Dataset.

Depletion of Cbk1 prevents cdc15-2 cell-separation rescue after TORC1 inactivation

Digital.CSIC. Repositorio Institucional del CSIC
oai:digital.csic.es:10261/360848
Digital.CSIC. Repositorio Institucional del CSIC
  • Foltman, Magdalena
  • Méndez, Iván
  • Bech-Serra, Joan J.
  • de la Torre, Carolina
  • Brace, Jennifer L.
  • Weiss, Eric L.
  • Lucas, María
  • Queralt, Ethel
  • Sánchez-Díaz, Alberto
(A) Schematic illustration of the “auxin inducible degron” system. (i) The Cbk1 auxin degron strain (cbk1-aid, where aid denotes the auxin inducible degron) contains a version of Cbk1 in which the auxin inducible degron was fused to the C-terminus of CBK1 in its own locus. Protein expression is under CBK1 promoter and Cbk1-aid is able to perform wt Cbk1’s functions. The fusion was carried out in a yeast strain in which the F-box protein Tir1 is expressed constitutively under the control of ADH1 promoter. Tir1 binds to SCF and forms the E3 ubiquitin ligase SCF-TIR1 that recruits the E2 ubiquitin conjugating enzyme [38]. (ii) Following the addition of auxins, the F-box Tir1 is able to specifically recognise the aid tag fused to Cbk1. Then, E2 ubiquitin conjugating enzyme polyubiquitylates aid, which rapidly promotes the degradation of Cbk1-aid by the proteasome (iii). This system allows to study the immediate biological consequences after Cbk1 depletion. (B) ADH-TIR1 (YJW15) and cbk1-aid ADH-TIR1 (YMF3657) cells were grown in YPD before the addition of NAA and IAA auxins. Samples were taken at the indicated times to determine cell-cycle progression by flow cytometry. (C) Schematic representation of experimental set-up in which cells were grown in YPD and arrested in late anaphase by shifting the temperature to 37 °C before the addition of NAA and IAA auxins for 50 min. Next, cells were incubated with DMSO (i) or rapamycin (ii) for 20 min while still arrested in anaphase. Cells were released in the absence (i) or presence (ii) of rapamycin, and with the NAA and IAA auxins present in medium throughout the rest of the experiment. (D) cbk1-aid cdc15-2 (YMF3866) cells were grown as represented in C. Samples were taken at shown times to determine cell-cycle progression by flow cytometry ((i) and (ii)) and cell morphology by light microscopy in the absence (iii) or in the presence (iv) of rapamycin at 120 min after the release from late anaphase arrest. Scale bars indicate 5 μm. (E) (i) To study functionally compromised versions of Cbk1, we initially expressed both Cbk1 fused to aid and the altered Cbk1 protein whose sequence had been integrated as an extra copy together with 5′ and 3′ UTRs. Cells are not defective under permissive conditions as viability is supported by Cbk1-aid, whereas after the addition of auxins ((ii) restrictive conditions) Cbk1-aid is depleted and functional consequences of compromised Cbk1 expression could be determined. (F) cbk1-D475A cbk1-aid cdc15-2 (YMF4047) cells were cultured an analysed as in D. (G) As control, CBK1 cbk1-aid cdc15-2 (YMF4082) cells were grown and analysed as in D. FACS graphs can be found in the supplementary FACS file (S1 File)., pbio.3002263.s002.pdf, Peer reviewed
 
DOI: http://hdl.handle.net/10261/360848
Digital.CSIC. Repositorio Institucional del CSIC
oai:digital.csic.es:10261/360848

HANDLE: http://hdl.handle.net/10261/360848
Digital.CSIC. Repositorio Institucional del CSIC
oai:digital.csic.es:10261/360848
 
Ver en: http://hdl.handle.net/10261/360848
Digital.CSIC. Repositorio Institucional del CSIC
oai:digital.csic.es:10261/360848

Digital.CSIC. Repositorio Institucional del CSIC
oai:digital.csic.es:10261/360848
Dataset. 2023

DEPLETION OF CBK1 PREVENTS CDC15-2 CELL-SEPARATION RESCUE AFTER TORC1 INACTIVATION

Digital.CSIC. Repositorio Institucional del CSIC
  • Foltman, Magdalena
  • Méndez, Iván
  • Bech-Serra, Joan J.
  • de la Torre, Carolina
  • Brace, Jennifer L.
  • Weiss, Eric L.
  • Lucas, María
  • Queralt, Ethel
  • Sánchez-Díaz, Alberto
(A) Schematic illustration of the “auxin inducible degron” system. (i) The Cbk1 auxin degron strain (cbk1-aid, where aid denotes the auxin inducible degron) contains a version of Cbk1 in which the auxin inducible degron was fused to the C-terminus of CBK1 in its own locus. Protein expression is under CBK1 promoter and Cbk1-aid is able to perform wt Cbk1’s functions. The fusion was carried out in a yeast strain in which the F-box protein Tir1 is expressed constitutively under the control of ADH1 promoter. Tir1 binds to SCF and forms the E3 ubiquitin ligase SCF-TIR1 that recruits the E2 ubiquitin conjugating enzyme [38]. (ii) Following the addition of auxins, the F-box Tir1 is able to specifically recognise the aid tag fused to Cbk1. Then, E2 ubiquitin conjugating enzyme polyubiquitylates aid, which rapidly promotes the degradation of Cbk1-aid by the proteasome (iii). This system allows to study the immediate biological consequences after Cbk1 depletion. (B) ADH-TIR1 (YJW15) and cbk1-aid ADH-TIR1 (YMF3657) cells were grown in YPD before the addition of NAA and IAA auxins. Samples were taken at the indicated times to determine cell-cycle progression by flow cytometry. (C) Schematic representation of experimental set-up in which cells were grown in YPD and arrested in late anaphase by shifting the temperature to 37 °C before the addition of NAA and IAA auxins for 50 min. Next, cells were incubated with DMSO (i) or rapamycin (ii) for 20 min while still arrested in anaphase. Cells were released in the absence (i) or presence (ii) of rapamycin, and with the NAA and IAA auxins present in medium throughout the rest of the experiment. (D) cbk1-aid cdc15-2 (YMF3866) cells were grown as represented in C. Samples were taken at shown times to determine cell-cycle progression by flow cytometry ((i) and (ii)) and cell morphology by light microscopy in the absence (iii) or in the presence (iv) of rapamycin at 120 min after the release from late anaphase arrest. Scale bars indicate 5 μm. (E) (i) To study functionally compromised versions of Cbk1, we initially expressed both Cbk1 fused to aid and the altered Cbk1 protein whose sequence had been integrated as an extra copy together with 5′ and 3′ UTRs. Cells are not defective under permissive conditions as viability is supported by Cbk1-aid, whereas after the addition of auxins ((ii) restrictive conditions) Cbk1-aid is depleted and functional consequences of compromised Cbk1 expression could be determined. (F) cbk1-D475A cbk1-aid cdc15-2 (YMF4047) cells were cultured an analysed as in D. (G) As control, CBK1 cbk1-aid cdc15-2 (YMF4082) cells were grown and analysed as in D. FACS graphs can be found in the supplementary FACS file (S1 File)., pbio.3002263.s002.pdf, Peer reviewed




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