Abstract
BackgroundSelective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.Methodology/Principal FindingsIn order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.Conclusions/SignificanceOur work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.
Highlights
The ubiquitin-26S proteasome system (UPS) is the major regulator to control the abundance of key factors and enzymes in all eukaryotes [1]
Plasmids YN-APC10 and YC-dsRNA-Binding Protein 4 (DRB4) were cobombarded into etiolated mustard hypocotyls, and a strong YFP signal was observed in the nucleus of examined cells (Figure 1C)
We describe for the first time a substrate of the plant Anaphase Promoting Complex/Cyclosome (APC/C) which is not involved in the regulation of cell cycle
Summary
The ubiquitin-26S proteasome system (UPS) is the major regulator to control the abundance of key factors and enzymes in all eukaryotes [1]. The E3 enzymes ( called Ubiquitin protein Ligases) play the central role in this mechanism as they recognise and select substrates. The Anaphase Promoting Complex/Cyclosome (APC/C) is a conserved multisubunit E3 ligase, composed of at least 11 core subunits and a coactivator protein from the CDC20/FIZZY or CDH1/FIZZYRELATED families [4,5]. APC2 and APC11 constitute the catalytic module of the enzyme, whereas CDC20 and CDH1 have been shown to bind and recruit substrates. Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. The APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Its role in post-mitotic cells remains elusive and only a few substrates have been characterized
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