Fig. 4.

Degron protection assay reveals protein topology. a A degron-tagged clathrin reporter ZF1::mCh::CHC-1 initially localizes to the plasma membrane (arrowhead) and intracellular puncta (n = 9). Scale bar: 10 µm. b After tat-5 knockdown, ZF1::mCh::CHC-1 is enriched at the plasma membrane (arrowhead, n = 14). c After expression of ZIF-1 begins in anterior cells, ZF1::mCh::CHC-1 is degraded throughout the anterior cells in control embryos (n = 19). d Although ZF1::mCh::CHC-1 is still enriched at the plasma membrane in posterior cells (arrowhead) in tat-5 RNAi-treated embryos, ZF1::mCh::CHC-1 is lost from the plasma membrane in anterior cells (arrow, n = 18), indicating that clathrin is accessible to ubiquitin ligases. GFP::PH::ZF1 labels the plasma membrane and extracellular vesicles. e Quantification of clathrin enrichment on a posterior cell contact (EMS:P2) or anterior cell contact (ABa:ABp) compared to the neighbouring cytoplasm at the four- and six-cell stage from two independent experiments. ZF1::mCh::CHC-1 fluorescence was significantly increased at the posterior EMS:P2 cell contact (***p < 0.001 using Student’s t-test, ctrl n = 19, tat-5 RNAi n = 18). No change was observed at anterior cell contacts (p > 0.05). Bars represent mean ± s.e.m. Source data are provided as a Source Data file. f If clathrin were in extracellular vesicles, ZF1::mCh::CHC-1 would be protected from ZIF-1-mediated degradation. However, as clathrin is inside the plasma membrane, ZF1::mCh::CHC-1 is accessible to ZIF-1-mediated degradation