Abstract
Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles can be established independent of proteolysis mediated by the APC and SCF enzymes.
Highlights
Proper execution of the eukaryotic cell division cycle depends heavily on ubiquitin-mediated proteolysis, involving the conjugation of polyubiquitin chains to substrate proteins by E3 ubiquitin ligases and their subsequent recognition and degradation by the 26S proteasome [1]
We describe an unconventional proteolytic mechanism, independent of Skp1/cullin/F-box protein complex (SCF) and anaphase-promoting complex (APC), that helps establish the strict cell cycle expression profile of the APC inhibitor Acm1 in budding yeast
Synchronized dbf2-2 cultures were released from a-factor-induced G1 arrest into fresh medium at 37uC so they would arrest in late anaphase, a point where APCCdc20 has been activated and has targeted its substrates for degradation (Figure 1A and 1B)
Summary
Proper execution of the eukaryotic cell division cycle depends heavily on ubiquitin-mediated proteolysis, involving the conjugation of polyubiquitin chains to substrate proteins by E3 ubiquitin ligases and their subsequent recognition and degradation by the 26S proteasome [1]. Two large ubiquitin ligase complexes are responsible for the majority of regulated proteolysis during the cell division cycle [2,4,5]. In contrast to SCF, the activity of APC is cell cycle-regulated by several mechanisms including phosphorylation of, and inhibitor binding to, its activator proteins Cdc and Cdh1 [9]. Following conjugation of polyubiquitin chains to substrate lysines by SCF and APC, recognition by the 26S proteasome results in their irreversible degradation, and helps drive the cell cycle forward
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