19 Apr 2023
by Richa

Summary- paper 42: The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast

Irene Pazos, Marta Puig-Tintó, Laura Betancur, Jorge Cordero, Nereida Jiménez-Menéndez, Marc Abella, Altair C Hernández, Ana G Duran, Emi Adachi-Fernández, Carla Belmonte-Mateos, Susana Sabido-Bozo, Sébastien Tosi, Akiko Nezu, Baldomero Oliva, Julien Colombelli, Todd R Graham, Tamotsu Yoshimori, Manuel Muñiz, Maho Hamasaki, Oriol Gallego

EMBO Reports, 2023

Questions/gaps addressed:

  • Eight Multisubunit tethering complexes (MTCs) have been described in yeast, all conserved in humans: COG, Dsl1, CORVET, HOPS, GARP, exocyst, TRAPPII and TRAPPIII.

  • What are the broad interaction partners of multiple MTCs?

Key methods:

  • Mining published genetic interaction datasets for multiple MTCs. Selected 426 proteins for subsequent protein–protein interaction test by PICT assay (why use genetic interaction datasets for this shortlist?).

  • PICT (Protein interactions from Imaging of Complexes after Translocation) assay based on the rapamycin-induced heterodimerization of the FK506-binding protein (FKBP) and the FKBP-rapamycin binding (FRB) domain in a tor1-1 bkg. The anchor is at the spindle pole body (Tub4-RFP-FKBP). This assay cannot distinguish between direct and indirect interactions. PICT screen identified seven candidates, one of which is Drs2 which binds both TRPAII and III.

  • Tested interaction between Drs2 and other P4-ATPases and all the MTCs again. Multiple MTCs bound Drs2.

  • TRAPIII is required for Atg9 trafficking to the PAS, and hence needed for Cvt pathway. Drs2 is essential for cell growth below 21°C. Tested if Drs2 is required for Cvt (measured by Ape1 precessing by WBs)? Yes, but only at low temp, while Trs85 (TRAPIII) is required at all temps.

  • Electron microscopy (EM) to look at Cvt flux. Don’t detect any Cvt body-like structures in the vacuoles of pep4∆ drs2∆.

  • Correlative Light and Electron Microscopy (CLEM) to look at Ape1-GFP aggregates in drs2∆. No Ape1-GFP aggregates in the vacuole of drs2∆ cells.

  • Drs2 is a lipid flippase is involved in multiple endosome and Golgi transport steps. Blocking transport from early endosomes to TGN (rcy1∆), blocking AP-1 pathway from TGN to early endosomes and the exit of the high-density class of vesicles from the TGN (apl4∆), blocking ALP pathway from TGN to the vacuole (apl5∆), or blocking CPY pathway from TGN to late endosomes (gga1 gga2) do not disrupt Ape1 processing. So, none of these roles of Drs2 likely impair Ape1 processing.

  • Drs2 activity mutants: PS flipping defective (drs2-GA), ATPase-dead (drs2-D560N) show no defect in Ape1 processing. No defect in cho1∆ (required for synthesis of PS) also had no defect- so PS is not required for the Cvt pathway.

  • C terminal tail mutants of Drs2: LSSLVMK motif-Gea2 interacting motif (GIM), another conserved motif (modulates autoinhibitory conformation), two NPFXD motifs endocytosis signals: none of these affect Ape1 processing.

  • N terminal tail mutants of Drs2: found a conserved stretch of 15 amino acids I(S/R)TTK motif (residues 198–212 in Drs2) in all the P4- ATPases bind MTCs (Dnf1, Dnf2, and Drs2) but absent in Neo1 and Dnf3. The stretch defines a cavity of 18.8 Å in the solved Drs2 structure. drs2-5A mutant lacking the motif grows at low temp, but is defective in Ape1 processing.

  • Competition assay by overexpressing the N tail of Drs2 results in some inhibition of Ape1 processing even in the presence of WT Drs2.

  • Cross-linking-mass spectrometry (XL-MS) immunoprecipitation to look at Drs2 interactors (wt vs 5A mutant)- disuccinimidyl sulfoxide (DSSO), a cross-linker of 10.1 Å in length. drs2-5A-GFP destabilized interaction with Trs85 (TRAPIII), Trs65, Trs120, Trs130 (specific subunits of TRAPPII) and Vps53 (GARP).

  • Live cell microscopy to track Atg9 (fast and slow moving puncta). In drs2∆ and drs2-5A cells, the low mobility population increases.

Major takeaways:

  • Drs2 and other P4 ATPases can function as regulatory partners of MTCs. MTCs bind the N-terminal cavity of P4-ATPases that nest the somewhat conserved I(S/R)TTK motif in the N tails, and this mechanism may be conserved.