Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.

BubR1 Promotes Bub3-Dependent APC/C Inhibition during Spindle Assembly Checkpoint Signaling.

The spindle assembly checkpoint (SAC) prevents premature sister chromatid separation during mitosis. Phosphorylation of unattached kinetochores by the Mps1 kinase promotes recruitment of SAC machinery that catalyzes assembly of the SAC effector mitotic checkpoint complex (MCC). The SAC protein Bub3 is a phospho-amino acid adaptor that forms structurally related stable complexes with functionally distinct paralogs named Bub1 and BubR1. A short motif ("loop") of Bub1, but not the equivalent loop of BubR1, enhances binding of Bub3 to kinetochore phospho-targets. Here, we asked whether the BubR1 loop directs Bub3 to different phospho-targets. The BubR1 loop is essential for SAC function and cannot be removed or replaced with the Bub1 loop. BubR1 loop mutants bind Bub3 and are normally incorporated in MCC in vitro but have reduced ability to inhibit the MCC target anaphase-promoting complex (APC/C), suggesting that BubR1:Bub3 recognition and inhibition of APC/C requires phosphorylation. Thus, small sequence differences in Bub1 and BubR1 direct Bub3 to different phosphorylated targets in the SAC signaling cascade.

A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint.

The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. Bub1 and BubR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment. Because both Bub1 and BubR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that Bub1, but not BubR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bub1 onto BubR1 promotes Bub1-independent kinetochore recruitment of BubR1. This gain-of-function BubR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubR1 relies on direct hetero-dimerization with Bub1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

KI motifs of human Knl1 enhance assembly of comprehensive spindle checkpoint complexes around MELT repeats.

BACKGROUND: The KMN network, a ten-subunit protein complex, mediates the interaction of kinetochores with spindle microtubules and recruits spindle assembly checkpoint (SAC) constituents to halt cells in mitosis until attainment of sister chromatid biorientation. Two types of motifs in the KMN subunit Knl1 interact with SAC proteins. Lys-Ile (KI) motifs, found in vertebrates, interact with the TPR motifs of Bub1 and BubR1. Met-Glu-Leu-Thr (MELT) repeats, ubiquitous in evolution, recruit the Bub3/Bub1 complex in a phosphorylation-dependent manner. The exact contributions of KI and MELT motifs to SAC signaling and chromosome alignment are unclear. RESULTS: We report here that KI motifs cooperate strongly with the neighboring single MELT motif in the N-terminal 250 residues (Knl1(1-250)) of human Knl1 to seed a comprehensive assembly of SAC proteins. In cells depleted of endogenous Knl1, kinetochore-targeted Knl1(1-250) suffices to restore SAC and chromosome alignment. Individual MELT repeats outside of Knl1(1-250), which lack flanking KI motifs, establish qualitatively similar sets of interactions, but less efficiently. CONCLUSIONS: MELT sequences on Knl1 emerge from our analysis as the platforms on which SAC complexes become assembled. Our results show that KI motifs are enhancers of MELT function in assembling SAC signaling complexes, and that they might have evolved to limit the expansion of MELT motifs by providing a more robust mechanism of SAC signaling around a single MELT. We shed light on the mechanism of Bub1 and BubR1 recruitment and identify crucial questions for future studies.

Panta rhei: the APC/C at steady state.

The anaphase-promoting complex or cyclosome (APC/C) is a conserved, multisubunit E3 ubiquitin (Ub) ligase that is active both in dividing and in postmitotic cells. Its contributions to life are especially well studied in the domain of cell division, in which the APC/C lies at the epicenter of a regulatory network that controls the directionality and timing of cell cycle events. Biochemical and structural work is shedding light on the overall organization of APC/C subunits and on the mechanism of substrate recognition and Ub chain initiation and extension as well as on the molecular mechanisms of a checkpoint that seizes control of APC/C activity during mitosis. Here, we review how these recent advancements are modifying our understanding of the APC/C.

Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling.

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELT(P)) then promote recruitment of downstream signaling components. How MELT(P) motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed ß-propeller, is the MELT(P) reader. It contains an exceptionally well-conserved interface that docks the MELT(P) sequence on the side of the ß-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores. DOI:http://dx.doi.org/10.7554/eLife.01030.001.

Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex.

Once all chromosomes are connected to the mitotic spindle (bioriented), anaphase is initiated by the protein ubiquitylation activity of the anaphase-promoting complex/cyclosome (APC/C) and its coactivator Cdc20 (APC/C(Cdc20)). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/C(Cdc20). We used single-particle electron microscopy to obtain three-dimensional models of human APC/C in various functional states: bound to MCC, to Cdc20, or to neither (apo-APC/C). These experiments revealed that MCC associates with the Cdc20 binding site on APC/C, locks the otherwise flexible APC/C in a "closed" state, and prevents binding and ubiquitylation of a wide range of different APC/C substrates. These observations clarify the structural basis for the inhibition of APC/C by spindle checkpoint proteins.