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Fig. 9. Depletion of TSG101 disrupts trafficking of lysosomal precursors at the early endosome. (A) Lamin A or TSG101-depleted HeLa cells were pulse labelled with [³⁵S]methionine and chased as indicated in the presence of excess unlabelled methionine. Anti-cathepsin D immunoprecipitates from cell culture supernatants (top part) and cells (bottom part) were analysed by SDS-PAGE and fluorography. The migration positions of procathepsin D (P; 45 kDa), the intermediate cleaved form of cathepsin D (I; 40 kDa) and mature cathepsin D (M; 27 kDa) are indicated. Duplicate samples from a representative experiment are shown. (B) Phosphorimager data from cathepsin D pulse-chase experiments were analysed to determine the amounts of cathepsin D cleaved to the intermediate form (interm), cleaved to the mature form (mature) or secreted from cells (secreted) in TSG101-depleted cells relative to lamin A-depleted cells. Data from a 2-hour chase are shown as white bars, and those from a 4-hour chase as shaded bars. Data are the means ± s.e.m. from three independent experiments, each performed in duplicate. (C) Lamin A-depleted (upper panels) or TSG101-depleted (centre panels) cells were fixed and stained for mannose 6-phosphate receptor (M6PR; green), and GM130 or EEA1 (red) as indicated. Inserts are magnified twofold relative to the main images. Alternatively, TSG101-depleted cells (lower panels) were allowed to internalise Alexa Fluor488-labelled EGF for 3 hours at 37°C prior to fixation, then examined for EGF (green) and mannose 6-phosphate receptor (red). Bar, 10 µm.

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