Apg2 is a novel protein required for the cytoplasm to vacuole targeting, autophagy, and pexophagy pathways

Abstract

To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions. Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway. Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole. Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut, and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways. In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes. Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are needed for vacuolar import through both the Cvt pathway and autophagy. Biochemical studies revealed that Apg2 is a peripheral membrane protein. Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.

FIG. 1. Apg2 is required for both the Cvt and autophagy pathways

A, cloning and characterization of APG2 (YNL242w). Wild type (WT; SEY6210), apg2 (MT2–4-3), apg2Δ (ynl242wΔ; CWY1), and the apg2Δ strain transformed with single copy (CEN; pYCG_YNL242w) or multicopy (2 µm; pAPG2(426)) plasmids encoding APG2 were grown to A₆₀₀ = 1.0 in SMD. Cells were precipitated with 10% trichloroacetic acid, lysed with glass beads, and analyzed by immunoblot using antiserum against Ape1 as described under “Experimental Procedures.” The APG2 gene complements the prApe1 accumulation phenotype of the apg2Δ strain. B, Apg2 is essential for survival during nitrogen starvation. Wild type (SEY6210), apg2Δ (CWY1), and the apg2Δ strain transformed with an APG2 centromeric plasmid (pYCG_YNL242w) were grown in SMD and shifted to SD-N as described under “Experimental Procedures.” The APG2 gene complements the starvation-sensitive phenotype of the apg2Δ mutant.

FIG. 2. Analysis of the apg2Δ strain indicates that Apg2 is required for the vesicle formation/completion step

A, precursor Ape1 in the apg2Δ strain is protease-accessible. Spheroplasts isolated from apg2Δ, apg7Δ, and ypt7Δ cells were osmotically lysed (T, total) and centrifuged at 13,000 × g to obtain low-speed supernatant (S) and pellet (P) fractions. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described under “Experimental Procedures.” B, precursor Ape1 in the apg2Δ strain accumulated in a membrane-associated float fraction. Spheroplasts from the apg2Δ strain were osmotically lysed (T, total) and separated into supernatant (S) and pellet (P) fractions by centrifugation at 13,000 × g for 10 min. The pellet fraction was resuspended in 15% Ficoll 400 in the absence or presence of 0.2% Triton X-100, and overlaid with 13 and 2% Ficoll 400 in a step gradient. The gradients were centrifuged at 13,000 × g for 10 min at room temperature. Three fractions were collected including float (F), non-float (NF) and pellet (P2) fractions as described under “Experimental Procedures.” All samples were trichloroacetic acid precipitated, acetone washed twice, resolved by SDS-polyacrylamide gel electrophoresis, and probed by immunoblot with antiserum against Ape1.

FIG. 3. Antiserum against Apg2 specifically detects an ~180-kDa protein

Antiserum to Apg2 was prepared as described under “Experimental Procedures.” Wild type (WT; SEY6210), apg2 (MT2–4-3), apg2Δ (CWY1), and the apg2Δ strain transformed with single copy (CEN; pYCG_YNL242w) or multicopy (2µ; pAPG2(426)) plasmids encoding APG2 were grown to A₆₀₀ = 1.0 in SMD. Cells lysates were resolved by SDS-polyacrylamide gel electrophoresis and analyzed with antiserum against Apg2. The positions of molecular weight markers are indicated on the right.

FIG. 4. Biosynthesis of Apg2

A, Apg2 is a stable peripheral membrane protein. Wild type (SEY6210) cells expressing APG2 from a multicopy plasmid were labeled for 30 min, followed by a non-radioactive chase. Crude cell extracts were collected as described under “Experimental Procedures” at the indicated chase time points, and analyzed by immunoprecipitation with antiserum to Apg2. There was no detectable change in the electrophoretic mobility of Apg2 over the 2-h time course. The asterisk marks a background band that cross-reacts with the anti-Apg2 serum. B, Apg2 exists in both soluble and pelletable pools. Wild type (SEY6210) cells were spheroplasted at mid-log phase and osmotically lysed as described under “Experimental Procedures” in PS200 buffer without or with 5 mm MgCl₂. The total cell lysate (T) was separated into low-speed supernatant (S13) and pellet (P13) fractions by centrifugation at 13,000 × g. The S13 fraction was further separated by high-speed centrifugation at 100,000 × g for 30 min to generate high-speed supernatant (S100) and pellet (P100) fractions. Fractions were trichloroacetic acid precipitated and examined by immunoblot with antiserum against Apg2 and the cytosolic marker Pgk1. C, biochemical characterization of pelletable Apg2. Spheroplasts from wild type (SEY6210) cells were osmotically lysed as described under “Experimental Procedures.” A total membrane pellet was obtained by highspeed centrifugation (100,000 × g) of the crude cell lysate and resuspended in buffer alone or buffer containing 1 m KCl, 0.1 m Na₂CO₃ (pH 10.5), 3 m urea, or 1% Triton X-100 and further separated into supernatant (S) and pellet (P) fractions as described under “Experimental Procedures.” Samples were resolved by SDS-polyacrylamide gel electrophoresis and detected by immunoblot with antibodies or antiserum to Pho8 (integral vacuolar membrane protein) and Apg2.

FIG. 5. GFPApg2 localization pattern

A and B, the apg2Δ (CWY1) and apg9Δ (JKY007) strains were transformed with a plasmid expressing GFPApg2 and grown in nutrient-rich conditions (SMD) or shifted to nitrogen-starvation conditions (SD-N) for 4 h and examined with a Nikon E-800 fluorescence microscope. A, GFPApg2 displays both a cytosolic and punctate, perivacuolar distribution in the apg2Δ strain. B, GFPApg2 displays a largely diffuse, cytosolic pattern in the apg9Δ strain in both SMD (shown) and SD-N (data not shown) conditions. Fluorescent (GFP) panels are shown on the left for each strain, Nomarski panels are shown in the middle, and an overlay of both panels is shown on the right. C, Apg2 and GFPApg2 are not induced under starvation conditions and are stable in the absence of Apg9. The apg2Δ (CWY1) and apg9Δ (JKY007) strains transformed with a plasmid expressing GFPApg2, and the non-transformed apg9Δ and wild type (SEY6210) strains were grown in SMD or shifted to SD-N as described in part A. Protein extracts were prepared and resolved by SDS-polyacrylamide gel electrophoresis. GFPApg2 and Apg2 were detected by Western blot using antiserum against Apg2.

FIG. 6. Apg2 co-localized with the Apg9 compartment

The wild type (SEY6210) strain transformed with the APG9 2µ plasmid was grown in SMD to A₆₀₀ = 1.0, converted to spheroplasts, and osmotically lysed. Crude cell lysates were pre-cleared, and centrifuged to obtain a total membrane pellet by centrifugation at 100,000 × g for 20 min. Pellets were resuspended in lysis buffer and loaded on an OptiPrep density gradient ranging from 0 to 66%, and centrifuged for 16 h at 100,000 × g as described under “Experimental Procedures.” A total of 14 fractions were collected from the top of the gradient and examined by immunoblot with antibodies or antiserum against A, Pho8 (vacuole), Dpm1 (endoplasmic reticulum), Mnn1 (Golgi), Pep12 (endosome), and Apg2; and B, Apg2 and Apg9.

FIG. 7. The Apg2 protein interacts with Apg9

A, the apg9Δ (JKY007) strain was transformed with the ProtA or ProtA-APG9 plasmids as indicated. Wild type (SEY6210) or apg9Δ cells were grown in SMD, and protein extracts were prepared and analyzed by immunoblot with antiserum against Ape1. The ProtA-Apg9 fusion protein complements the prApe1 accumulation defect. B, cell lysates were prepared from wild type (SEY6210) cells overexpressing Apg2 and protein A (ProtA) or protein A-Apg9 (ProtA-APG9) in the absence or presence of Nonidet P-40 detergent as described under “Experimental Procedures.” Lysates were incubated with Dynabeads M-500 cross-linked to human IgG. Protein A-Apg9 and associated proteins were isolated by collecting the Dynabeads from the crude cell lysates by magnetic separation (Pull-Down). Proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by immunoblot with antiserum against Apg2. Protein A-Apg9 was detected in the blot due to the affinity between the protein A moiety and rabbit IgG. The positions of Apg2 and protein A-Apg9 are indicated. Input lanes corresponded to 2.5% of cell lysates used for each pull-down reaction. Apg2 is pulled down by the ProtA-Apg9 fusion but not by protein A alone.

FIG. 8. The apg2Δ mutant is unable to degrade peroxisomes

A, The apg2Δ mutant is defective in the ability to degrade Fox3. Wild type (WCGa) and apg2Δ (CWY2) strains were grown in YPD to mid-log phase, shifted to YTO medium for peroxisome induction, and then shifted to degradation medium (SD-N) as described under “Experimental Procedures.” Samples were taken at the indicated times after shift to SD-N, trichloroacetic acid precipitated, resolved by SDS-polyacrylamide gel electrophoresis, and probed by immunoblot with Fox3 antiserum. B, a GFP-SKL peroxisome-targeted fusion protein is maintained in the apg2Δ mutant following a shift to pexophagy conditions. Wild type (WCGa) and apg2Δ (CWY2) strains harboring the pCuGFPSKL(416) plasmid were grown in SMD-Ura to an A₆₀₀ = 1.0 and then shifted to peroxisome-inducing conditions as described in “A.” Following a shift to SD-N, samples were removed at the indicated times and examined by fluorescence microscopy as described under “Experimental Procedures.” Fluorescent (GFP) panels are shown on the left for each strain and Nomarski panels are shown on the right.