The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast

Abstract

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

Keywords: Atg9; Cvt pathway; Drs2; TRAPPIII; vesicle transport.

Figure 1. PICT assay and protein–protein interactions relevant for MTCs function

1. A

   Illustration of the PICT assay (left). Most efficient baits used to anchor each MTC (in parenthesis) are listed (top right) (see Dataset EV2).

2. B

   Summary of detected protein–protein interactions and Fold change obtained (see Dataset EV1). Human homologs are indicated in parenthesis.

3. C

   PICT assay for the interactions between indicated pairs of proteins tagged to FRB and GFP. Representative images before and after adding rapamycin (RAP) of RFP‐tagged anchor and GFP‐tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9 μm. Scale bar, 5 μm.

4. D

   Network of interactions between MTCs and P4‐ATPases. MTCs are represented by blue boxes and P4‐ATPases by orange oval circles. Black lines show the interactions found with PICT.

Figure EV1. PICT assay for the interactions between TRAPPIII and proteins located at the TGN

Representative images of the PICT assay obtained from the screening shown in Fig 1 (Dataset EV1) for the interaction of Trs85‐FRB (TRAPPIII) and Sec7‐GFP or Vps10‐GFP after adding rapamycin (RAP). RFP‐tagged anchor and GFP‐tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9 μm. Scale bar, 5 μm.Source data are available online for this figure.

Figure 2. Drs2 is critical for the biogenesis of the Cvt vesicle

1. A

   Ape1 processing was analyzed by Western blot in the indicated strains and temperatures and normalized to the processing achieved in wild‐type cells ± SD, n = 3.

2. B

   Aggregation of Ape1. Representative images of Ape1‐GFP in wild‐type, trs85Δ and drs2Δ cells. BF, brightfield images. Scale bar, 5 μm.

3. C

   PICT assay for the interaction of Trs85‐FRB with Drs2‐GFP or Bet5‐GFP. The interaction score was normalized to the measurement at 23°C. Error bars: mean ± SD, n = 3. Asterisk indicates significant difference as determined by a two‐tailed Student's t‐test (P < 0.05).

4. D

   Representative EM images of pep4Δ and pep4Δdrs2Δ strains. Black squares show a zoom‐in in the vacuole. White arrowheads point to Cvt bodies.

5. E

   Representative CLEM images of drs2Δ cells. Ape1‐GFP (top) correlates with a membrane‐free ribosome exclusion area (middle). Black square (bottom) shows a zoom‐in at the position correlating with Ape1‐GFP (white arrowhead).

Data information: n = biological replicate.

Figure EV2. Drs2 is essential for the Cvt pathway as the temperature decreases

Ape1 processing was analyzed by Western blot in the indicated strains.

1. A

   drs2Δ cells were grown at 30°C and then cultured for 2 h at the indicated temperatures.

2. B

   Wild‐type, trs85Δ and drs2Δ cells were grown at 30°C and then cultured for 2 h at 16°C.

3. C

   Analysis of Ape1 processing in drs2Δ cells where either the Cvt pathway (atg19Δ) or nonselective autophagy (atg17Δ) were blocked.

4. D

   drs2Δ cells were grown at 30°C and then cultured for 1, 2 or 4 h at 23°C.

5. E

   Wild‐type, trs85Δ and drs2Δ cells were grown at 30°C and then cultured for 4 h at 23°C without a nitrogen source to analyze the effect of nonselective autophagy.

Data information: (A, D) Below, Ape1 processing was normalized to the processing achieved after 2 h at 23°C. (B, C, E) Below, Ape1 processing was normalized to the processing achieved in wild‐type cells. Source data are available online for this figure.

Figure 3. Drs2 role in the Cvt pathway is independent from its known mechanisms

1. A–E

   (A, C, E) Ape1 processing was analyzed by Western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild‐type Drs2 ± SD, n ≥ 3 biological replicates. (B) Representation of the main structural features of Drs2. Each color in the top bar indicates the location of the features in the sequence of wild‐type Drs2. Gray boxes depict transmembrane domains. Below, a summary of the mutants tested and an enlarged view of their C‐terminal tail. QQ > GA and D > N mutations are depicted in red. Numbers indicate the residues position in Drs2 sequence. (D) Ape1 processing was analyzed by Western blot in the indicated strains, and normalized to the processing achieved in wild‐type cells ± SD, n = 3 biological replicates.

Figure 4. The I(S/R)TTK motif is required for the interaction between Drs2 and TRAPPIII and its function in the Cvt pathway

1. A

   Representation of the 15 amino acid (aa) cavity (purple) in the N‐terminal tail of P4‐ATPases. Gray boxes depict transmembrane domains. Black dashed box shows the sequence coding for the cavity (left). Yellow background highlights the I(S/R)TTK motif in Drs2, Dnf1 and Dnf2.

2. B

   The I(S/R)TTK motif in the Drs2‐Cdc50 structure (PDB ID: 6ROH; Timcenko et al, ; Data ref: Timcenko et al, 2019b). Black box zooms in the cavity of Drs2 (purple) and the I(S/R)TTK motif (yellow). Dark blue line depicts the unstructured N‐terminal region.

3. C

   Cold‐sensitive assay for the indicated strains. Cells streaked onto YPD plates were incubated at 30 and 18°C for 3 days.

4. D

   Ape1 processing was analyzed by Western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild‐type Drs2 ± SD, n = 3 biological replicates.

Figure EV3. Modeling of the N‐terminal cavity of P4‐ATPases

Structural representation of the 15 aa cavity (purple) in the N‐terminal tail of Dnf1 (15.5 × 9.4 Å), Dnf2 (11.6 × 9.5 Å), Dnf3 (13.5 × 15.5 Å) and Neo1 (18.1 × 13.8 Å). Drs2 was used as a template PDB ID: 6ROH:A (Timcenko et al, ; Data ref: Timcenko et al, 2019b). The I(S/R)TTK motif present in Dnf1 and Dnf2 is highlighted in yellow. Structural representation of the 15 aa cavity (purple) in the N‐terminal tail of the human ATP8A2 (17.7 × 20.5 Å). ATP8A1 was used as a template (PDB ID: 6K7N; Hiraizumi et al, ; Data ref: Hiraizumi et al, 2019b). The ISTAK motif is highlighted in yellow. The only mismatch with the I(S/R)TTK motif is labeled in blue.

Figure EV4. The role of the I(S/R)TTK motif

1. A

   Representative images (top) and quantification (bottom) of the co‐localization between Sec7‐RFP and Drs2‐GFP or drs2‐5A‐GFP in drs2Δ cells. White arrowheads point to Sec7‐RFP clusters co‐localizing with Drs2‐GFP or drs2‐5A‐GFP. Scale bar, 5 μm. Values were normalized to the measurements in cells expressing wild‐type Drs2.

2. B

   Graphic representation of the 26 proteins whose interaction with Drs2 is diminished by the mutation of the I(S/R)TTK motif in XL‐MS experiments. Binding to Atg9 as reported in SGD (Data ref: Saccharomyces Genome Database, 2021). BFDR, Bayesian False Discovery Rate.

3. C

   Interaction between Trs130‐FRB and Drs2‐GFP or drs2‐5A‐GFP analyzed by PICT. The Interaction score was normalized to the measurement of Drs2‐GFP.

4. D

   Interaction of either Trs85‐FRB or Vps53‐FRB with Dnf1‐GFP, dnf1‐5A‐GFP, Dnf2‐GFP or dnf2‐5A‐GFP analyzed by PICT. The Interaction score was normalized to the measurement of Dnf1‐GFP or Dnf2‐GFP.

5. E

   Representative images of Dnf1‐GFP, dnf1‐5A‐GFP, Dnf2‐GFP or dnf2‐5A‐GFP.

Data information: (A, C, E) Error bars: mean ± SD, n = 3 biological replicates. Asterisk indicates significant difference as determined by a two‐tailed Student's t‐test (*P < 0.05; **P < 0.01). Source data are available online for this figure.

Figure 5. The I(S/R)TTK motif and the interaction between MTCs and P4‐ATPases

1. A

   Ape1 processing was analyzed by Western blot in cells overexpressing N‐terminal Drs2 (1–212 aa) and normalized to the processing achieved in cells with an empty vector ± SD, n = 3.

2. B

   Network of interactions between MTCs and P4‐ATPases (lines) adapted from Fig 1D. XL‐MS detected proximal interactions that require the I(S/R)TTK motif (thick maroon lines). The interaction with COG could not be recapitulated by XL‐MS (dashed line). P4‐ATPases that rely on the I(S/R)TTK motif to bind MTCs are annotated (right column).

3. C

   Left, PICT assay for the interaction of Trs85‐FRB (TRAPPIII) and Drs2‐GFP or drs2‐5A‐GFP after adding rapamycin (RAP). The Interaction score was normalized to the measurement of Drs2‐GFP. Error bars: mean ± SD, n = 3. Asterisk indicates significant difference as determined by a two‐tailed Student's t‐test (**P < 0.01). Right, representative images of the PICT assay. RFP‐tagged anchor and GFP‐tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9 μm. Scale bar, 5 μm.

Data information: n = biological replicate.

Figure 6. I(S/R)TTK motif of Drs2 plays a central role in the transport of Atg9

1. A

   Histograms representing the mean speed of Atg9 puncta observed in wild‐type, drs2Δ and drs2‐5A cells. The histograms were fitted to a mixture of Gaussian distributions with two components; the percentage of each population and the means of the fitting curves are indicated on top of the plot.

2. B

   Representative images (left) and quantification (right) of the co‐localization between Atg9‐GFP and Ape1‐mCherry (upper and middle row, respectively) in cells expressing wild‐type Drs2 or drs2‐5A. White arrowheads point to Ape1‐mCherry clusters co‐localizing with Atg9‐GFP.

3. C

   Representative images (left) and quantification (right) of the PICT assay for the interaction of Trs85‐FRB (TRAPPIII) and Atg9‐GFP in cells expressing wild‐type Drs2 or drs2‐5A after adding rapamycin (RAP). RFP‐tagged anchor and GFP‐tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9 μm.

4. D

   Model for the interplay between Drs2 and TRAPPIII.

Data information: B, C Values were normalized to the measurements in cells expressing wild‐type Drs2. Error bars: mean ± SD, n = 3 biological replicates. Asterisks indicate significant difference as determined by a two‐tailed Student's t‐test (**P < 0.01). Scale bar, 5 μm.

Figure EV5. GFP‐tagged Drs2 do not localize with the PAS marker Atg11

Representative images of the co‐localization between Atg11 tagged to mCherry and Drs2‐GFP in cells lacking the kinase Atg1, required for the Cvt vesicle formation (n = 3). Arrowhead points to an Atg11‐mCherry spot. Scale bar, 5 μm.Source data are available online for this figure.