Figure 5.

Vps35p is required for retrieval of A-ALP and Vps10p from the prevacuolar compartment to the Golgi. vps27Δ (AHY65), vps27Δ vps35Δ (AHY68), and vps27Δ vps35–101 (AHY69) strains containing CEN plasmids directing expression of A-ALP (pAH16) and containing the VPS27 gene under GAL1 promoter control (pHY5) were propagated in media containing raffinose as a carbon source. At the 0-min time point the cultures were adjusted to 2% galactose to induce expression of VPS27. After 0 and 90 min after galactose addition, cells were fixed, spheroplasted, and costained with antibodies against ALP and Vps10p. After subsequent treatment with fluorochrome-conjugated secondary antibodies, the cells were viewed by DIC optics and by epifluorescence through filters specific for FITC and Texas Red.