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The APC11 RING-H2 finger mediates E2-dependent ubiquitination
J D Leverson1, C A Joazeiro, A M Page
1Molecular Biology and Virology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
Abstract:
Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.
Insights
The RING-H2 finger protein Apc11 is the minimal ubiquitin ligase for the anaphase-promoting complex (APC), essential for cell viability. Apc11 recruits E2 enzymes and mediates ubiquitination of APC substrates.
Area of Science:
- Cellular biology
- Molecular mechanisms of protein degradation
- Ubiquitin-proteasome system
Background:
- Polyubiquitination targets proteins for 26S proteasome degradation via E1, E2, and E3 enzymes.
- The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase crucial for cell cycle progression.
Purpose of the Study:
- To identify the minimal ubiquitin ligase component of the APC.
- To elucidate the role of Apc11 in APC/C ubiquitin ligase activity.
Main Methods:
- Investigated the essentiality of the Apc11p RING-H2 finger for yeast cell viability.
- Used purified recombinant proteins to demonstrate Apc11p interaction with Ubc4 (E2).
- Assessed Apc11p's ability to mediate ubiquitination of substrates like Clb2p in vitro.
Main Results:
- Apc11p's RING-H2 finger integrity is vital for budding yeast cell viability.
- Apc11p directly interacts with the Ubc4 E2 enzyme.
- Apc11p catalyzes E1- and E2-dependent ubiquitination of substrates in vitro, independent of Apc2p.
Conclusions:
- Apc11 defines the minimal ubiquitin ligase activity of the APC.
- Apc11p likely recruits E2 enzymes and facilitates ubiquitin transfer to APC substrates in vivo.