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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

Efficient degradation of mitotic exit substrates requires UBE2S. U2OS cells were transfected with indicated constructs together with control or UBE2S siRNA and imaged by fluorescence time-lapse microscopy from 24 to 48 h after transfection. Fluorescence intensity of Venus over time, in individual mitotic cells, was quantified and plotted as a function of anaphase onset. (A) In vivo degradation curve (left) shows averaged intensities normalized to anaphase ± SD (n ≥ 5); degradation rate curve (right) shows the change in rate over time, derived from degradation curves as described in Min et al. (2013). (B) Degradation curves for indicated substrates under conditions of control (GL2i) or Ube2S knockdown (Ube2Si). (C) Degradation rate curves for mitotic exit substrates under control conditions. (D) Degradation rate curves for mitotic exit substrates after UBE2Si.
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Figure 3: Efficient degradation of mitotic exit substrates requires UBE2S. U2OS cells were transfected with indicated constructs together with control or UBE2S siRNA and imaged by fluorescence time-lapse microscopy from 24 to 48 h after transfection. Fluorescence intensity of Venus over time, in individual mitotic cells, was quantified and plotted as a function of anaphase onset. (A) In vivo degradation curve (left) shows averaged intensities normalized to anaphase ± SD (n ≥ 5); degradation rate curve (right) shows the change in rate over time, derived from degradation curves as described in Min et al. (2013). (B) Degradation curves for indicated substrates under conditions of control (GL2i) or Ube2S knockdown (Ube2Si). (C) Degradation rate curves for mitotic exit substrates under control conditions. (D) Degradation rate curves for mitotic exit substrates after UBE2Si.

Mentions: We expressed fluorescent protein–tagged substrates and traced their levels in single cells in asynchronously proliferating cultures. We then normalized fluorescence levels to anaphase onset and in silico–synchronized cell traces. Knockdown of Ube2S did not alter the timing of anaphase onset (Supplemental Figure S3) or, for each of the substrates analyzed, the timing of degradation with respect to anaphase onset (Figure 3, A and B). However, degradation rates for all substrates were decreased under these conditions (Figure 3, A and B), suggesting that K11 linkages are important in efficient degradation of mitotic exit substrates. Moreover, in the absence of UBE2S, all substrates underwent degradation with similar kinetics despite distinct degradation kinetics under control conditions (Figure 3, C and D), indicating that K11 chains specify accelerated rates of degradation in a substrate-dependent manner.


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)

Efficient degradation of mitotic exit substrates requires UBE2S. U2OS cells were transfected with indicated constructs together with control or UBE2S siRNA and imaged by fluorescence time-lapse microscopy from 24 to 48 h after transfection. Fluorescence intensity of Venus over time, in individual mitotic cells, was quantified and plotted as a function of anaphase onset. (A) In vivo degradation curve (left) shows averaged intensities normalized to anaphase ± SD (n ≥ 5); degradation rate curve (right) shows the change in rate over time, derived from degradation curves as described in Min et al. (2013). (B) Degradation curves for indicated substrates under conditions of control (GL2i) or Ube2S knockdown (Ube2Si). (C) Degradation rate curves for mitotic exit substrates under control conditions. (D) Degradation rate curves for mitotic exit substrates after UBE2Si.
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Related In: Results  -  Collection

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Figure 3: Efficient degradation of mitotic exit substrates requires UBE2S. U2OS cells were transfected with indicated constructs together with control or UBE2S siRNA and imaged by fluorescence time-lapse microscopy from 24 to 48 h after transfection. Fluorescence intensity of Venus over time, in individual mitotic cells, was quantified and plotted as a function of anaphase onset. (A) In vivo degradation curve (left) shows averaged intensities normalized to anaphase ± SD (n ≥ 5); degradation rate curve (right) shows the change in rate over time, derived from degradation curves as described in Min et al. (2013). (B) Degradation curves for indicated substrates under conditions of control (GL2i) or Ube2S knockdown (Ube2Si). (C) Degradation rate curves for mitotic exit substrates under control conditions. (D) Degradation rate curves for mitotic exit substrates after UBE2Si.
Mentions: We expressed fluorescent protein–tagged substrates and traced their levels in single cells in asynchronously proliferating cultures. We then normalized fluorescence levels to anaphase onset and in silico–synchronized cell traces. Knockdown of Ube2S did not alter the timing of anaphase onset (Supplemental Figure S3) or, for each of the substrates analyzed, the timing of degradation with respect to anaphase onset (Figure 3, A and B). However, degradation rates for all substrates were decreased under these conditions (Figure 3, A and B), suggesting that K11 linkages are important in efficient degradation of mitotic exit substrates. Moreover, in the absence of UBE2S, all substrates underwent degradation with similar kinetics despite distinct degradation kinetics under control conditions (Figure 3, C and D), indicating that K11 chains specify accelerated rates of degradation in a substrate-dependent manner.

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