Atg9 vesicles recruit vesicle-tethering proteins Trs85 and Ypt1 to the autophagosome formation site

Abstract

Atg9 is a transmembrane protein that is essential for autophagy. In the budding yeast Saccharomyces cerevisiae, it has recently been revealed that Atg9 exists on cytoplasmic small vesicles termed Atg9 vesicles. To identify the components of Atg9 vesicles, we purified the Atg9 vesicles and subjected them to mass spectrometry. We found that their protein composition was distinct from other organellar membranes and that Atg9 and Atg27 in particular are major components of Atg9 vesicles. In addition to these two components, Trs85, a specific subunit of the transport protein particle III (TRAPPIII) complex, and the Rab GTPase Ypt1 were also identified. Trs85 directly interacts with Atg9, and the Trs85-containing TRAPPIII complex facilitates the association of Ypt1 onto Atg9 vesicles. We also showed that Trs85 and Ypt1 are localized to the preautophagosomal structure in an Atg9-dependent manner. Our data suggest that Atg9 vesicles recruit the TRAPPIII complex and Ypt1 to the preautophagosomal structure. The vesicle-tethering machinery consequently acts in the process of autophagosome formation.

FIGURE 1.

Purification and mass spectrometric analysis of Atg9 vesicles. A, an illustration of the two-step purification of Atg9 vesicles. See text for details. B, purification of Atg9 vesicles. Cells expressing Atg9-3xBAP (Atg9-3B) and Atg9-6xFLAG (Atg9-6F) or non-tagged Atg9 under growing conditions were disrupted using beads. Cell lysates (input (In)) were subjected to immunoisolation using anti-FLAG beads followed by elution with 3xFLAG peptide. The eluate (E1) was subjected to a second immunoisolation using streptavidin beads. Bound proteins were eluted by solubilization of membrane structures with Triton X-100 (E2), and the remaining proteins were recovered by denaturation with SDS sample buffer at 65 °C (E3). These fractions were subjected to immunoblotting using antibodies against Atg9, Vph1 (vacuole), Kex2 (late Golgi), Pgk1 (cytoplasm), Sed5 (early Golgi), Pep12 (endosome), Por1 (mitochondria), or Dpm1 (endoplasmic reticulum). Eluted fractions were concentrated 10-fold. Asterisks show bands corresponding to immunoglobulin. C, E1 fractions were subjected to negative staining with uranyl acetate and observed by electron microscopy. Arrowheads indicate isolated vesicles. Scale bars, 50 nm. D, eluted proteins (E2 and E3) were stained with SYPRO Ruby. In comparison with the negative control fractions (−), specific bands in the fractions of FLAG-tagged Atg9 (FLAG) were subjected to mass spectrometric analysis. A representative image is shown, and detected proteins are indicated by name at the corresponding positions. Asterisks show contaminating BSA and immunoglobulin. E, the co-immunoprecipitation assay for Trs85 and Ypt1. Cells expressing Trs85-6xHA or GFP-Ypt1 were harvested under growing or rapamycin-treated conditions. Atg9 vesicles were immunoisolated using anti-FLAG beads. Trs85-6xHA and GFP-Ypt1 were detected using anti-HA and anti-GFP antibodies, respectively. Immunoprecipitation (IP) fractions were concentrated 50-fold.

FIGURE 2.

Trs85 interacts with Atg9. A, co-immunoprecipitation experiments using anti-FLAG beads and cells expressing Atg9-6xFLAG or non-tagged Atg9. Cells were harvested after rapamycin treatment for 1 h. Immunoisolated Atg9 vesicles were solubilized with Triton X-100 (TX), and remaining proteins were recovered with SDS sample buffer (SB). Proteins were analyzed by immunoblotting. Triton X-100 and SDS sample buffer fractions were concentrated 50-fold. Asterisks show bands corresponding to immunoglobulin. B, two-hybrid assay between Atg9 and Trs85. N-terminal (N) and C-terminal (C) regions of Atg9 were fused with the Gal4 activation domain. The full length and N-terminal and C-terminal halves of Trs85 were fused with the DNA-binding domain. AH109 strains transformed with each vector (Vec) were grown on SD lacking either leucine and tryptophan (−LW) or histidine, leucine, and tryptophan (−HLW). C, cells expressing Atg9-6xFLAG and HA-tagged subunits of TRAPP complexes (lane 1, Bet3; lane 2, Trs31; lane 3, Trs33; lane 4, Trs65; lane 5, Trs85) were treated with rapamycin for 1 h and subjected to a co-immunoprecipitation assay. Immunoprecipitation (IP) fractions were concentrated 50-fold. Asterisks show bands corresponding to immunoglobulin. D, the in vitro binding assay using isolated Atg9 vesicles. Isolated Atg9 vesicles were incubated with Trs65- or Trs85-bound beads. Proteins that were unbound (Un) or bound (B) to the beads were subjected to immunoblotting. Trs65-TAP and Trs85-TAP were detected with HRP-conjugated anti-mouse IgG. As a negative control, cells not expressing TAP-tagged proteins were also subjected to the assay. E, schematic illustration of the in vitro binding assay. Isolated Atg9 vesicles were bound to Trs85 (TRAPPIII) beads but not to Trs65 (TRAPPII) beads. Atg9-3B, Atg9-3xBAP; Atg9-6F, Atg9-6xFLAG.

FIGURE 3.

The PAS localization of Trs85 is dependent on Atg9. A, the localization of Trs85-2xGFP in wild-type, atg9Δ, and atg14Δ cells. Cells were treated with rapamycin for 1 h. The percentages of the PAS localization of Trs85-2xGFP in 100 cells are shown in the bar graph. B and C, the localization of Atg9-2xGFP (B) and Atg14-2xGFP (C) in wild-type and trs85Δ cells under growing conditions. RFP-Ape1 was used as a PAS marker. Filled arrowheads and open arrowheads indicate dots that were colocalized and not colocalized, respectively. Scale bars, 3 μm.

FIGURE 4.

Ypt1 is localized on Atg9 vesicles and transported to the PAS in a Trs85-dependent manner. A, the localization of GFP-Ypt1 in wild-type, trs85Δ, and atg9Δ cells under rapamycin-treated conditions. RFP-Ape1 was used as a PAS marker. Filled arrowheads and open arrowheads indicate dots that were colocalized or not colocalized, respectively. The percentages of the PAS localization of GFP-Ypt1 in 100 cells are shown in the bar graph. Scale bars, 3 μm. B, co-immunoprecipitation experiments using wild-type and trs85Δ cells. Cells were treated with rapamycin for 1 h, and the Atg9 vesicles were immunoprecipitated using anti-FLAG beads. Immunoprecipitation (IP) fractions were concentrated 50-fold. The ratios of relative amounts of coprecipitated Ypt1 between wild-type and trs85Δ cells normalized to the amount of precipitated Atg9 are shown. C, schematic diagram of how Ypt1 is transported to the PAS and functions in autophagosome formation. In this model, the Trs85-containing TRAPPIII complex is localized onto Atg9 vesicles via the interaction between Trs85 and Atg9. Prenylated Ypt1 is associated with cytoplasmic Atg9 vesicles with the aid of the TRAPPIII complex. After the PAS localization of Atg9 vesicles, other Atg9 vesicles or other unidentified membrane structures are tethered to the Atg9 vesicle at the PAS by Ypt1 and the TRAPPIII complex.