Figure 1. PEX5 recycling recapitulated in Xenopus egg extract (A) Xenopus eggs were lysed by centrifugation to generate an extract (the indicated layer between the lipid on top and cellular debris on bottom). The extract was treated with dimethylsulfoxide (DMSO) or the indicated E1 enzyme inhibitors dissolved in DMSO (UBA1, ubiquitin-activating enzyme; NAE, NEDD8-activating enzyme). Import activity was then assessed by incubating with the fluorescent cargo mScarlet-SKL and imaging on a spinning disk confocal microscope; repeated rounds of import into peroxisomes result in the appearance of bright puncta. The number of puncta in an imaged field was quantified relative to that in the untreated reaction (n = 9 fields per reaction; bars specify the median). (B) Upper scheme illustrates the strategy to deplete free ubiquitin from extract by inhibiting deubiquitinating enzymes (DUBs) with ubiquitin vinyl sulfone (UbVS); lower scheme shows the approach for restoring peroxisome import activity. On the right, extract was treated with buffer (mock) or UbVS and then supplemented either with buffer or wild-type ubiquitin, wild-type PEX5, or both components together. Import activity was then assessed as above (n = 16 fields per reaction). (C) As in (B), except that reactions were supplemented with the indicated components in the absence or presence of the E1 inhibitors. Import activity was assessed as above (n = 16 fields per reaction). (D) As in (C). Ubiquitin mutant (ÎGG) lacks both C-terminal glycines. Import activity was assessed as above (n = 16 fields per reaction). All scale bars, 5 μm. See also Figure S1.
Image published in: Skowyra ML and Rapoport TA (2022)
Copyright © 2022. Image reproduced with permission of the Publisher, Elsevier B. V.
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