Extended Data Fig. 2: Use of a Tev protease assay shows an increased import rate of cargo into the enhanced capacity peroxisomes.

Scale bars in all microscopy images are 5 µm. (a) A TEV protease assay utilizes expression of RFP-TEV protease cleavage site-YFP-ePTS1 with varying expression levels of the TEV protease. At low expression of TEV protease, both RFP and YFP should be localized to the peroxisome as there is not enough TEV protease to cleave the fluorescent proteins before import to the peroxisome. At high expression of the TEV protease, there is competition between import rate and TEV cleavage. If TEV protease cleaves between the fluorescent proteins before import, only YFP will be imported to the peroxisome while RFP will remain cytosolic. At high TEV protease expression, WT capacity peroxisomes have a large quantity of RFP in the cytosol as visualized by the diffuse red fluorescence throughout the cell, meaning that TEV-catalyzed cleavage occurred before peroxisome import. In the enhanced peroxisome capacity strain, the bulk of the RFP is colocalized in the peroxisome, meaning faster import to the peroxisomes protects the fluorescent proteins from being cleaved from each other. Microscopy was performed two individual times with similar results. (b) Fluorescence microscopy of YFP-ePTS1 in WT, cells with the engineered TFs, and the ML predicted enhanced capacity peroxisome strain. The TFs gave singular, large peroxisomes compared to WT, while the enhanced capacity peroxisomes had many, bright peroxisomes that were not as large as the TFs, but led to an overall increase in functional capacity. Microscopy was performed two individual times with similar results.