Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.
Histones
/ genetics
Saccharomyces cerevisiae
/ genetics
Proliferating Cell Nuclear Antigen
/ genetics
Saccharomyces cerevisiae Proteins
/ metabolism
Ubiquitination
Ubiquitin
/ genetics
Deubiquitinating Enzymes
/ genetics
Ubiquitin-Conjugating Enzymes
/ metabolism
Ubiquitin Thiolesterase
/ metabolism
Transcription Factors
/ metabolism
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
04 10 2023
04 10 2023
Historique:
received:
21
05
2023
accepted:
30
09
2023
medline:
6
10
2023
pubmed:
5
10
2023
entrez:
4
10
2023
Statut:
epublish
Résumé
Post-translational covalent conjugation of ubiquitin onto proteins or ubiquitination is important in nearly all cellular processes. Steady-state ubiquitination of individual proteins in vivo is maintained by two countering enzymatic activities: conjugation of ubiquitin by E1, E2 and E3 enzymes and removal by deubiquitinases. Here, we deleted one or more genes encoding deubiquitinases in yeast and evaluated the requirements for ubiquitin conjugation onto a target protein. Our proof-of-principle studies demonstrate that absence of relevant deubiquitinase(s) provides a facile and versatile method that can be used to study the nuances of ubiquitin conjugation and deubiquitination of target proteins in vivo. We verified our method using mutants lacking the deubiquitinases Ubp8 and/or Ubp10 that remove ubiquitin from histone H2B or PCNA. Our studies reveal that the C-terminal coiled-domain of the adapter protein Lge1 and the C-terminal acidic tail of Rad6 E2 contribute to monoubiquitination of histone H2BK123, whereas the distal acidic residues of helix-4 of Rad6, but not the acidic tail, is required for monoubiquitination of PCNA. Further, charged substitution at alanine-120 in the H2B C-terminal helix adversely affected histone H2BK123 monoubiquitination by inhibiting Rad6-Bre1-mediated ubiquitin conjugation and by promoting Ubp8/Ubp10-mediated deubiquitination. In summary, absence of yeast deubiquitinases UBP8 and/or UBP10 allows uncovering the regulation of and requirements for ubiquitin addition and removal from their physiological substrates such as histone H2B or PCNA in vivo.
Identifiants
pubmed: 37794081
doi: 10.1038/s41598-023-43969-z
pii: 10.1038/s41598-023-43969-z
pmc: PMC10550974
doi:
Substances chimiques
Histones
0
Proliferating Cell Nuclear Antigen
0
Saccharomyces cerevisiae Proteins
0
Ubiquitin
0
Deubiquitinating Enzymes
EC 3.4.19.12
Ubiquitin-Conjugating Enzymes
EC 2.3.2.23
UBP10 protein, S cerevisiae
EC 3.4.19.12
Ubiquitin Thiolesterase
EC 3.4.19.12
Bre1 protein, S cerevisiae
0
Lge1 protein, S cerevisiae
0
Transcription Factors
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
16731Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM127783
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01GM127783
Pays : United States
Commentaires et corrections
Type : UpdateOf
Informations de copyright
© 2023. Springer Nature Limited.
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