Figure 5.

Inp1p acts as a molecular hinge between ER and peroxisomes. (A) Mating assay. Cells expressing mCherry-PTS1 and Inp1p-GFP and either Pex3p-W128L (MATa) or Pex3p-V81E (MATα) were mated to evaluate reconstitution of peroxisome tethering in the diploid cell. (B) Haploid cells used for mating in (A). Bar, 1 μm. (C) Time-lapse series of images of cells mated as depicted in (A). Time 0′ denotes cell fusion. MATa and MATα cells are labelled. Arrows highlight tethered peroxisomes in the zygote (105′) and its progeny (210′, 315′). Inserts show tethering complexes at high magnification. Bar, 3 μm. (D) Combined mating and split-GFP assay. Cells expressing mCherry-PTS1 and either Pex3p-W128L and Inp1p-½GFP (MATa) or Pex3p-V81E-½GFP and Inp1p (MATα) were mated to evaluate reconstitution of GFP fluorescence via interaction between Inp1p-½GFP in foci and Pex3p-V81E-½GFP on the surface of peroxisomes. (E) Images of cells at different times following the mating depicted in (D). Haploid cells are designated a and α. Zygotes are outlined. Diploid cells are unlabelled. Arrows highlight reconstitution of GFP fluorescence in tethering complexes. The insert shows a tethering complex at high magnification. Bar, 3 μm. See Supplementary Movie 5.