In Saccharomyces cerevisiae, surprisingly, the transmembrane protein Sbh2, which harbors an intramembrane degron, is a substrate of the ubiquitin-protein ligase Doa10.

Eps8, a bi-functional actin cytoskeleton remodeler, is a positive regulator of cell proliferation and motility. Here, we describe an unrecognized mechanism regulating Eps8 that is required for proper mitotic progression: While Eps8 is stable in G1 and S phase, its half-life drops sharply in G2. This requires G2-specific proteasomal degradation mediated by the Ubiquitin E3 ligase SCFFbxw5. Consistent with a short window of degradation, Eps8 disappears from the cell cortex early in mitosis, but reappears at the midzone of dividing cells. Failure to reduce Eps8 levels in G2 prolongs its localization at the cell cortex and markedly delays cell rounding and prometaphase duration. However, during late stages of mitosis and cytokinesis, Eps8 capping activity is required to prevent membrane blebbing and cell shape deformations. Our findings identify SCFFbxw5-driven fluctuation of Eps8 levels as an important mechanism that contributes to cell-shape changes during entry into – and exit from - mitosis.

Figure 4. Investigation of Sbh1/Sbh2 chimeric proteins. Both Sbh2 TM and ER-luminal regions contribute to the degron. (A) Schematic representation of Sbh1, Sbh2, and Sbh1/Sbh2 chimeric proteins used in this study. Sbh1-derived regions are depicted in light blue, and Sbh2-derived regions are shown in black. The nomenclature for Sbh1/Sbh2 chimeric proteins is as follows: "Sbh-XYZ" designates a chimeric protein in which the number at position X indicates the source of the cytosolic domain ("1": Sbh1; or "2": Sbh2), the number at position Y indicates the source of the TM helix, and the number at position Z indicates the source of the ER-luminal domain, respectively. (right) Comparison of Sbh1, Sbh2, and Sbh1/Sbh2 chimera TA sequences. Residues depicted bold in red represent identical residues shared between Sbh1 and Sbh2; residues depicted in light blue are Sbh1-specific residues, and residues depicted in black indicate residues specific for Sbh2; the black horizontal line indicates the TM helix, and the asterisks indicate Sbh2 residues Ser61 and Ser68, respectively. N, N terminal; C, C terminal. (B) Degradation of HA-Sbh-122. chx chase as in Fig. 1

Microtubule depolymerases of the kinesin-13 family play important roles in various cellular processes and are frequently overexpressed in different cancer types. Despite the importance of their correct abundance, remarkably little is known about how their levels are regulated in cells. Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCF Fbxw5 , including the kinesin-13 member Kif2c/MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2a and Kif2b become efficiently polyubiquitylated by neddylated SCF Fbxw5 and Cdc34, without requiring preceding modifications. In cells, SCF Fbxw5 targets MCAK for proteasomal degradation predominantly during G 2 . While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies and impairs ciliogenesis in the following G 1 /G 0 , which can be rescued by concomitant knockdown of MCAK, Kif2a or Kif2b. We thus propose a novel regulatory event of ciliogenesis that begins already within the G 2 phase of the preceding cell cycle.