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Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages.

Min M, Mevissen TE, De Luca M, Komander D, Lindon C - Mol. Biol. Cell (2015)

Bottom Line: In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis.All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin.Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Regulation of K11 chain assembly by APC/C-Cdh1. (A) U2OS-bioUb cells transfected with siRNA sequence against GL2 (control), Cdh1, or UBE2S were synchronized to mitotic exit. Whole-cell lysate was interrogated by K11 linkage–specific antibody and by biotin antibody to show total ubiquitin conjugates. (B–D) U2OS-bioUb cells were transfected with indicated Venus-tagged constructs together with control (GL2) or Cdh1 or UBE2S siRNA sequence, induced for bioUb expression for 44 h, and synchronized to mitotic exit. Cellular ubiquitination assays were carried out to detect total ubiquitin, K11 linkage, and K48 linkage on each substrate. Bar plot shows mean measurements from three repeats for AurA and AurB and two repeats for KIFC1, with SD plotted where three repeats are available. In vivo degradation assays were carried out in parallel (shown in Supplemental Figure S4), together with representative blots to validate depletions. (E) U2OS-AurA-Venus cells were transfected with indicated siRNAs and synchronized to prometaphase using a sequential thymidine and STLC block. Cells were then released into mitotic exit by treating with 300 nM CDK I/II inhibitor for 45 min before harvesting. Whole-cell lysates (left) or AurA-Venus pull downs (middle) were probed for ubiquitin conjugates. Whole-cell lysates were also tested for knockdown efficacies with indicated antibodies (right).
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Figure 4: Regulation of K11 chain assembly by APC/C-Cdh1. (A) U2OS-bioUb cells transfected with siRNA sequence against GL2 (control), Cdh1, or UBE2S were synchronized to mitotic exit. Whole-cell lysate was interrogated by K11 linkage–specific antibody and by biotin antibody to show total ubiquitin conjugates. (B–D) U2OS-bioUb cells were transfected with indicated Venus-tagged constructs together with control (GL2) or Cdh1 or UBE2S siRNA sequence, induced for bioUb expression for 44 h, and synchronized to mitotic exit. Cellular ubiquitination assays were carried out to detect total ubiquitin, K11 linkage, and K48 linkage on each substrate. Bar plot shows mean measurements from three repeats for AurA and AurB and two repeats for KIFC1, with SD plotted where three repeats are available. In vivo degradation assays were carried out in parallel (shown in Supplemental Figure S4), together with representative blots to validate depletions. (E) U2OS-AurA-Venus cells were transfected with indicated siRNAs and synchronized to prometaphase using a sequential thymidine and STLC block. Cells were then released into mitotic exit by treating with 300 nM CDK I/II inhibitor for 45 min before harvesting. Whole-cell lysates (left) or AurA-Venus pull downs (middle) were probed for ubiquitin conjugates. Whole-cell lysates were also tested for knockdown efficacies with indicated antibodies (right).

Mentions: Given that the subset of mitotic exit substrates of cellular proteolysis could be major contributors to the K11-linked polyubiquitin detected in Figure 1A, we hypothesized that Cdh1 activity accounted for the dramatic increase in K11 chain assembly at mitotic exit. Indeed, we observed a 60% reduction in K11 linkage after siRNA-mediated Cdh1 depletion (Cdh1i; Figure 4A), supporting the idea that K11 linkages could be the critical output of Cdh1 specificity in substrate degradation. Therefore we interrogated ubiquitin conjugates on individual substrates after Cdh1i. We found that, on the exclusively Cdh1-dependent substrates AurA and AurB (Floyd et al., 2008; Supplemental Figure S4, A–D), Cdh1i treatment abolished K11 chains as effectively as Ube2Si (Figure 4, B–C). By contrast, however, K11 chains on non–Cdh1-dependent substrate KIFC1 (Min et al., 2014; Supplemental Figure S4, E and F) were not largely affected by Cdh1i (Figure 4D).


Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages.

Min M, Mevissen TE, De Luca M, Komander D, Lindon C - Mol. Biol. Cell (2015)

Regulation of K11 chain assembly by APC/C-Cdh1. (A) U2OS-bioUb cells transfected with siRNA sequence against GL2 (control), Cdh1, or UBE2S were synchronized to mitotic exit. Whole-cell lysate was interrogated by K11 linkage–specific antibody and by biotin antibody to show total ubiquitin conjugates. (B–D) U2OS-bioUb cells were transfected with indicated Venus-tagged constructs together with control (GL2) or Cdh1 or UBE2S siRNA sequence, induced for bioUb expression for 44 h, and synchronized to mitotic exit. Cellular ubiquitination assays were carried out to detect total ubiquitin, K11 linkage, and K48 linkage on each substrate. Bar plot shows mean measurements from three repeats for AurA and AurB and two repeats for KIFC1, with SD plotted where three repeats are available. In vivo degradation assays were carried out in parallel (shown in Supplemental Figure S4), together with representative blots to validate depletions. (E) U2OS-AurA-Venus cells were transfected with indicated siRNAs and synchronized to prometaphase using a sequential thymidine and STLC block. Cells were then released into mitotic exit by treating with 300 nM CDK I/II inhibitor for 45 min before harvesting. Whole-cell lysates (left) or AurA-Venus pull downs (middle) were probed for ubiquitin conjugates. Whole-cell lysates were also tested for knockdown efficacies with indicated antibodies (right).
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Figure 4: Regulation of K11 chain assembly by APC/C-Cdh1. (A) U2OS-bioUb cells transfected with siRNA sequence against GL2 (control), Cdh1, or UBE2S were synchronized to mitotic exit. Whole-cell lysate was interrogated by K11 linkage–specific antibody and by biotin antibody to show total ubiquitin conjugates. (B–D) U2OS-bioUb cells were transfected with indicated Venus-tagged constructs together with control (GL2) or Cdh1 or UBE2S siRNA sequence, induced for bioUb expression for 44 h, and synchronized to mitotic exit. Cellular ubiquitination assays were carried out to detect total ubiquitin, K11 linkage, and K48 linkage on each substrate. Bar plot shows mean measurements from three repeats for AurA and AurB and two repeats for KIFC1, with SD plotted where three repeats are available. In vivo degradation assays were carried out in parallel (shown in Supplemental Figure S4), together with representative blots to validate depletions. (E) U2OS-AurA-Venus cells were transfected with indicated siRNAs and synchronized to prometaphase using a sequential thymidine and STLC block. Cells were then released into mitotic exit by treating with 300 nM CDK I/II inhibitor for 45 min before harvesting. Whole-cell lysates (left) or AurA-Venus pull downs (middle) were probed for ubiquitin conjugates. Whole-cell lysates were also tested for knockdown efficacies with indicated antibodies (right).
Mentions: Given that the subset of mitotic exit substrates of cellular proteolysis could be major contributors to the K11-linked polyubiquitin detected in Figure 1A, we hypothesized that Cdh1 activity accounted for the dramatic increase in K11 chain assembly at mitotic exit. Indeed, we observed a 60% reduction in K11 linkage after siRNA-mediated Cdh1 depletion (Cdh1i; Figure 4A), supporting the idea that K11 linkages could be the critical output of Cdh1 specificity in substrate degradation. Therefore we interrogated ubiquitin conjugates on individual substrates after Cdh1i. We found that, on the exclusively Cdh1-dependent substrates AurA and AurB (Floyd et al., 2008; Supplemental Figure S4, A–D), Cdh1i treatment abolished K11 chains as effectively as Ube2Si (Figure 4, B–C). By contrast, however, K11 chains on non–Cdh1-dependent substrate KIFC1 (Min et al., 2014; Supplemental Figure S4, E and F) were not largely affected by Cdh1i (Figure 4D).

Bottom Line: In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis.All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin.Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.

No MeSH data available.


Related in: MedlinePlus