FIG. 1.

Identification of YPR128cp as a peroxisomal membrane protein in S. cerevisiae. (A) Subcellular fractionation of wild-type cells expressing NH-YPR128cp. Oleate-grown cells were fractionated by differential centrifugation of a homogenate (H) into a 17,000 × g pellet (P) and supernatant (S). The upper panel shows the activity of 3-hydroxyacyl-CoA dehydrogenase (3HAD), a peroxisomal marker, whereas the lower panel shows the NH-YPR128cp fusion protein as detected on Western blot using an antibody against the NH tag. (B) The 17,000 × g pellet (P) was further fractionated by Nycodenz equilibrium density gradient centrifugation (fractions 1 to 15). Mitochondrial (M) and peroxisomal (P) matrix markers are fumarase (▴) and 3-hydroxyacyl-CoA dehydrogenase (3HAD) (●), respectively (upper panel), and NH-YPR128cp was detected by immunoblot analysis (lower panel). (C) Immunogold electron micrograph showing association of NH-YPR128cp with the peroxisomal membrane. NH-YPR128cp was visualized using specific antibodies against the NH epitope and protein A-gold particles.