Extended Data Fig. 3: The orientation of chauffeured Yea4p was screened by a TEV protease assay.
From: Functional targeting of membrane transporters and enzymes to peroxisomes
Yea4p cargo was tracked by a CFP, and the chauffeur was fused to an RFP. (a) YFP followed by a TEV protease cleavage site was fused to the N-terminus of Yea4p. With and without TEV protease induction (with either 100 nM or 1000 nM β-estradiol), the YFP signal of this N-terminal fusion remained punctate and localized at the peroxisome, consistent with protection from the TEV protease from having a lumen-facing orientation throughout trafficking and within the peroxisome. (b) The C-terminus of Yea4p-2xSH3 was fused with a TEV protease cleavage site followed by a YFP. With this fusion and induction of TEV protease, the release of YFP to the cytoplasm was observed, indicating a cytosolic-oriented C-terminus. Based on traces of Yea4p-CFP signal residing on the ER membrane and the incomplete TEV protease cleavage at 100 nM β-estradiol, the YFP fusion may partially interfere with SH3 chauffeuring and TEV protease cleavage through steric hindrance. A higher concentration of 1000 nM β-estradiol resulted in more complete cleavage of YFP from peroxisomes. All scale bars represent 5 µm.
