Ypt1 recruits the Atg1 kinase to the preautophagosomal structure

Abstract

When macroautophagy, a catabolic process that rids the cells of unwanted proteins, is initiated, 30-60 nm Atg9 vesicles move from the Golgi to the preautophagosomal structure (PAS) to initiate autophagosome formation. The Rab GTPase Ypt1 and its mammalian homolog Rab1 regulate macroautophagy and two other trafficking events: endoplasmic reticulum-Golgi and intra-Golgi traffic. How a Rab, which localizes to three distinct cellular locations, achieves specificity is unknown. Here we show that transport protein particle III (TRAPPIII), a conserved autophagy-specific guanine nucleotide exchange factor for Ypt1/Rab1, is recruited to the PAS by Atg17. We also show that activated Ypt1 recruits the putative membrane curvature sensor Atg1 to the PAS, bringing it into proximity to its binding partner Atg17. Since Atg17 resides at the PAS, these events ensure that Atg1 will specifically localize to the PAS and not to the other compartments where Ypt1 resides. We propose that Ypt1 regulates Atg9 vesicle tethering by modulating the delivery of Atg1 to the PAS. These events appear to be conserved in higher cells.

Keywords: GEF; membrane tethering.

Fig. 1.

TRAPPIII contains seven subunits and is conserved from yeast to man. (A, Left) Trs85–TAP was purified from yeast as described before (30). Purified TAP-tagged Trs85 yields a complex that contains seven major subunits. (Right) Mass spectrometry analysis of purified TAP-tagged Trs85 from yeast. (B) NIH 3T3 lysate was fractionated on a Superdex-200 column as described in Materials and Methods and immunoblotted for mTrs85, mTrs130, and mTrs20. (C) mTrs120 (Left) or mTrs85 (Right) were immunoprecipitated from lysates, and the immunoprecipitates were blotted for the presence of mTrs85, mTrs130, mTrs120, and mTrs20. Similar results were obtained when HeLa or COS-7 cells were used for the precipitations.

Fig. 2.

The localization of Trs85–3XGFP to the PAS is disrupted in the atg17Δ mutant. (A) Cells expressing Trs85–3XGFP and Ape1–RFP were grown to log phase in synthetic complete (SC)–Leu medium, pelleted, and resuspended in SD-N medium for 4 h. (Scale bar, 2 μm.) (B) Quantitation of the data in A, and several other atg mutants, in our laboratory strain background, which is derived from S288C. The PAS targeting index (PTI) was calculated in 50 cells by multiplying the percent of cells that contain colocalized Trs85–3XGFP and Ape1–RFP to the total Trs85–3XGFP signal that resides at the PAS. The PTI in wild-type was set at 1.00. Error bars represent SEM, n = 150 cells from three separate experiments. ***P < 0.001 Student t test. (C) Trs85 specifically binds to Atg17. Yeast lysate containing Trs85–myc (Top) or Trs130–myc (Bottom) was incubated with glutathione-Sepharose beads. Bound protein was eluted and analyzed.

Fig. 3.

The recruitment of Atg1 to the PAS is reduced in the ypt1–2 mutant. (A) Cells were grown at 25 °C and shifted to SD-N medium for 0, 3, and 5 h. Protein extracts were assayed for vacuolar alkaline phosphatase activity as described in Materials and Methods. (B) The localization of Atg1–GFP to the PAS is disrupted in the ypt1–2 mutant. Wild-type and ypt1–2 cells expressing Atg1–GFP and Ape1–RFP were grown to log phase and shifted to SD-N medium as in Fig. 2. (Scale bar, 2 μm.) (C) The PAS localization of Atg13–GFP, Atg17–GFP, Atg29–GFP, and Atg31–GFP is not disrupted in the ypt1–2 mutant. Cells were grown and analyzed as in B. The PTI in wild-type was set at 1.00. Error bars represent SEM, n = 150 cells from three separate experiments. ***P < 0.001 Student t test. The slight increase seen with Atg13–GFP, Atg17–GFP, and Atg29–GFP is not statistically significant.

Fig. 4.

The overexpression of Ypt1 increases the recruitment of Atg1–GFP to the PAS. (A) Autophagy was induced as described in Fig. 2 in cells harboring Atg1–GFP and a 2 µm vector, without or with YPT1, and the recruitment of Atg1–GFP, Atg13–GFP, Atg17–GFP, Atg29–GFP, and Atg31–GFP to the PAS was measured. Error bars represent SEM, n = 150 cells from three separate experiments. **P < 0.01 Student t test. (B) YPT1 overexpression enhances autophagy. Cells were grown in SC–Ura medium at 25 °C and shifted to SD-N medium for 0, 2, and 4 h. Protein extracts were assayed for vacuolar alkaline phosphatase activity. (C) Atg1 kinase activity is not affected in the ypt1–2 mutant. Wild-type and ypt1–2 cells were treated for 30 min at 30 °C without (Left) or with rapamycin (Center) and assayed for activity using MBP as substrate. The atg13Δ mutant was used as a negative control. The data in the center panel are quantitated on the right. Error bars represent SD, n = 3 from three separate experiments.

Fig. 5.

The interaction between Atg1 and activated Ypt1 is enhanced in rapamycin-treated cells. (A) Atg1–HA was integrated into wild-type or trs85Δ cells. (Left) Lysed spheroplasts were treated with or without rapamycin, immunoprecipitated with anti-HA antibody, and immunoblotted. (Right) Lysates were immunoblotted with anti-Ypt1 antibody. (B) Atg1–HA preferentially coprecipitates with the GTP form of Ypt1. (Left) Cells expressing the GTP (Q67L) or GDP (S22N) form of Ypt1 were converted to spheroplasts, treated with rapamycin, and processed as in A. (Right) Lysates were immunoblotted with anti-Ypt1 antibody.

Fig. 6.

Ypt1 binds to the N terminus of Atg1. (A) Purified Ypt1–His₆ was incubated for 3 h at 4 °C with equimolar amounts (0.1 µM) of immobilized GST, GST–Atg1 (aa 1–500), or GST–Atg1 (aa 501–897). The beads were pelleted, washed, and bound protein was eluted and analyzed by Western blotting. (B) GST–Atg1 (aa 1–500), but not GST or GST–Atg1 (aa 501–897), can phosphorylate MBP. (Upper) ³²P-labeled MBP is shown in lane 2. (Lower) Ponceau S staining of MBP in each sample. Equal amounts of GST and GST fusion proteins were used in each sample. (C) Rab1 coprecipitates with Ulk1 from HeLa cell lysates. HeLa cell lysates were incubated for 2 h at 4 °C with anti-Ulk1 antibody or rabbit IgG, followed by the addition of Protein-A–conjugated agarose beads for an additional hour. The beads were pelleted, washed, and bound protein was eluted and analyzed by Western blotting. The induction of autophagy with rapamycin did not stimulate the coprecipitation of Rab1 with Ulk1. (D) (1) TRAPPIII binds to Atg17 and activates Ypt1 (2). Activated Ypt1 recruits Atg1 (3). Dimeric Atg1 tethers Atg9 vesicles to each other or to other membranes.