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. 1998 Aug 10;142(3):651–663. doi: 10.1083/jcb.142.3.651

Figure 2.

Figure 2

Figure 2

Figure 2

(A) Localization of ALP, (F/A)A-ALP, and (F/A)RS-ALP in vps45Δ cells. NBY83 (vps45Δ-X pho8Δ-X pep4Δ-X) harboring pSN92 (ALP; a, d, and g), pSN100 ((F/A)A-ALP; b, e, and h) or pSN123 ((F/A)RS-ALP; c, f, and i) were prepared for double labeling indirect immunofluorescence using the α-ALP mAb, 1D3-A10 (a–c) and affinity-purified antibodies against the 100-kD subunit of the V-ATPase, Vph1p (d–f), as described in Materials and Methods. Cells were also visualized using DIC microscopy (g–i). (B) Kinetics of processing of ALP, A-ALP, (F/A)A-ALP, RS-ALP, and (F/A)RS-ALP in wild-type and vps45Δ cells. AACY28 (wild type) and NBY68 (vps45Δ-X) pho8Δ-X PEP4 cells harboring pSN92 (ALP), pSN55 (A-ALP), pSN100 ((F/A)A-ALP), pSN97 (RS-ALP), or pSN123 ((F/A)RS-ALP) were labeled with [35S]Met for 10 min and chased by adding unlabeled methionine and cysteine, each to a final concentration of 50 μg/ ml. At the indicated times, proteins were immunoprecipitated from cell extracts using polyclonal antibodies against ALP. The resulting immunoprecipitates were subjected to SDS-PAGE and fluorography. The products of PEP4-dependent proteolysis are indicated using asterisks. (C) Model depicting the pathways taken by CPY and ALP to the vacuole. CPY reaches the vacuole by firstly transiting through a prevacuolar/endosomal compartment (PVC). Entry of proteins into this compartment requires the product of VPS45. Exit of proteins from the PVC, both back to the TGN and on to the vacuole requires the product of VPS27. ALP follows an alternative pathway to the vacuole bypassing the trafficking intermediates defined by mutations in VPS45 and VPS27. The large shaded arrow depicts the retrograde membrane trafficking pathway proposed to be taken by RS-ALP out of the vacuole to reach the PVC.