The yeast adaptor protein complex, AP-3, is essential for the efficient delivery of alkaline phosphatase by the alternate pathway to the vacuole

Abstract

A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast and in animals. To gain insight into the role of yeast AP-3, a genetic strategy was devised to isolate gene products that are required in the absence of the AP-3 mu chain encoded by APM3. One gene identified by this synthetic lethal screen was VPS45. The Vps pathway defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is transported via an alternate, intracellular route. This suggested that the apm3-Delta vps45 synthetic phenotype could be caused by a block in both the alternate and the Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing and missorting of ALP. ALP is no longer localized to the vacuole membrane by immunofluorescence, but is found in small punctate structures throughout the cell. This pattern is distinct from the Golgi marker Kex2p, which is unaffected in AP-3 mutants. We also show that in the apm3-Delta mutant some ALP is delivered to the vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way to the vacuole or destined for recycling to the Golgi. Surprisingly, in AP-3 class E vps double mutants these proteins reappear on the vacuole. We suggest that some AP-3-dependent cargo proteins that regulate late steps in Golgi to vacuole transport are diverted into the Vps pathway allowing completion of transfer to the vacuole in the class E vps mutant.

Figure 1

Precursor CPY is secreted from mts1-1 cells. Cells were pulse labeled with [³⁵S]methionine and [³⁵S]cysteine for 10 min and then chased for 30 min with excess cold amino acids. CPY was then immunoprecipitated from internal (I) and external (E) fractions. Strains indicated are: wt, SL1652 + pDS24 (pAPM3, CEN); apm3-Δ, SL1652; mts1-1, SL2284; mts2-1, SL2286. p2CPY indicates precursor CPY; mCPY indicates mature CPY.

Figure 2

Proposed sorting steps blocked by vps and other trafficking mutants analyzed in Table II. The sorting steps shown and the placement and grouping of mutants are based on a large number of studies (Raymond et al., 1992; Stack et al., 1995, and citations therein). In the Vps pathway cargo molecules are sorted at the late Golgi/TGN into transport vesicles that fuse with a PVC/ endosomal compartment. This latter step is blocked in many of the original class D vps mutants, such as vps45. Exit from the PVC for transport to the vacuole and for recycling back to the Golgi is thought to require the function of the class E mutants. Class C vps mutants block the final step of transport to the vacuole. Some of the sorting mutants analyzed in Table II are indicated with a question mark or are not shown in the figure (vps8, vps39, pep8, and dnm1). In these cases the placement in the sorting pathways is more ambiguous. Mutants showing synthetic growth phenotypes with apm3-Δ are indicated with an asterisk.

Figure 3

ALP processing is defective in AP-3 mutant cells. (A) Pulse-chase immunoprecipitation of ALP from isogenic wt (SL1652 + pDS24 [pAPM3, CEN]) and apm3-Δ (SL1652) cells. Cells were labeled with [³⁵S]methionine and [³⁵S]cysteine for 10 min, chased for indicated times (min), and subjected to IP with ALP antiserum. (B) Steady state levels of ALP forms in cells of the indicated genotypes (wt, SL1463; pho8-Δ, SNY17; aps3-Δ, LRB739; apm3-Δ, SL1652; apl5-Δ, HPY20; apl6-Δ, LRB858). Cells were lysed and prepared for immunoblotting with affinity-purified ALP antibodies. pALP, precursor ALP; mALP, mature ALP; *ALP, soluble lumenal ALP are indicated.

Figure 4

Fractionation of ALP forms in AP-3 mutant cells. Cells from an isogenic pair of strains (wt, SL1652 + pDS24 [pAPM3, CEN]; apm3-Δ, SL1652) were subjected to subcellular fractionation, followed by immunoblot analysis with anti-ALP antibodies. ALP forms are as indicated in Fig. 3. S13K, 13,000 g supernatant; S100K, 100,000 g supernatant; P100K, 100,000 g pellet.

Figure 5

Immunofluorescence localization of ALP in AP-3 mutant cells. wt (SL1653 + pDS24 [pAPM3, CEN]) and apm3-Δ (SL1653) cells were prepared for immunofluorescence. The V-ATPase was detected with monoclonal antibody 13D11 and goat anti–mouse Cy3. ALP was visualized with affinity-purified ALP antibodies and goat anti–rabbit FITC secondary.

Figure 8

Loss of AP-3 function results in disappearance of the class E PVC. ALP, V-ATPase, and Kex2p were localized by immunofluorescence as described in Figs. 5 and 6. (A) Localization of ALP and the V-ATPase in the class E prevacuole in vps4 strain, SF838-1D. (B) vps4 (SF838-1D + YCpKX22) cells stained for Kex2p and the V-ATPase. (C) Localization of ALP and the V-ATPase in the vps4 apm3-Δ strain, SL2845. (D) Colocalization of Kex2p and the V-ATPase to the vacuole in vps4 apm3-Δ cells (SL2845 + YCpKX22).

Figure 8

Loss of AP-3 function results in disappearance of the class E PVC. ALP, V-ATPase, and Kex2p were localized by immunofluorescence as described in Figs. 5 and 6. (A) Localization of ALP and the V-ATPase in the class E prevacuole in vps4 strain, SF838-1D. (B) vps4 (SF838-1D + YCpKX22) cells stained for Kex2p and the V-ATPase. (C) Localization of ALP and the V-ATPase in the vps4 apm3-Δ strain, SL2845. (D) Colocalization of Kex2p and the V-ATPase to the vacuole in vps4 apm3-Δ cells (SL2845 + YCpKX22).

Figure 6

Golgi staining is normal in AP-3 mutant cells. wt (SL1463 + YCpKX22) and apm3-Δ (SL1652 + YCpKX22) cells were stained with affinity-purified Kex2p antibodies followed by goat anti–rabbit FITC secondary. The plasmid YCpKX22 was used for overexpression of Kex2p to enable intracellular visualization as described previously (Wilcox et al., 1992).

Figure 7

ALP reaches the vacuole via the Vps Pathway in AP-3 mutant cells. Isogenic pairs of strains were shifted to 37°C for 30 min, pulse labeled for 10 min, chased for the indicated times (min), and then subjected to IP with ALP antiserum. (A) apm3-Δ (SL2767 + pEND4, CEN) and apm3-Δ end4-1 (SL2767). (B) apm3-Δ (SL1904) and apm3-Δ vps45-13 (SL2956).

Figure 9

Models for the Vps-dependent and alternative ALP pathways to the vacuole. (A) Proteins such as the Vps10p CPY sorting receptor, Kex2p and the vacuolar ATPase membrane protein, Vph1p, exit the late Golgi in transport vesicles, which then fuse with the prevacuole. This fusion is dependent upon the class D Vps proteins, such as Vps45p. In a step dependent upon the class E Vps proteins (e.g., Vps4p), late Golgi proteins and Vps10p are recycled to the Golgi from the PVC, whereas resident vacuolar proteins, such as Vph1p and soluble vacuolar hydrolases continue onto the vacuole in a step dependent on late acting Vps proteins (e.g., Vps33p and Vam3p). As shown in this study, ALP transport through the alternative route to the vacuole is dependent upon AP-3 function. It is not clear how many intermediate steps are required for transport of ALP to the vacuole and at what step AP-3 actually functions. Shown here is the possibility that AP-3 is involved in cargo selection at the late Golgi and that Vps41p/Vam2p acts at second, later step in the ALP pathway. Final delivery of ALP to the vacuole is also dependent on the Vps33p and Vam3p. The vacuolar t-SNARE, Vam3p, may also transit the alternative ALP pathway to the vacuole. (B) Much of the existing information on transport of cargo molecules to the vacuole is consistent with a model in which the Vps prevacuole identified in class E mutants is an early endosomal compartment that intersects with endocytic traffic. In this case, convergence of the alternative ALP and Vps pathways would occur at a late endosome. The final delivery of constituents from both the Vps and ALP pathways would still be dependent upon the class C and other late acting Vps components. The possibility that AP-3 is involved in recycling from an endosomal compartment back to the Golgi is also indicated in this model (see Discussion).