MDDS-causing point mutations abolish WSC function and complex assembly. (A) The wsc deletion mutant was complemented with the indicated version of wsc–gfp and the distribution of HEX assemblies was quantified in apical and subapical compartments. Standard deviation is shown. These mutants do not dramatically alter steady-state levels of protein as revealed by a Western blot of extracts from the indicated strains (inset). (B) Capping is abolished by the R102W mutation. The indicated versions of WSC–GFP were localized using laser-scanning confocal microscopy. RFP-PTS1 reveals the peroxisome matrix. Arrow points to weak capping occasionally observed in the R102W mutant. Bar, 5 μm. (C) Evidence that WSC and HEX physically interact and effect of the R102W mutation on this interaction. Detergent extracts were prepared from strains expressing WSC-HA or WSC-R102W-HA from the native wsc promoter and anti-HA antibodies were used to precipitate WSC. Anti-HEX antibodies reveal the degree of HEX coprecipitation. (D) The R102W mutation affects WSC–WSC interaction. Extracts obtained from strains expressing the indicated versions of WSC were examined for WSC coprecipitation as described in Fig. 6 C. (E) Aberrant WSC–WSC complexes in the R102W mutant. Extracts were prepared from the indicated strains, treated with detergent, and separated by density gradient centrifugation, as in Fig. 6 E, using buffer H or buffer N.