A. Expression of Ypt1 from a CEN plasmid results in a 10-fold increase of its level. The levels of Ypt1 protein in cells expressing it from its endogenous locus or a CEN plasmid were determined using immuno-blot analysis and anti-Ypt1 antibodies. Cells were transformed with a CEN plasmid expressing from left to right: yEVenus-Ypt1, empty plasmid (Ø), and Ypt1 (from its native promoter and terminator). Shown top to bottom: yEVenus-Ypt1, Ypt1, G6PDH (loading control), quantification of yEVenus-Ypt1 (left lane), and Ypt1 expressed as fold of endogenous level. B. Expression of Ypt1 from a 2μ plasmid results in a ~15-fold increase of its level. The level of Ypt1 was determined as described for panel A. Cells were transformed with a 2μ plasmid expressing from left to right: empty plasmid (Ø), Ypt1 and Ypt1-GTP (Ypt1-Q67L). Shown top to bottom: Ypt1, G6PDH (loading control), quantification of Ypt1 expressed as fold of endogenous level. C. The effect of expression of Ypt1 from a CEN plasmid on the co-localization of Cop1 and Sec7. Cells expressing Cop1-mRFP and Sec7-EGFP from their endogenous loci were transformed with a CEN plasmid (from panel A): empty (top) and for Ypt1 expression (bottom) and visualized by live-cell microscopy. Shown from left to right: DIC, Sec7, Cop1, and merge (yellow). D. The effect of expression of Ypt1 from a 2μ plasmid on the co-localization of Cop1 and Sec7. Cells expressing Cop1-mRFP and Sec7-EGFP from their endogenous loci were transformed with a 2μ plasmid (from panel B): empty (top), Ypt1 (middle) and Ypt1-GTP (bottom), and visualized by live-cell microscopy. Shown from left to right: DIC, Cop1, Sec7 and merge (yellow). Panels C–D: white arrows point to co-localized signal; bar, 5μm. E. The number of Sec7 puncta does not change upon overexpression of Ypt1 (blue bars) or Ypt31 (green bars). F. Co-localization (%) of Cop1 and Sec7 increases upon overexpression of Ypt1 (blue bars) but not Ypt31 (green bars). Shown from left to right in panels E–F: no plasmid (−), empty CEN and 2μ plasmids, expression of wild-type Ypt from CEN, 2μ, and Ypt-GTP from 2μ plasmids. Error bars and +/− represent STDEV. Information about Ypt1 is from this figure, and Ypt31 is from Figure S3A. Quantifications from two independent experiments for Ypt1 and Ypt31 are detailed in Figures S2C and S3C, respectively