Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae

Abstract

One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. alpha-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.

Fig. 1. Biosynthesis of Ams1

A, antiserum to Ams1 recognizes a 122-kDa band. Wild type (SEY6210; lane 1), ams1Δ (lane 2), and wild type cells harboring AMS1 on a CEN or 2μ plasmid (lanes 3 and 4) were grown in SMD to the early log phase. Ams1 expressed from plasmids is detected in a dosage-dependent manner. Plasmid-containing strains were induced with 50 μm copper sulfate for 40 min prior to harvest. Cells were lysed with glass beads and run on an 8% SDS-PAGE gel for immunoblot detection using anti-Ams1 antiserum. 1.0 A₆₀₀ equivalent units of extract was loaded in lanes 1 and 2 and 0.25 units in lanes 3 and 4. B, Pep4-dependent Ams1 cleavage is not a direct indication of vacuolar delivery. Steady-state levels of Ams1 are shown at various conditions for several strains. Strains in lanes 1–4 contain the CEN AMS1 plasmid pCuAMS1(414). Lane 1, wild type cells grown to early log phase in SMD and induced for 40 min with 50 μm copper sulfate. Lanes 2 and 3, wild type cells shifted 12 or 24 h to SGd (containing 10 μm copper sulfate), respectively. The level of Ams1 is increased, and a 73-kDa cleavage product (indicated by the asterisk) appears as cells are deprived of glucose (lanes 2 and 3). Lane 4, pep4Δ cells shifted 24 h to SGd (containing 10 μm copper sulfate). Lack of the 73-kDa species indicates this cleavage event is Pep4-dependent. Lane 5, ams1Δ cells shifted 24 h to SGd (containing 10 μm copper sulfate) is shown as a control for cross-reactive protein recognized by the anti-Ams1 antiserum. WT, wild type.

Fig. 2. Vacuole delivery of Ams1 is defective in cvt, apg, and aut mutants

A, mutants specific to the Cvt pathway do not import Ams1 into the vacuole. Vacuoles were isolated on Ficoll step gradients as described under “Experimental Procedures.” Activity assays of Ams1 and marker proteins show equivalent recovery of Ams1 and CPY-invertase in the vacuole fraction of wild type (WT) cells, but not in the vacuole fraction purified from the cvt3 and cvt9 mutant strains. CPY-invertase (vacuole marker), white bar; Ams1, black bar; α-glucosidase (cytosol marker), light gray bar; NADPH-cytochrome c reductase (ER marker), dark gray bar. Vacuoles were purified at least four times for each strain shown, and the total enzyme activity recovered in the vacuole fraction was divided by the total activity loaded on the gradient to obtain the percentage of recovery. Average recovery values are shown with error calculated as the standard deviation. B, mutants defective in both autophagy and the Cvt pathway are also defective in vacuolar delivery of Ams1. Ams1 is not recovered in the vacuole fractions from apg1, apg7, apg9Δ, apg14, and aut7 strains. Percentage of recovery was as determined for A. The cvt17 mutant accumulates Cvt vesicles within the vacuole, and shows equivalent vacuole recovery of Ams1 due to the accumulation of Ams1 within these vesicles.

Fig. 3. Ams1 transits to the vacuole as an oligomer

A, wild type (WT) cells harboring the CEN Ams1 plasmid pCuAMS1(414) were grown to early log phase and induced with 50 μm copper sulfate for 1 h. Strain MHY11 (AMS1∷AMS1-HA) was grown to early log phase and harvested directly. 20 A₆₀₀ units of cells were lysed with glass beads, mixed with βOG detergent (2% final concentration), and loaded on a 20–50% glycerol gradient. Ten fractions of 200 μl were collected from the top and examined by immunoblot. Immunoblots were probed with anti-Ams1, anti-HA, or anti-API antisera and then quantitated using the Vistra detection reagents as described under “Experimental Procedures.” Ams1 peaked as an oligomeric complex in fraction 6, whereas mAPI peaked at fraction 7 (7). Protein standards run on an identical gradient included: bovine serum albumin (66 kDa, fraction 3), aldolase (158 kDa, fraction 4), catalase (240 kDa, fraction 5), apoferritin (450 kDa, fraction 6), urease (545 kDa, fraction 6), and thyroglobulin (669 kDa, fraction 6). Note that the diffuse band in fractions 3 and 4 of the gradient detected with peptide antiserum is a cross-reacting contaminant and was not included in the quantification. B and C, oligomerization of Ams1 occurs in the cytosol and is maintained during vacuolar delivery. Native protein extracts from the apg7 and cvt17 mutant strains harboring pCuAMS1(414) were analyzed by glycerol gradients as in A. Ams1 is an oligomer in apg7, a mutant that is defective in vacuolar delivery of Ams1, and in cvt17 that is defective in the breakdown of Cvt bodies.

Fig. 4. Ams1 is delivered to the vacuole by autophagy

The Ams1YFP fusion protein was expressed in cvt17, pep4Δ, apg1, and apg7 cells and visualized by confocal microscopy using the GFP channel as described under “Experimental Procedures.” Vacuoles were labeled with FM 4–64, and cells were starved in SD–N for 12 h. Ams1YFP accumulates in subvacuolar vesicles of cvt17 and pep4Δ strains under nitrogen starvation conditions. Ams1 is not delivered to the vacuole in apg1 and apg7 strains that show only cytosolic localization of Ams1YFP.

Fig. 5. Model for Ams1 delivery to the vacuole

Like prAPI, Ams1 assembles into an oligomer in the cytosol and is sequestered by membrane that forms either a Cvt vesicle (nutrient-rich conditions) or an autophagosome (starvation conditions). It is not known whether Ams1 associates with the prAPI-containing Cvt complex. The completed vesicle targets to and fuses with the vacuole, releasing the Cvt body or autophagic body into the vacuole lumen. Subsequent vesicle breakdown releases the cargo. Unlike prAPI, proteolytic maturation of Ams1 does not occur concomitant with vacuolar import. Oligomeric Ams1 becomes peripherally associated with the vacuole membrane.