Extended Data Fig. 2: Activity and localization of trafficked candidate UDP-glucose transporters.
From: Functional targeting of membrane transporters and enzymes to peroxisomes
(a) Localization of non-trafficked Yea4p was compared to the ER labeled with a YFP (Venus-HDEL). In the dead SH3 peptide (dSH3) control and cases of inefficient trafficking, Yea4p co-localized with the ER-localized YFP signal as expected. (b) Esculin standard curve. Fluorescence at 460 nm plotted against esculin concentration generated the standard curve used to interpolate esculin titers for esculetin-to-esculin bioconversion assays. (c) Trafficked Yea4p activity was analyzed using the esculetin-to-esculin assay. Yea4p was trafficked via the engineered SH3 domain-SH3 peptide ligand protein-protein interaction. In the direct fusion strategies, the native protein truncations tPex22 and tPex15 were fused to either the N- or C-terminus of Yea4p, respectively. While tPex22-Yea4p and Yea4p-tPex15 resulted in weak and strong peroxisome localization (Fig. 1), respectively, low levels of activity were observed for both fusions. One-way ANOVA was used to compare the sample data between each group of 4 biological replicates. (d) NSTs trafficked to the peroxisome via the chauffeur strategy were tested for their UDP-glucose transport activities on the peroxisome membrane. Previous studies suggest that Yea4p and AtUTR1 exhibit transport activities for UDP-glucose while Hut1p and Ymd8p only exhibit marginal transport activities toward the co-factor. CDT2 is an unrelated membrane protein from Neurospora crassa that transports cellodextrins and is used as a negative control. (e) AtUTR1 trafficked with an active SH3 domain traffics some of the membrane protein to the peroxisome, while the dSH3 control results in protein that does not appear to associate with a yeast membrane. All scale bars represent 5 µm. All data shown are the mean ± s.e.m. of four biological replicates and all subfigures use the following significance: * denotes p-value < 0.05; ** p-value < 0.01; **** p-value < 0.001.
