yeast histone H3 endopeptidase activity had not been identified. In searching for a yeast histone H3 endopeptidase, we found that yeast vacuolar protein Prb1 is present in the cellular fraction enriched for the H3 N-terminus endopeptidase activity and this endopeptidase activity is lost in the PRB1 deletion mutant (prb1Δ). In addition, like Cathepsin L and GDH, purified Prb1 from yeast cleaves H3 between Lys23 and Ala24 in the N-terminus in vitro as shown by Edman degradation. In conclusion, our data argue that PRB1 is required for clipping of the histone H3 N-terminal tail in Saccharomyces cerevisiae.
Highlights
Eukaryotic chromosomal DNA is packaged in nucleosomes which contain 2 copies each of the core histones H2A, H2B, H3 and H4 [1]
We found that three peaks of cleavage activity were present in the fractions separated by hydrophobic interaction chromatography (HIC) as shown in Figure 1b and that all of them can be inhibited by the serine protease inhibitor PMSF (Figure 1c, lanes 3, 6 and 9) but not by the aspartyl protease inhibitor Pepstatin A (Figure 1c, lane 4, 7 and 10)
Our findings agree with previous studies by demonstrating that the yeast H3 N-terminus endopeptidase activity comes from a serine protease and its endopeptidase activity can be inhibited by PMSF [16]
Summary
Eukaryotic chromosomal DNA is packaged in nucleosomes which contain 2 copies each of the core histones H2A, H2B, H3 and H4 [1]. The histone H3 N-terminal tail is the target of many such modifications that are involved in gene activity [6,7,8], silencing of transcription and higher order structure of heterochromatin [9,10,11], replication [12], nucleosome assembly [13,14] and chromatin remodeling [15]. In Saccharomyces cerevisiae, a serine protease activity is present that cleaves the histone H3 tail after Ala in sporulation and stationary phase, and preventing truncation using a mutation H3 Q19A, L20A results in decreased transcription of selected yeast genes in stationary phase [16]. A search of 21 viable deletion strains lacking different serine proteases found that none of the deletion strains lost the ability to clip H3, prompting the suggestion that redundant enzymes were involved [16]
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