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. 2010 Apr 9;285(15):11476-88.
doi: 10.1074/jbc.M109.080374. Epub 2010 Feb 12.

Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy

Usha Nair  1 , Yang Cao, Zhiping Xie, Daniel J Klionsky
Affiliations

Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy

Usha Nair et al. J Biol Chem. .

Abstract

Atg18 and Atg21 are homologous WD-40 repeat proteins that bind phosphoinositides via a novel conserved Phe-Arg-Arg-Gly motif and function in autophagy-related pathways. Atg18 is required for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas Atg21 is only required for the Cvt pathway. Currently, the functions of both proteins are poorly understood. Here, we examined the relationship between the phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding abilities of Atg18 and Atg21 and autophagy by expressing variants of these proteins that have mutations in their phosphoinositide-binding motifs. Cells expressing PtdIns(3)P-binding mutants of both these proteins showed highly reduced autophagy. Furthermore, the localization of components of two related ubiquitin-like protein conjugation systems, Atg8 and Atg16, to the phagophore assembly site is affected. Consistent with the aberrant localization of the above Atg proteins, precursor Ape1, a cargo of the Cvt pathway and autophagy, is partially protease-sensitive in starvation conditions. This finding suggests a requirement for the PtdIns(3)P binding capability of Atg18 and Atg21 in efficient completion of the sequestering autophagic vesicles. Finally, using a multiple knock-out strain, we found that Atg18 and Atg21 facilitate the recruitment of Atg8-PE to the site of autophagosome formation and protect it from premature cleavage by Atg4, which represents a key aspect of post-translational autophagy regulation. Taken together, our results suggest that PtdIns(3)P binding by at least Atg18 or Atg21 is required for robust autophagic activity and that the PtdIns(3)P-binding motifs of Atg18 and Atg21 can compensate for one another in the recruitment of Atg components that are dependent on PtdIns(3)P for their phagophore assembly site association.

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Figures

FIGURE 1.
FIGURE 1.
Mutations in the PtdIns(3)P-binding motifs of both Atg18 and Atg21 result in a drastic reduction of autophagy. A, Pho8Δ60 activity is severely reduced in cells expressing Atg18FKKG-PA and Atg21FKKG. The atg18Δ atg21Δ pho8Δ60 (YCY31) cells were transformed with the following: empty vectors, centromeric plasmids expressing wild-type Atg18-PA and Atg21, or two plasmids bearing Atg18-PA and Atg21 constructs with mutations in their lipid binding domains (Atg18FKKG-PA and Atg21FKKG (denoted as Atg18KK and Atg21KK, respectively)). Cells were grown in nutrient-rich medium to mid-log phase and then shifted to starvation conditions for 4 h in SD-N medium. The Pho8Δ60-specific activity (nmol/min/mg) was measured as described under “Experimental Procedures.” The results represent the mean ± S.D. of three independent experiments. B, atg18Δ atg21Δ cells were transformed with the indicated plasmids and analyzed for Pho8Δ60-specific activity after 4 h in SD-N. The activity measured from cells expressing wild-type Atg18-PA and Atg21 was set to 100%, and the other values were normalized. C, GFP-Atg8 processing is severely affected in cells expressing Atg18 and Atg21 PtdIns(3)P-binding mutants. The atg18Δ atg21Δ (YCY28) strain was transformed with the combination of plasmids as in A. Cells were grown in nutrient-rich medium until mid-log phase (0-h time point) and then shifted to nitrogen-starvation conditions for 2 or 4 h. Aliquots were collected at the indicated time points and analyzed by immunoblotting using anti-yellow fluorescent protein antibody (which detects GFP). The positions of full-length GFP-Atg8 and free GFP are indicated.
FIGURE 2.
FIGURE 2.
Mutations in the Atg18 and Atg21 PtdIns(3)P-binding motifs resulted in fewer autophagosomes. A, wild-type (atg18Δ atg21Δ pep4Δ vps4Δ; UNY110 cells expressing Atg18-PA and Atg21), experimental (atg18Δ atg21Δ pep4Δ vps4Δ cells expressing Atg18FKKG-PA and Atg21FKKG), and negative control (UNY110 cells bearing empty vectors) strains were grown to early log phase in SMD lacking uracil and leucine, shifted to SD-N for 4 h to induce autophagy, fixed in potassium permanganate, and examined by electron microscopy. Scale bar, 0.5 μm. B, quantification of the average number of autophagic bodies per vacuole. The error bars represent standard error of the mean. C, quantification of autophagic body accumulation. The number of autophagic bodies accumulated was determined from cells containing similarly sized vacuoles. D, quantification of autophagic body size. The average diameters of cross-sections of autophagic bodies are shown; error bars represent mean ± S.E.; n > 175.
FIGURE 3.
FIGURE 3.
Protease protection assay reveals that atg18Δ atg21Δ pep4Δ cells expressing the PtdIns(3)P-binding mutants of Atg18-PA and Atg21 are defective in autophagosome completion. A, mutations in the PtdIns(3)P-binding motifs of both Atg18 or Atg21 result in a reduction in prApe1 maturation during starvation conditions. atg18Δ atg21Δ pho8Δ60 (YCY31) cells bearing empty vectors, Atg18-PA and Atg21 (wild type (WT)), or Atg18FKKG-PA and Atg21FKKG (KK) centromeric plasmids were grown to mid-log phase in rich medium and then shifted to SD-N for 2 h. Protein extracts were prepared and examined by SDS-PAGE. The precursor and mature forms of Ape1 were examined by immunoblotting with anti-Ape1 antiserum. B–D, under starvation conditions, the majority of precursor Ape1 is protease-sensitive in atg18Δ atg21Δ pep4Δ cells expressing Atg18FKKG-PA and Atg21FKKG. atg18Δ atg21Δ pep4Δ (YCY38) cells bearing empty vectors (B), Atg18-PA and Atg21 (wild type; C), or Atg18FKKG-PA and Atg21FKKG (FKKG; D) were grown to mid-log phase in rich medium and converted to spheroplasts. To induce autophagy, the spheroplasts were starved for 1 h in SD-N medium supplemented with 1.2 m sorbitol. The spheroplasts were collected by centrifugation, resuspended in osmotically balanced lysis buffer, and then disrupted as described under “Experimental Procedures.” The lysate was clarified using a low speed centrifugation step to remove unbroken cells. The resulting total lysate (T) was further separated into 5,000 × g lysate (S5) and pellet (P5) fractions. The P5 fraction from each strain was divided into four parts and subjected to either no treatment, treatment with 0.4% Triton X-100 (TX), or treatment with proteinase K (PK) in the presence or absence of 0.4% Triton X-100 and analyzed by immunoblot using anti-Ape1 antiserum. Lysis conditions were verified by immunoblot analysis using the anti-Pgk1 and anti-Ape1 antisera. To verify that the lysis method did not disrupt the integrity of organellar membranes in the P5 fractions, maturation of the precursor form of Prc1 was examined by immunoblot analysis using anti-Prc1 antibody.
FIGURE 4.
FIGURE 4.
Atg18FKKG and Atg21FKKG mutants are defective in the recruitment of Atg8 to the PAS in nutrient-rich medium and in both its recruitment to and dissociation from the PAS under starvation conditions. A, GFP-Atg8 localization patterns were monitored by fluorescence microscopy in nutrient-rich conditions (SMD) and after a shift to starvation medium (SD-N) for 4 h in wild-type (WT; SEY6210), atg18Δ (YCY26), atg21Δ (YCY14), atg18Δ atg21Δ (YCY28), or atg18Δ atg21Δ cells expressing Atg18FKKG-PA and Atg21FKKG. DIC, differential interference contrast. Scale bar, 2.5 μm. B, quantification of perivacuolar GFP-Atg8 punctate dots under nutrient-rich conditions in the cells from A. C, percentage of cells showing perivacuolar, GFP-Atg8 punctate dots (white bars) and vacuolar GFP staining (black bars) under nitrogen starvation conditions in the cells from A. Three independent experiments with ∼100 cells for each strain were analyzed for scoring the percentage of cells with GFP-Atg8 puncta. Error bars represent mean ± S.D.
FIGURE 5.
FIGURE 5.
Atg18 and Atg21 lipid-binding mutants are defective in the recruitment of Atg16 to the PAS under starvation conditions. Each of the following strains was chromosomally tagged with Atg16-GFP. A and B, wild-type (WT, SEY6210), atg18Δ (YCY26), atg21Δ (YCY14), and atg18Δ atg21Δ (YCY28). C and D, atg18Δ atg21Δ strain was additionally transformed with plasmids expressing wild-type Atg18-PA and Atg21, the FKKG mutants, or empty vectors. A and C, Atg16-GFP localization pattern was examined by fluorescence microscopy; B and D, percentage of cells showing Atg16-GFP puncta in each of these strains after a 4-h shift to starvation conditions was quantified. The quantification shown here is from three independent experiments. Approximately 500 cells for each strain were analyzed for scoring the percentage of cells with fluorescent Atg16-GFP PAS puncta. Error bars represent the mean ± S.E. DIC, differential interference contrast. Scale bar, 2.5 μm.
FIGURE 6.
FIGURE 6.
Atg18-Atg2 and Atg21 protect Atg8 from Atg4-mediated cleavage. A, MKO strain expressing Atg3 and GFP-Atg8ΔR, which lacks the C-terminal arginine residue of Atg8, was transformed with plasmids expressing different combinations of Atg proteins as indicated. The GFP-Atg8ΔR localization pattern in these different strains was monitored in nutrient-rich conditions by fluorescence microscopy. Similar results were observed with cells shifted to starvation conditions for 4 h. DIC, differential interference contrast. Scale bar, 2.5 μm. B, percentage of cells with fluorescent GFP-Atg8ΔR puncta were scored from three independent experiments. Error bars represent mean ± S.D. C, lipidation of Atg8 is affected in the MKO strain expressing FKKG mutants of Atg18 and Atg21. Atg8ΔR was expressed from the lys2 locus of an MKO strain bearing ATG3. This strain was transformed with empty vectors or the indicated plasmids. pep4Δ, atg8Δ, or atg4Δ strains were used as controls. Cells were grown to mid-log phase and collected, and protein extracts were subjected to SDS-PAGE followed by immunoblotting with anti-Atg8 antibody.
FIGURE 7.
FIGURE 7.
Model for the function of Atg18 and Atg21 in regulating Atg4 access to Atg8–PE. Atg4 initially cleaves the C-terminal arginine of Atg8 (not depicted). Atg8 is subsequently conjugated to PE through the action of Atg7 and Atg3. The PtdIns 3-kinase complex generates PtdIns(3)P that allows recruitment of Atg18 (and its binding partner Atg2) and Atg21, which in turn are needed for efficient recruitment of Atg8 and Atg16; Atg18 and Atg21 also regulate the dissociation of these proteins from the PAS/phagophore. Atg16 at the PAS and subsequent cleavage by Atg4 at other sites may limit Atg8–PE to the PAS and the forming phagophore; the presence of Atg18 and Atg21 blocks access of Atg4 to Atg8–PE at the PAS/phagophore, preventing a premature second cleavage event. Following completion of the autophagosome, Atg18 and Atg21, along with the Atg12–Atg5-Atg16 complex (which is present as a tetramer) dissociate in a process that is probably regulated by Atg1, allowing Atg4 to cleave Atg8 from PE on the surface of the autophagosome.
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