The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway

Abstract

The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals that the dome-shaped Trs85 subunit associates primarily with the Trs20 subunit of TRAPPI. We further demonstrate that TRAPPIII binds the coat protein complex (COP) II coat subunit Sec23. The COPII coat facilitates the budding and targeting of ER-derived vesicles with their acceptor compartment. We provide evidence that COPII-coated vesicles and the ER-Golgi fusion machinery are needed for macroautophagy. Our results imply that TRAPPIII binds to COPII vesicles at the phagophore assembly site and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. These events are conserved in yeast to mammals.

Fig. 1.

Single-particle EM analysis of the yeast TRAPPIII complex. (A) Representative EM image of negatively stained TRAPPIII complex produced in E. coli showing monodispersed, elongated particles. (B) A class average of recombinant TRAPPIII revealing a banana-shaped complex ∼23 nm in length (see Fig. S1C for all class averages). The densities representing TRAPPI and Trs85 are labeled. Side length of image is 36 nm. Inset shows a projection average of TRAPPI (12). (C) A class average of TRAPPIII purified from S. cerevisiae using a TAP tag on Trs85 shows the same structural features as the recombinant complex in B (see Fig. S2C for all class averages). Side length of image is 36 nm. (D) Orthogonal views of the TRAPPIII density map at 22 Å resolution obtained by random conical tilt reconstruction. (Scale bar: A, 100 nm; D, 5 nm.)

Fig. 2.

Subunit organization of the TRAPPIII complex. (A) Representative EM image of negatively stained Trs85/Trs20/Trs31/Trs23 subcomplex. (Scale bar: 100 nm.) Inset shows a class average of the subcomplex with individual subunits labeled (see Fig. S3B for all class averages). Side length of image is 36 nm. (B) Atomic model of the TRAPPI complex (generated from three crystal structures; PDB ID codes 2J3T, 2J3W, and 3CUE) fit into the EM density map of TRAPPIII; TRAPPI subunits are labeled in black, and the TRAPPIII-specific Trs85 subunit in red. (C) Close-up view of the interface between Trs85 and the Trs20 and Trs31 subunits of TRAPPI. Residue Asp-46 in Trs20 is shown in red; mutation of the corresponding aspartate in the mammalian protein causes SEDL.

Fig. 3.

Mapping the binding site for Ypt1 in TRAPPIII. (A) Atomic model of Ypt1 bound to TRAPPI (generated from three crystal structures; PDB ID codes 2J3T, 2J3W, and 3CUE) was fit into the EM density map of TRAPPIII. (B) Representative EM image of negatively stained TRAPPIII incubated with a GST-Ypt1 fusion protein. Circles indicate TRAPPIII particles with bound GST-Ypt1. (Scale bar: 100 nm.) Inset shows a class average of TRAPPIII with bound GST-Ypt1 (see Fig. S5B for all class averages). Side length of Inset is 36 nm. (C) Recombinant TRAPPIII binds to Sec23, but not to Sec13. Purified TRAPPIII was incubated for 3 h at 4 °C with equimolar amounts (100 nM) of immobilized GST, GST-Sec23, or GST-Sec13. The beads were pelleted, washed, and bound protein was eluted and analyzed by Western blot analysis.

Fig. 4.

Sec13 and Sec23 accumulate at the PAS when autophagy is blocked. (A) Log phase wild-type (WT), atg1Δ, and atg13Δ cells expressing Sec13-GFP and Ape1-RFP were pelleted, resuspended in SD-N medium, and incubated at 25 °C for 4 h before they were examined by fluorescence microscopy. Arrowheads point to Sec13-GFP puncta, and arrows point to Ape1-RFP. (B) One hundred fifty cells from three separate experiments were examined to calculate the percentage of Ape I-RFP puncta that colocalize with (Left) or lie adjacent to (Right) Sec13-GFP puncta. Error bars represent SEM. **P < 0.01, ***P < 0.001 Student’s t test. (C) WT, atg1Δ, and atg13Δ cells expressing Sec23-GFP and Ape1-RFP were treated as in A. Arrowheads point to Sec23-GFP puncta, and arrows point to Ape1-RFP. (D) One hundred fifty cells from three separate experiments were examined to calculate the percent of Ape I-RFP puncta that colocalize with (Left) or lie adjacent to (Right) Sec23-GFP puncta. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001 Student’s t test. (E) Log-phase WT and uso1-1 cells expressing Sec13-GFP and Ape1-RFP were resuspended in SD-N medium and incubated at 37 °C for 2 h before they were examined by fluorescence microscopy. Arrowheads point to Sec13-GFP puncta, and arrows point to Ape1-RFP. (F) One hundred fifty cells from three separate experiments were examined to calculate the percent of Ape I-RFP puncta that colocalize with (Left) or lie adjacent to (Right) Sec13-GFP puncta. Error bars represent SEM. (G) WT and uso1-1 cells expressing Sec23-GFP and Ape1-RFP were treated the same as cells in E. Arrowheads point to Sec23-GFP puncta, and arrows point to Ape1-RFP. (H) One hundred fifty cells from three separate experiments were examined to calculate the percent of Ape I-RFP puncta that colocalize with (Left) or lie adjacent to (Right) Sec23-GFP puncta. Error bars represent SEM. (Scale bars: 2 μm.)

Fig. 5.

Macroautophagy is blocked in the sec12-4, bos1-1, bet1-1, sed5-1, and sly1^ts mutants, but not in the uso1-1 mutant. Cells were grown to log phase in YPD medium at 25 °C, shifted to SD-N medium at 37 °C for 0, 1, and 2 h, and lysed with glass beads. An equal amount of protein was assayed for vacuolar alkaline phosphatase activity. Protein concentration was measured by using the Bradford assay.

Fig. 6.

A model for the early steps of phagophore formation (1). Atg17 recruits TRAPPIII to the PAS, where it binds to a COPII coated vesicle via an interaction with Sec23 (2). TRAPPIII activates Ypt1, and activated Ypt1 recruits its effector, Atg1, to tether a COPII vesicle to an Atg9 vesicle (3).