Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4.
Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Geetha
G
Hewawasam
Manjunatha
M
Shivaraju
Mark
M
Mattingly
Swaminathan
S
Venkatesh
Skylar
S
Martin-Brown
Laurence
L
Florens
Jerry L
JL
Workman
Jennifer L
JL
Gerton
D002503:Centromere / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D006657:Histones; D006801:Humans; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
C092746:CSE4 protein, S cerevisiae; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006657:Histones; D010445:Peptide Elongation Factors; D020033:Protein Isoforms; D029701:Saccharomyces cerevisiae Proteins; C521684:Scm3 protein, S cerevisiae; C554184:Psh1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases