Proximity Labeling Reveals Spatial Regulation of the Anaphase-Promoting Complex/Cyclosome by a Microtubule Adaptor

Abstract

The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination events. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. We describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. Microtubule-localized proximity biotinylation tools revealed that PLEKHA5 depletion decreased APC/C association with the microtubule cytoskeleton, which prevented efficient loading of APC/C with its coactivator CDC20 and led to reduced APC/C E3 ligase activity. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5–APC/C interaction in microtubules. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

“…EGFP-tagged PLEKHA4 and PLEKHA5 constructs with a CMV promoter were generated in our previous studies 17 , 20 . mScarlet-i-PLEKHA5 WW was made by inserting the cDNA sequence encoding the tandem WW domain (residue 1–105) into the CMV-mScarlet-i-C1 vector (Addgene 85044) using BamHI and SalI.…”

“…HeLa cell line with PLK1 C-terminally tagged by GFP at the endogenous locus via CRISPR/Cas9 was a gift from Prof. Iain Cheeseman (MIT). H2B-mCherry-expressing Flp-In T-Rex HeLa cells were made in our previous study 17 . To generate a stable cell line expressing Lyn 10 -TurboID-V5 under doxycycline induction (2.5 μg/mL), pSBtet-Lyn 10 -TurboID-V5-puro and pCMV(CAT)T7-SB100 (Addgene 34879) were co-transfected into Flp-In T-Rex HeLa cells in a 19:1 ratio, and cells were selected with 2 μg/mL puromycin treatment beginning 24 h after transfection and continuing for 2 days.…”

“…During interphase, PLEKHA5 localizes to the microtubule cytoskeleton via N-terminal tandem WW domains [17][18][19] (Figure 1A). However, we observed that during mitosis, a substantial pool of EGFP-PLEKHA5 FL (FL, full-length) localized to the plasma membrane (PM), in addition to the mitotic spindle (Figure 1B).…”