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Fig. 4. Ent3p and Ent5p were not involved in transport of all cargo proteins between the TGN and endosomes. Retrograde transport from the late endosome to the TGN was monitored using A-ALP (A) and the CPY receptor Vps10p (B). Both proteins contain a retrieval signal for recycling from the late endosome to the TGN. A-ALP consists of the cytosolic domain of dipeptidyl aminopeptidase A (DPAP A), which localizes to the TGN, fused to the transmembrane and luminal domain of alkaline phosphatase (ALP) for detection (Nothwehr et al., 1993). The stability of these proteins was not strongly reduced indicating that their trafficking was not significantly affected by the lack of Ent3p or Ent5p [The percentage of A-ALP remaining compared to the amount after the 5 minutes chase: 60 minutes chase, WT 68% (s.d. ±24, n=3), ent3
65% (s.d. ±14), ent5 56% (s.d. ±10) and 120 minutes chase, WT 39% (s.d. ±17), ent3 35% (s.d. ±15), ent5 24% (s.d. ±17). The percentage of intact Vps10p compared to total Vps10p after 3 hours of chase was: WT 58% (±2.2%, n=2), ent3 69% (±0.9%), ent5 65% (±0.5%) and ent3 ent5 61% (±0.3%).] Cells were pulse labeled at 37°C and chased for the indicated time periods before immunoprecipitation. Vps10p was immunoprecipitated with antisera directed against Vps10p from strains SEY6211, SCY2, SCY25 and SCY26. A-ALP was immunoprecipitated with antisera directed against ALP in cells deleted for PHO8, the gene encoding ALP to avoid immunoprecipitation of ALP (strains: SNY18, BKY25, BKY26 all with the CEN plasmid pSN55 encoding A-ALP). pA-ALP, Golgi proA-ALP; mA-ALP, mature A-ALP processed in the vacuole; Vps10p*, Vps10p cleaved in the vacuole.
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