Fig. 1.

Mdh2 is localized to the cytosol and to peroxisomes. (A) When endogenous GFP–MDH2 is expressed under its native promoter, the GFP–Mdh2 protein is dually localized to the cytosol and to peroxisomes, as seen by colocalization with Pex3–mCherry (in oleate the colocalization is demonstrated by white arrows). Mdh2 expression levels are higher on oleate medium due to the removal of the glucose-induced repression. Therefore, a longer imaging exposure was used for glucose-grown cells. Hundreds of cells were imaged in three technical repeats. (B) Mdh2–GFP (expressed under its native promoter) is no longer localized to puncta in the absence of peroxisomes (Δpex19), proving that the puncta represent peroxisomes. Hundreds of cells were imaged in three technical repeats. (C) Mdh2–GFP (expressed under its native promoter) is present both in the cytosolic fraction (S) and in the organellar pellet (P) of fractionated cells. T, total. Fox3 (also known as Pot1, peroxisomal 3-oxoacyl-CoA thiolase, a PTS2 protein), Fox1 (also known as Pox1, acyl-CoA oxidase), Pcs60 (oxalyl-CoA synthetase, a PTS1 protein) and Pex11 (a peroxisomal membrane protein; arrowhead) were used as peroxisomal markers. Por1 (mitochondrial porin) was used as a mitochondrial marker, and Pgk1 (phosphoglycerate kinase 1) as a cytosolic marker. n=2. (D) Density gradient of the postnuclear supernatant (PNS) shows that Mdh2–GFP (expressed under its native promoter) can be found in peroxisomal fractions (marked with a dashed line). Fox3 was used as peroxisomal marker, Por1 was used as a mitochondrial marker and Pgk1 as a cytosolic marker. n=2.