Novel PtdIns(3)P-binding protein Etf1 functions as an effector of the Vps34 PtdIns 3-kinase in autophagy

Abstract

Autophagy is the process whereby cytoplasmic cargo (e.g., protein and organelles) are sequestered within a double membrane-enclosed transport vesicle and degraded after vesicle fusion with the vacuole/lysosome. Current evidence suggests that the Vps34 phosphatidylinositol 3-kinase is essential for macroautophagy, a starvation-induced autophagy pathway (Kihara et al., 2001). Here, we characterize a requirement for Vps34 in constitutive autophagy by the cytoplasm-to-vacuole targeting (Cvt) pathway. First, we show that transient disruption of phosphatidylinositol (PtdIns) 3-phosphate (PtdIns[3]P) synthesis through inactivation of temperature-sensitive Vps34 or its upstream activator, Vps15, blocks the Cvt and macroautophagy pathways. Yet, PtdIns(3)P-binding FYVE domain-containing proteins, which mediate carboxypeptidase Y (CPY) transport to the vacuole by the CPY pathway, do not account for the requirement of Vps34 in autophagy. Using a genetic selection designed to isolate PtdIns(3)P-binding effectors of Vps34, we identify Etf1, an uncharacterized type II transmembrane protein. Although Etf1 does not contain a known 3-phosphoinositide-binding domain (i.e., FYVE or Phox), we find that Etf1 interacts with PtdIns(3)P and that this interaction requires a basic amino acid motif (KKPAKK) within the cytosolic region of the protein. Moreover, deletion of ETF1 or mutation of the KKPAKK motif results in strong sorting defects in the Cvt pathway but not in macroautophagy or in CPY sorting. We propose that Vps34 regulates the CPY, Cvt, and macroautophagy pathways through distinct sets of PtdIns(3)P-binding effectors and that Vps34 promotes protein trafficking in the Cvt pathway through activation/localization of the effector protein Etf1.

Figure 1.

Multicopy ETF1 specifically enhances CPY missorting in the vps34^tsf strain. (A) Wild-type (WT), vps34 ^tsf, vps15 ^tsf, vac1 ^tsf, or vps27 ^(C176A) strains were grown overnight at 26°C and pulse labeled with [³⁵S]methionine for 10 min at 38°C. After a 1- or 2-h chase with or without 0.2 μg/ml rapamycin, labeled cells were lysed and immunoprecipitated for API. Immunoprecipitates were resolved by SDS-PAGE and fluorography, revealing API maturation defects in vps34 ^tsf and vps15 ^tsf but not vac1 ^tsf or vps27 ^(C176A) strains at 38°C. (B) Wild-type and vps34 ^tsf cells were grown, pulse labeled with [³⁵S]methionine for 10 min, and chased for 1 or 2 h under permissive conditions for the vps34 ^tsf (30°C). API was immunoprecipitated and assayed as described above. (C) Wild-type, vps34 ^tsf, pep12 ^tsf (CBY9), and vam3 ^tsf cells transformed with a 2 μm vector or multicopy ETF1 (as indicated) were grown overnight at 27°C, shifted to 33°C for ∼4 h. Each strain was pulse labeled with [³⁵S]methionine for 10 min and chased for 30 min at 33°C. CPY immunoprecipitates derived each strain were analyzed by SDS-PAGE and fluorography, revealing that multicopy ETF1 synthetically enhances CPY missorting in the vps34 ^tsf but not wild-type, pep12 ^tsf, or vam3 ^tsf cells.

Figure 2.

ETF1 encodes a type II transmembrane protein. (A) Analysis of the primary amino acid sequence using MTOP indicates that Etf1 is a type II membrane-spanning protein with a single transmembrane (TM) region (aa 128–144). A basic cluster (aa 113–118: KKPAKK) is present within the cytoplasmic region of Etf1. (B) Wild-type or etf1Δ-7 (etf1Δ) cells were converted to spheroplasts, labeled with [³⁵S]methionine for 20 min, and osmotically lysed. The lysates were either subjected to 1% Triton, 0.1 M Na₂CO₃, 1 M NaCl, 2 M urea, or left mock treated on ice for 10 min. Lysates were then centrifuged at 100,000 g to yield a high speed membrane pellet (P) and supernatant (S). Etf1 was immunoprecipitated from each fraction and analyzed by SDS-PAGE and fluorography.

Figure 3.

Etf1 associates with PtdIns(3)P. (A) MBP-Etf1 (∼ 100 ng) purified from E. coli was incubated with nitrocellulose strips spotted with 60 pmol of the indicated lipid. After extensive washing of the nitrocellulose, immunoblot analysis revealed that Etf1 interacts with PtdIns(3)P. (B) Affinities of Etf1 and the EEA1–FYVE domain for PtdIns(3)P were compared by incubating ∼100 ng of GST–Etf1 or GST–EEA1–FYVE with 60, 30, or 15 pmol of the indicated PI spotted on nitrocellulose. Monoclonal antibody specific to GST was used to detect protein–lipid interactions. Quantification of protein bound to each PI was estimated by the Scatchard method and expressed as an association constant (K _A). (C) 25 μg of liposomes comprised equally of phosphatidylcholine (PC), phosphatidylserine (PS), and PtdIns were supplemented with either PtdIns(3)P or PtdIns(4,5)P₂ (2% final molar concentration) and incubated with 1 μg of purified MBP–Etf1 or MBP–Etf1^{(K113–118A)}. The relative amounts pelletable/liposome-bound Etf1 was compared with 10% of the total quantity Etf1 added to the binding reaction by SDS-PAGE and Western blot. (D) PtdIns(3)P or PtdIns(4,5)P₂ (60 to 15 pmol) were spotted onto nitrocellulose and incubated with 100 ng of purified MBP-Etf1 or MBP-Etf1^{(K113–118A)}. The relative affinity of Etf1 for each PI was determined by immunoblot analysis. The above experiments were repeated with independent PI stocks; representative examples are shown.

Figure 4.

Multicopy etf1^{(K113–118A)} fails to elicit dominant-negative CPY missorting defects in the vps34^tsf. vps34 ^tsf (DDY3407) cells transformed with multicopy ETF1 or etf1 ^{(K113–118A)} were grown at 27°C and shifted to 33°C for ∼4 h. After a 10-min pulse label with [³⁵S]methionine and a 30-min chase at 33°C, cells were subjected to CPY and Etf1 immunoprecipitation and analyzed by SDS-PAGE/fluorography.

Figure 5.

ETF1 is required for Cvt pathway trafficking. Cells were grown overnight at 26°C and pulse labeled with [³⁵S]methionine for 10 min at 38°C. (A) After a 30 min chase, labeled wild-type, vps34 ^tsf, and etf1Δ cells were lysed and immunoprecipitated for CPY. Samples were resolved by SDS-PAGE and fluorography. (B) After 1- or 2-h chases in the presence or absence of 0.2 μg/ml rapamycin, API immunoprecipitates derived from labeled wild-type, vps34 ^tsf, etf1Δ, and etf1 ^{(K113–118A)} strains were analyzed by SDS-PAGE and fluorography, revealing defects in API maturation in the etf1Δ cells by the Cvt pathway but not macroautophagy.

Figure 6.

etf1 Δ cells exhibit autophagic bodies within the lumen of the vacuole by EM. Cells were grown to mid-log phase at 26°C and treated with 0.2 μg/ml rapamycin at either 26 or 38°C for 2 h. Strains were then fixed with 3% glutaraldehyde, converted to spheroplasts, and visualized by EM. (A) vps34 ^tsf at 26°C. (B) vps34 ^tsf at 38°C. (C) ypt7 ^tsf at 26°C. (D) ypt7 ^tsf at 38°C. (E) etf1Δ. Arrows indicate autophagosomes and autophagic bodies. N, nucleus; V, vacuole; m, mitochondria. Bar, 0.5 μm.

Figure 7.

Vps34 and Etf1 each regulate the formation/sequestration of the Cvt vesicle. Wild-type, vps34 ^tsf, and etf1Δ spheroplasts were pulse labeled with [³⁵S]methionine for 10 min at 38°C and chased 20 min. Spheroplasts were osmotically lysed and separated by a 5,000 g spin into a low speed pellet (P) and soluble (S) fractions. (A) Analysis of API immunoprecipitates of the P and S fractions by SDS-PAGE and fluorography indicate that Vps34 and Etf1 are not required for recruitment of prAPI to Cvt vesicles. (B) P fractions derived from wild-type, vps34 ^tsf, etf1Δ, and ypt7 ^tsf cells were either proteinase K or mock treated and then immunoprecipitated for API. SDS-PAGE/fluorography revealed that the P fraction pool of prAPI is protease accessible in both the vps34 ^tsf and etf1Δ cells. (C) Detergent extracts of strains overexpressing GFP, GFP-AUT7, and/or ETF1 were subjected to native immunoprecipitation using polyclonal antiserum specific to GFP or Etf1 as indicated. Immunoprecipitates were resolved by SDS-PAGE and analyzed for the presence of Etf1 by Western blot. For direct immunoprecipitation of Etf1 using Etf1 antiserum, an ∼15% load is shown.

Figure 8.

Etf1 localization by immunofluorescence. Wild-type and etf1Δ cells were grown at 26°C and converted to spheroplasts. After fixation with 4% formaldehyde, spheroplasts were immobilized on multiwell slides and probed with affinity-purified Etf1 polyclonal antibody as described in Materials and methods. Etf1 immunofluorescence and an overlay of fluorescence and Nomarski images are presented.

Figure 9.

Distinct sets of PtdIns(3)P-binding effectors mediate the signaling effects of the Vps34 PtdIns 3-kinase. Etf1 serves as a transmembrane effector of Vps34 in the formation of Cvt vesicles through its basic amino acid cluster (KKPAKK). This suggests that an unidentified PtdIns(3)P-binding protein carries out the formation of autophagosomes in the macroautophagy pathway. The PX protein Vam7, vacuolar t-SNARE Vam3, and rab GTPase Ypt7 are key mediators in the vacuolar fusion of autophagic transport intermediates of both the Cvt and macroautophagy pathways.