Figure 5. HEX controls WSC localization... | Rockefeller University Press

Figure 5.

$Figure 5. HEX controls WSC localization and the development of large peroxisomes. (A) The localization of WSC–GFP was examined in the presence (wt) and absence (Δhex) of HEX. In wild-type hyphae, WSC is enriched in the WB membrane and is mostly excluded from abundant cytosolic peroxisomes. In the absence of hex, WSC is localized to small cytosolic peroxisomes. Bar, 10 μm. Magnified views of the region are indicated by a black square and shown below. (B) WSC cofractionates with thiolase in the absence of HEX. An organellar fraction was obtained from the hex deletion strain expressing WSC-HA and separated by density gradient centrifugation. The bottom (B) and top (T) of the gradient are indicated. (C) The detergent insolubility of WSC depends on HEX. An organellar fraction from wild-type and hex deletion strains was treated as indicated and fractionated into pellet (P) and supernatant (S) fractions by centrifugation at 100,000 g for 45 min. (D) Large apical peroxisomes are no longer observed in the hex deletion strain. GFP-PTS1–expressing wild-type and hex deletion strains were examined by laser-scanning confocal microscopy and peroxisome size was estimated using morphometric analysis as described in Materials and methods. Peroxisome size is plotted against mean GFP intensity (arbitrary units), which provides an estimate of GFP-PTS1 density in the peroxisome matrix. The inset panels show a representative image of the indicated strains as well as a Western blot showing that both strains express equal levels of GFP-PTS1.$

HEX controls WSC localization and the development of large peroxisomes. (A) The localization of WSC–GFP was examined in the presence (wt) and absence (Δhex) of HEX. In wild-type hyphae, WSC is enriched in the WB membrane and is mostly excluded from abundant cytosolic peroxisomes. In the absence of hex, WSC is localized to small cytosolic peroxisomes. Bar, 10 μm. Magnified views of the region are indicated by a black square and shown below. (B) WSC cofractionates with thiolase in the absence of HEX. An organellar fraction was obtained from the hex deletion strain expressing WSC-HA and separated by density gradient centrifugation. The bottom (B) and top (T) of the gradient are indicated. (C) The detergent insolubility of WSC depends on HEX. An organellar fraction from wild-type and hex deletion strains was treated as indicated and fractionated into pellet (P) and supernatant (S) fractions by centrifugation at 100,000 g for 45 min. (D) Large apical peroxisomes are no longer observed in the hex deletion strain. GFP-PTS1–expressing wild-type and hex deletion strains were examined by laser-scanning confocal microscopy and peroxisome size was estimated using morphometric analysis as described in Materials and methods. Peroxisome size is plotted against mean GFP intensity (arbitrary units), which provides an estimate of GFP-PTS1 density in the peroxisome matrix. The inset panels show a representative image of the indicated strains as well as a Western blot showing that both strains express equal levels of GFP-PTS1.