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Fig. 6. SKD1(E235Q) increases the membrane association of lyst and enlarges lysosomes, but neither SKD1 nor SBP1 can decrease the size of lysosome in CHS fibroblast. (A) The pAb against murine Beige protein can detect human lyst protein on immunoblotting. Total cell lysates prepared from control human fibroblast (GM05655) and fibroblast cells obtained from a CHS patient (GM02075) were subjected to 5% SDS-PAGE and processed for immunoblotting. (B) PNS, cytosol (sup.) and membrane (ppt.) fractions prepared from control and GFP SKD1(E235Q) adenovirus-infected U251 cells were subjected to immunoblotting analysis with anti-lyst antibody. The numbers above each lane represent the volume ratio of all fractions loaded on the SDS gel. Notice, a significant amount of lyst was associated with the membrane in the cells expressing GFP-SKD1(E235Q). (C) Overexpression of GFP SKD1(E235Q) in control fibroblast cells (asterisks in a and b) formed enlarged lysosomes (arrows in a and b), which resembled the phenotype seen in CHS fibroblasts (arrows in c-f). However, neither wild-type GFP SKD1 (asterisks in c and d) nor GFP-SBP1 (asterisks in e and f) can decrease the size of giant-lysosomes in CHS fibroblasts. Bar, 20 µm. (D) Transfection of GFP SKD1(E235Q) in CHS fibroblasts did not inhibit the formation of or redistribution of SBP1 to E235Q compartments (asterisk indicated transfected cell). Scale bar: 20 µm.

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