Figure 6. WSC overproduction promotes... | Rockefeller University Press

Figure 6.

$Figure 6. WSC overproduction promotes cortical association in the absence of HEX. (A) WSC can target peroxisomes to the cell cortex in the absence of hex. WSC–GFP was overproduced from the strong hex promoter (WSC op). Bar, 10 μm. Magnified views of the region are indicated by a black square and shown below. (B) Overproduced WSC–GFP cofractionates with thiolase. Fractionation was done as described for Fig. 4 A. The bottom (B) and top (T) of the gradient are indicated. (C) WSC is in oligomers. WSC-HA and WSC–GFP were coexpressed or WSC–GFP was expressed alone in the wsc deletion strain. Detergent extracts from these strains were precipitated with anti-HA, and Western blotting with anti-GFP reveals the degree of WSC–GFP coprecipitation. WSC–GFP alone provides a control for the specificity of anti-HA precipitation. (D) Peroxisomes from the hex deletion (Δhex) overproducing WSC–GFP contain detergent-insoluble WSC–GFP–enriched membrane domains. An organellar fraction from the indicated strain was examined by epifluorescence microscopy in the absence (buffer) and presence (1% TX-100) of detergent. Bar, 1 μm. (E) Detergent-insoluble WSC–GFP complexes sediment to densities higher than the peroxisomes in which they form (compare with B). The organellar fraction was treated with 1% Triton X-100 and separated as described in Fig. 6 B. Porin and thiolase provide controls for detergent extraction and, in a separate experiment, PEX14-GFP reveals the behavior of another PMP (bottom). The bottom and top of the gradient are indicated. (F) The WSC complex promotes BiFC. The indicated constructs were coexpressed in the wsc mutant background and the resulting strain was examined for YFP fluorescence. Arrows point to mature WBs. Bar, 10 μm.$

WSC overproduction promotes cortical association in the absence of HEX. (A) WSC can target peroxisomes to the cell cortex in the absence of hex. WSC–GFP was overproduced from the strong hex promoter (WSC op). Bar, 10 μm. Magnified views of the region are indicated by a black square and shown below. (B) Overproduced WSC–GFP cofractionates with thiolase. Fractionation was done as described for Fig. 4 A. The bottom (B) and top (T) of the gradient are indicated. (C) WSC is in oligomers. WSC-HA and WSC–GFP were coexpressed or WSC–GFP was expressed alone in the wsc deletion strain. Detergent extracts from these strains were precipitated with anti-HA, and Western blotting with anti-GFP reveals the degree of WSC–GFP coprecipitation. WSC–GFP alone provides a control for the specificity of anti-HA precipitation. (D) Peroxisomes from the hex deletion (Δhex) overproducing WSC–GFP contain detergent-insoluble WSC–GFP–enriched membrane domains. An organellar fraction from the indicated strain was examined by epifluorescence microscopy in the absence (buffer) and presence (1% TX-100) of detergent. Bar, 1 μm. (E) Detergent-insoluble WSC–GFP complexes sediment to densities higher than the peroxisomes in which they form (compare with B). The organellar fraction was treated with 1% Triton X-100 and separated as described in Fig. 6 B. Porin and thiolase provide controls for detergent extraction and, in a separate experiment, PEX14-GFP reveals the behavior of another PMP (bottom). The bottom and top of the gradient are indicated. (F) The WSC complex promotes BiFC. The indicated constructs were coexpressed in the wsc mutant background and the resulting strain was examined for YFP fluorescence. Arrows point to mature WBs. Bar, 10 μm.