Dnm1p-dependent peroxisome fission requires Caf4p, Mdv1p and Fis1p.

Strains and plasmids

Yeast strains were derivatives of BY4741 ( MATA his3 -Δ 1 ​ leu2 -Δ 0 ​ met15 -Δ 0 ​ ura3 -Δ 0 ) or BY4742 ( MATa his3 -Δ 1 ​ leu2 -Δ 0 ​ lys2 -Δ 0 ​ ura3 -Δ 0 ) obtained from the EUROSCARF consortium. Double or triple gene deletions were made by replacing the entire coding sequence of the mutated genes with a marker ( Schizosaccharomyces pombe HIS5 , or the Klebsiella pneumoniae hygromycin B phosphotransferase gene cassette that confers resistance to Hygromycin B ( Goldstein and McCusker, 1999 ). dnm1 Δ / vps1 Δ, mdv1 Δ / vps1 Δ, caf4 Δ / vps1 Δ, mdv1 Δ / caf4 Δ/ vps1 Δ, num1 Δ/ vps1 Δ and Δ fis1 Δ / vps1 were generated by replacing the VPS1 reading frame with the HIS5 cassette in the dnm1 Δ, mdv1 Δ, caf4 Δ, mdv1 Δ / caf4 Δ, num1 Δ and fis1 Δ, respectively. The MGM1, ATP7 and ATP17 open reading frames were replaced by that of the Hygromycin cassette to generate dnm1 Δ / vps1 Δ / mgm1 Δ, dnm1 Δ / vps1 Δ / atp7 Δ, dnm1 Δ / vps1 Δ / atp17 Δ, / vps1 Δ / atp7 Δ, / vps1 Δ / atp17 Δ and dnm1 Δ / atp7 Δ,. The mdv1 Δ / caf4 Δ double mutant was constructed by replacing the CAF4 open reading frame with that of the Hygromycin cassette in the mdv1 Δ mutant. URA3 and LEU2 centromere plasmids were derived from Ycplac33 and Ycplac111 ( Gietz and Sugino, 1988 ). GFP-PTS1 is a peroxisomal luminal GFP marker protein appended with the well-characterised peroxisomal targeting signal type 1 (PTS1) ( Gould et al., 1988 ). A far-red peroxisomal luminal marker was made by appending a variant of the Heteractis crispa Chromoprotein (HcRed) with the PTS1. As source of HcRed we used HcRed-Tandem with optimised yeast codon usage (Evrogen, Moscow, Russia). Constitutive expression of GFP-PTS1 and HcRed-PTS1 was under control of the TPI1 promoter and the HIS3 promoter, respectively. Dnm1p overexpression was achieved using the TPI1 promoter. All constitutive expression constructs contained the PGK1 terminator. The Fis1-Pex15p fusion protein was expressed from a construct containing the FIS1 promoter, and the fusion protein contained the cytoplasmic domain of Fis1p and the C-terminal tail anchor of Pex15. Caf4p and Mdv1p were N-terminally tagged with GFP. Conditional expression constructs contained the GAL1 promoter. In order to reduce the half-life of the transcript we replaced the PGK1 terminator with the MFA2 terminator ( Duttagupta et al., 2003 ; LaGrandeur and Parker, 1999 ) Growth conditions and mating assay For all experiments, cells were grown overnight in selective glucose medium. For analysis of phenotypes by microscopy, cells were subsequently diluted to OD 0.1 in 2% glucose medium + casamino acids and grown for 2 - 3 cell divisions (4 - 6 h), so that phenotypes were analysed under conditions whereby cells are actively maintaining their peroxisome number. In the case of the mgm1Δ and ATP synthase mutants, peroxisome number varied significantly depending on growth media but reproducible results were obtained using 2% glucose + casamino acid medium. Cells were fixed (see below) for 5 minutes before imaging. For the experiment described in Fig. 3 , an overnight culture was used to inoculate selective galactose medium at an OD 600 of 0.1 to allow induction of reporter proteins for 3 h. Cells were then switched to selective glucose medium for 2 hours, to shut down expression of the gal-inducible reporter protein, before mating. For mating, cells were collected by filtration onto a 0.22 micron Millipore nitrocellulose filter (type GS, 25 mm diameter) and this filter was incubated, cells side up, on a pre-warmed YPD plate at 30°C. 1×10 7 cells of each strain were collected per 25 mm filter. After 2 hours, cells were harvested by vortexing the filter in selective glucose medium, and fixed for 5 min by adding formaldehyde to 3.6%. Free formaldehyde groups were quenched in 0.1 M ammonium chloride/1xPBS. Cells were imaged within 1 h of fixing as loss of fluorescence intensity and increase of autofluorescence was seen in fixed cells left for extended periods. For each experiment, >100 cells were examined and images are representative of findings. For Fig, 1 , budding cells were counted as single cells.

FM4-64 vacuole staining

Cells were grown in 2% glucose selective medium to log phase (OD 0.5). 1 ml of log phase cells were pelleted, resuspended in YPD containing 20 μM FM 4-64 and incubated at 30°C for 15 min. They were then pelleted, and resuspended in 1 ml of YPD and incubated at 30°C for 30 min. Subsequently the cells were washed in 1 ml of water before being resuspended in 2% glucose medium, ready for imaging. Image acquision Live and fixed cells were analysed at with an Axiovert 200M (Zeiss) equipped with Exfo X-cite 120 excitation light source, band pass filters (Zeiss and Chroma) and alpha Plan-Fluar 100 x/1.45 NA or A-Plan 40 X/0.65 NA Ph2 objective lens (Zeiss) and Hamamatsu Orca ER digital camera. Image acquisition was performed using Openlab software (Improvision) at 21°C. Fluorescence images were collected as 0.2 μ m z-stacks and merged into one plain after contrast enhancing in Openlab, and processed further in Photoshop except when stated differently in text or figure legends. Bright field images were collected in one plain. In bright field image was added into the blue channel in Adobe Photoshop. The level of the bright field images was modified and the image was blurred, sharpened and blurred again before one more round of level adjustment so that only the circumference of the cell was visible.