The P4-ATPase Drs2 Interacts with and Stabilizes the Multisubunit Tethering Complex TRAPPIII in Yeast

Article The P4-ATPase Drs2interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast Irene Pazos 1 , Marta Puig-Tint o 1 , Laura Betancur 1 , Jorge Cordero 1 , Nereida Jim enez-Men endez 1 , Marc Abella 1 , Altair C Hern andez 1 , Ana G Duran 1 , Emi Adachi-Fern andez 1 , Carla Belmonte-Mateos 1 , Susana Sabido-Bozo 2,3 ,S  ebastien Tosi 4 , Akiko Nezu 5,6 , Baldomero Oliva 1,7 , Julien Colombelli 4 , Todd R Graham 8 , Tamotsu Yoshimori 5,6 , Manuel Mu~ niz 2,3 , Maho Hamasaki 5,6 & Oriol Gallego 1,* 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 Drs2as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N- terminal tail of Drs2stabilizes TRAPPIII on membranes loaded with Atg9and is required for Atg9delivery 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 Dnf1and Dnf2with MTCs, suggesting a broader role of P4-ATPases in MTC regulation. Keywords Atg9; Cvt pathway; Drs2; TRAPPIII; vesicle transport Subject Categories Autophagy & Cell Death; Membrane & Trafficking DOI 10.15252/embr.202256134 | Received 16 September 2022 | Revised 10 February 2023 | Accepted 17 February 2023 EMBO Reports (2023)e56134 Introduction Multisubunit tethering complexes (MTCs) are a group of protein complexes essential for vesicle transport. Multisubunit tethering complexes mediate the tethering of the vesicle to the acceptor membrane through long-range interactions that precede vesicle fusion and cargo delivery at the destination of the transport pathway (Yu & Hughson, 2010; Bro ¨cker et al,2010). Eight MTCs have been described in yeast, all conserved in humans (Dubuke & Munson, 2016). The conserved oligomeric Golgi (COG) complex is involved in intra-Golgi retrograde transport (Suvorova et al,2002). Dsl1 is responsible for tethering COPI vesicles derived from the Golgi to the endoplasmic reticulum (ER) (Ren et al,2009). The class C core vacuole/endosome tethering (CORVET) complex and the homotypic fusion and vacuole protein sorting (HOPS) complex act at the endosomal/vacuolar pathway where CORVET tethers vesicles at early endosomes and HOPS functions in late endosomes and the vacuole (Balderhaar & Ungermann, 2013). The Golgi-associated retrograde protein (GARP) complex tethers vesicles derived from endosomes to the trans-Golgi network (TGN) (Conibear et al,2002). The exocyst is responsible for tethering secretory vesicles to the plasma membrane (Heider et al,2016). Finally, the transport protein particle (TRAPP) complexes have historically been classified as MTCs despite their major role as guanine nucleotide exchange factors for Rab GTPases (Yu & Liang, 2012). The transport protein particle comes in two flavors: TRAPPII and TRAPPIII (Thomas et al,2018). Both complexes have a heterohexamer core called TRAPPI. TRAPPII, with four more specific subunits (Yip et al,2010), activates Ypt31/32 to regulate intra-Golgi transport (Morozova et al,2006). TRAPPIII, with the sole-specific subunit Trs85 (Tan et al,2013), activates Ypt1 to 1Department of Medicine and Life Sciences (MELIS), Pompeu Fabra University (UPF), Barcelona, Spain 2Department of Cell Biology, University of Seville, Seville, Spain 3Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Roc ıo/CSIC/Universidad de Sevilla, Seville, Spain 4Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 5Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan 6Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan 7Structural Bioinformatics Lab (GRIB-IMIM), Barcelona, Spain 8Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA *Corresponding author. Tel: +34 933160849; E-mail: [email protected] Ó2023 The Authors. Published under the terms of the CC BY NC ND 4.0license EMBO reports e56134 |2023 1of 18 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License regulate the transport of ER-derived COPII vesicles to the Golgi (Tan et al,2013). The function of MTCs requires the interaction with other components of the trafficking machinery such as GTPases, SNAREs, and Sec1/Munc18 (SM) proteins (Bro ¨cker et al,2010; Yu & Hughson, 2010). However, studies have been centered on the mechanism of individual MTCs, which limited the understanding of the molecular bases that play a general role in MTCs function. To shed light on the molecular mechanisms that regulate MTCs, we have systematically searched for interactions that are functionally relevant for these protein complexes in yeast. Our approach combines genetic interactions and PICT (Protein interactions from Imaging Complexes after Translocation), a live-cell imaging method that can detect low abundant interactions (Gallego et al,2013; Torreira et al,2017). We found that MTCs involved in the anterograde and retrograde transport at the Golgi (COG, GARP, TRAPPII, and TRAPPIII) bind Type IV P-type ATPases (P4-ATPases), a family of lipid flippases that maintain membrane lipid asymmetry by translocating and accumulating phospholipids to the cytosolic leaflet. COG, GARP, TRAPPII, and TRAPIII are involved in the transport of the Autophagy-related protein 9 (Atg9) (Reggiori et al,2003; Shirahama-Noda et al,2013; Zou et al,2013; Wang et al,2017), a transmembrane lipid scramblase (Matoba et al,2020; Sawa- Makarska et al,2020) essential for the Cytoplasm-to-vacuole targeting (Cvt) pathway. The Cvt pathway is used as a model to study selective autophagy, a set of pathways that target the degradation of cellular components in a specific manner to maintain the cell fitness. The Cvt pathway initiates with the construction of the PAS (pre-autophagosomal structure). Then, a double membrane elongates from the PAS to enwrap the precursor Ape1 (prApe1) aminopeptidase until the double membrane closes generating the Cvt vesicle. After fusion and internalization in the vacuole, the prApe1 is cleaved to mature Ape1 (mApe1) (Lynch-Day & Klionsky, 2010; Guimaraes et al,2015). In growing cells, Atg9 cycles in cytoplasmic vesicles between Atg9 reservoirs at the endocytic compartments and the Golgi (Yamamoto et al,2012). However, when the Cvt pathway is triggered, Atg9 is transported to the PAS, a step that is strictly required for the de novo formation of the Cvt vesicle (Shirahama- Noda et al,2013). Among the MTCs involved in the transport of Atg9, the function of TRAPPIII is essential for the Cvt pathway. TRAPPIII is responsible for Atg9 vesicular transport from early endosomes to the Golgi, allowing Atg9 transport to the PAS (Shirahama-Noda et al,2013). Thus, in trs85Δcells, Atg9 does not reach the PAS, the biogenesis of the Cvt vesicle is blocked and the prApe1 accumulates in large cytosolic aggregates (Meiling-Wesse et al,2005; Shirahama-Noda et al, 2013). The mechanisms regulating the function of TRAPIII during the transport of Atg9 vesicles from the reservoirs remain elusive (Yamamoto et al,2012). We challenged the functional relevance of the interactions between MTCs and P4-ATPases by studying the role of the TRAPPIII-Drs2 assembly in the transport of Atg9. Drs2 is a P4- ATPase located at the TGN and early endosomes that flips phosphatidylserine (PS) specifically (Natarajan et al,2004; Hanamatsu et al, 2014). Here, we show that Drs2 regulates TRAPPIII in the transport of Atg9, a role that is independent of Drs2 reported functions. Crosslinking and competitive assays demonstrate that proximal binding to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9, which is necessary for the transport of Atg9 for the early stages of the Cvt pathway. We demonstrate that Drs2 is required for the biogenesis of Atg9 vesicles from the reservoirs through a mechanism that requires the I(S/R)TTK motif located in its N-terminal tail. This motif is conserved in other flippases and is crucial to sustain the network of interactions between P4-ATPases and MTCs. Results Protein–protein interactions relevant for MTCs function In an effort to thoroughly explore the space of protein interactions that are relevant for MTCs function, we conducted a genome-wide search based on reported genetic interactions in yeast (Costanzo et al,2011; Data ref: Saccharomyces Genome Database, 2013). Genes coding for MTCs subunits establish 2,587 genetic interactions with 1,283 other genes. Based on the probability of these genetic interactions to occur randomly, we selected those gene products that are more likely to be functionally linked with MTCs and that have not been shown to bind MTCs before. Overall, we selected 426 proteins for subsequent protein–protein interaction screening (Dataset EV1, see Materials and Methods). We then used the PICT assay to screen interactions directly in living cells (Gallego et al,2013; Torreira et al,2017; Fig 1A). PICT is based on the rapamycin-induced heterodimerization of the FK506- binding protein (FKBP) and the FKBP-rapamycin binding (FRB) domain (Chen et al,1995; Gallego et al,2013). The addition of rapamycin to the media induces the translocation of proteins tagged with FRB (bait-FRB) to intracellular anchoring platforms tagged with FKBP and RFP (anchor-RFP-FKBP). In our strains rapamycin has no effect on endogenous TORC1 as this has been mutated to the rapamycin-insensitive form tor1-1 (see Materials and Methods). If a GFP-tagged protein (prey-GFP) interacts with the bait-FRB, it will build-up at the anchor upon addition of rapamycin, which can be quantified by the increment in the co-localization of the GFP and RFP signals. In a previous study, we enhanced PICT sensitivity up to 200-fold by engineering yeast cells to express anchoring platforms at the spindle pole body (Tub4-RFP-FKBP). By tagging Tub4 with RFP and FKBP, the resulting cells harbor one or two anchoring platforms only. Thus, even low abundant complexes accumulate enough prey-GFP in the anchoring platforms to be efficiently detected and quantified (Torreira et al,2017). The PICT assay cannot discern between direct and indirect interactions. However, among the 426 proteins screened, there is extensive redundancy of the subcellular localization. The selective binding patterns detected minimize the possibility that the PICT assay generally reports unspecific interactions resulting from the anchoring of entire membrane compartments. The screen identified seven proteins, all conserved in humans, which establish eight protein–protein interactions with MTCs (Fig 1B and C). The COG complex binds Snc2, an exocytic SNARE known to traffic through the Golgi (Protopopov et al,1993). CORVET binds Hse1, Bro1, Vps45, and Vps9, all involved in the sorting of vacuolar proteins. These interactions provide molecular bases to the mechanism that underlies the interplay between CORVET and the Vps9-Vps21-Vps45 module (Balderhaar et al,2013; Zhou et al,2017). We also found that TRAPPII and TRAPPIII bind 2of 18 EMBO reports e56134 |2023 Ó2023 The Authors EMBO reports Irene Pazos et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Drs2, while GARP interacts with Lem3. Drs2 is one of the five yeast P4-ATPases together with Dnf1, Dnf2, Dnf3, and Neo1. Except for the last one, all P4-ATPases act as heterodimers with its ß-subunit: Lem3 for Dnf1 and Dnf2; Cdc50 for Drs2; and Crf1 for Dnf3 (Saito et al,2004; Sebastian et al,2012). Thus, the screen identified three MTCs that could bind to P4-ATPases, suggesting a general mechanism underlying these interactions (Dataset EV1 and Fig 1B and C). The network of interactions between MTCs and P4-ATPases To gain a more comprehensive view of the relationship between MTCs and P4-ATPases, we expanded the study of interactions to all MTCs and P4-ATPases (Fig 1D). Detected binding events define a network with specific configuration of interactions. GARP and TRAPPIII concentrate most of the interactions with the P4-ATPases Drs2, Dnf1, and Dnf2, flippases that circulate between the plasma membrane and the Golgi. These interactions provide a molecular basis for the recycling of Dnf1 and Dnf2 mediated by GARP (Eising et al,2019). Note that Dnf1 and Dnf2, known to have redundant functions (Hua et al,2002), also show identical binding specificity with MTCs. In addition to GARP, TRAPII, and TRAPPIII, Drs2 also interacts with COG, being the flippase that shows the highest number of interactions with MTCs. Overall, MTCs involved in trafficking pathways at the Golgi show preference to bind Drs2, Dnf1, and Dnf2, whereas we could not detect binding with other proteins found at this organelle (i.e., Sec7 and Vps10, Fig EV1). These results raise the possibility that binding to P4-ATPases might be involved in the molecular mechanisms that COG, GARP, TRAPII, and TRAPIII use to control vesicle transport in the Golgi. To better understand the molecular mechanism that NeoI PICT assay -RAP Trs85-FRB - Drs2-GFP +RAP +RAP -RAP Trs130-FRB - Drs2-GFP +RAP Prey-GFP -RAP Vps53-FRB - Lem3-GFP +RAP Anchor- RFP-FKBP Merge Anchor-RFP-FKBP Bait-FRB (MTC) Cog6 (COG) Dsl1 (Dsl1) Sec3 (Exocyst) Vps53 (GARP) Vam6 (HOPS) Trs130 (TRAPPII) Trs85 (TRAPPIII) Vps3 (CORVET) Prey-GFP 426 proteins selected from genetic interactions MTCs P4-ATPases Prey GFP Anchor FKBP FRB RFP Bait Anchor Prey FRB FKBP GFP Bait RFP GARP TRAPPII TRAPPIII Dnf2 Dnf3 DSL1 Exocyst CORVET HOPS Drs2 COG Dnf1 Neo1 A C D B MTC Prey (Human homologs) COG Snc2 (VAMP1) CORVET Hse1 (STAM1/STAM2) Bro1 (PDCD61P) Vps45 (VPS45) Vps9 (RabGEF1) GARP Lem3 (TMEM30A/TMEM30B) Drs2 (ATP8A1/ATP8A2)TRAPPII Drs2 (ATP8A1/ATP8A2) Interactions with MTCs CORVET CORVET CORVET TRAPPIII Fold change 200 ms Fold change 1500 ms 38.21 27.24 0 1072.33 273.4 122.09 23.33 82.04 10.73 0 7.23 5.68 258.36 39.51 28.38 25.42 Figure 1. PICT assay and protein–protein interactions relevant for MTCs function. A Illustration of the PICT assay (left). Most efficient baits used to anchor each MTC (in parenthesis) are listed (top right) (see Dataset EV2). B Summary of detected protein–protein interactions and Fold change obtained (see Dataset EV1). Human homologs are indicated in parenthesis. 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, 0.9μm. Scale bar, 5μm. 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. Ó2023 The Authors EMBO reports e56134 |2023 3of 18 Irene Pazos et al EMBO reports 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License regulates MTCs function, we further investigated the interactions with Drs2, the only P4-ATPase whose atomic structure was available at the time of this study (Bai et al,2019; Timcenko et al, 2019a). Drs2regulates the biogenesis of the Cvt vesicle We investigated the functional relationship between TRAPPIII and Drs2. In cells lacking TRAPPIII subunit Trs85 (trs85Δ), Atg9 is not delivered to the PAS, the biogenesis of the Cvt vesicle is blocked, and Ape1 is accumulated in large cytosolic aggregates (Meiling- Wesse et al,2005; Shirahama-Noda et al,2013; Fig 2A and B). The cellular relevance of Drs2 varies at different growing temperatures: Drs2 is essential for cell growth below 21°C (although cells remain viable) (Chen et al,1999), whereas the flippase is dispensable at higher temperatures. Therefore, we first analyzed the contribution of Drs2 in the Cvt pathway at different temperatures. While Ape1 processing is fully blocked in trs85Δcells incubated at any of the temperatures tested, cells lacking Drs2 (drs2Δ) show a progressive inhibition of Ape1 maturation when decreasing the temperature. Thus, drs2Δcells process Ape1 near to normality at 37°C, while we could only detect traces of mApe1 in cells incubated at 16°C (Fig 2A and EV2A and B). Accordingly with a specific role of Drs2 at low temperatures, the processing of Ape1 in wild-type cells at 16°C was normal (Fig EV2B). We continued studying the interplay between Drs2 and TRAPPIII at 23°C to avoid undesired effects derived from impaired cell fitness. At this temperature, drs2Δcells mature 12% of the aminopeptidase (Fig 2A). We confirmed that such levels of mApe1 represent the steady-state levels of the Cvt pathway in mutant cells incubated at 23°C by analyzing Ape1 processing in cells lacking Drs2 and Atg19 (i.e., specific receptor for the Cvt pathway), Drs2 and Atg17 (i.e., specific scaffold for nonselective autophagy) and in a time course assay (Fig EV2C and D). Consistently with a functional relationship between TRAPPIII and Drs2, cells lacking either Trs85 or Drs2 accumulate similar Ape1-GFP aggregates (Fig 2B). PICT assay showed that, concomitantly to an exacerbation of the Drs2 role in the Cvt pathway, the Drs2-TRAPPIII interaction is boosted under colder conditions: at 23°C the Interaction score between Drs2 and Trs85 is 24% higher than at 37°C (Fig 2C), while no significant difference could be detected for the interaction between Trs85 and Bet5, a subunit of TRAPPI core. This observation suggests that the cell regulates the interplay between Drs2 and TRAPPIII in response to temperature shifts. We then used electron microscopy (EM) to study the ultrastructure of the Cvt pathway in the absence of Drs2. To figure out whether defects in Ape1 processing result from a defective vacuolar function, we mutated Pep4. Pep4 is a protease required for the processing of the Cvt vesicle once it is internalized in the vacuole (Baba et al,1997). While 89.3% of the pep4Δcells presented Cvt bodies inside the vacuole, we could not detect any Cvt body-like structure in the vacuoles of pep4Δdrs2Δcells (Fig 2D), indicating that Ape1 processing defect originates before the Cvt cargo is delivered to the vacuole. We then analyzed Ape1-GFP aggregates in drs2Δcells with Correlative Light and Electron Microscopy (CLEM) (Kukulski et al, 2011). 94.8% of the Ape1-GFP spots that were analyzed correlated with a ribosome exclusion area of amorphous shape and that was devoid of double membrane or Cvt vesicle-like structure (Fig 2E). In agreement, we could not detect Ape1-GFP aggregates in the vacuole of drs2Δcells stained with FM4-64 and imaged by fluorescent microscopy (Movie EV1). Overall, these results demonstrate that Drs2 is necessary for the early stages of the biogenesis of the Cvt vesicle, a role that is further underscored by the enrichment of DRS2 genetic interactions with genes coding for proteins involved in autophagy (51 genes of 571, P=7×10 −5 ). The role of Drs2in the Cvt pathway is independent from known mechanisms Drs2 is a lipid flippase with multiple functions in vesicle transport at the interface of endosomes and the Golgi (Natarajan et al,2004; Hanamatsu et al,2014). We used yeast genetics to investigate the possibility that defects in the Cvt pathway in drs2Δcells result indirectly from the perturbation of other processes regulated by the flippase. However, Ape1 processing was not hindered in cells lacking Rcy1 (i.e., cells where the transport from early endosomes to TGN is blocked; Furuta et al,2007; Hanamatsu et al,2014), Apl4 (i.e., cells where the AP-1 pathway from TGN to early endosomes and the exit of the high-density class of vesicles from the TGN are blocked; Hinners, 2003; Liu et al,2008), Apl5 (i.e., cells where ALP pathway from TGN to the vacuole is blocked; Odorizzi et al,1998) or both Gga1 and Gga2 (i.e., cells where CPY pathway from TGN to late endosomes is blocked; Sakane et al,2006; Fig 3A). Thus, genetic data suggest that the biogenesis of the Cvt vesicle does not involve the pathways where Drs2 has been reported to participate. We then explored the molecular mechanism of Drs2 that is required for the biogenesis of the Cvt vesicle. We analyzed the Ape1 processing in drs2Δcells expressing different constructs of Drs2 (Fig 3B). All the constructs employed in this study to express Drs2 used the control of DRS2 promoter. We first targeted the PS flipping activity of Drs2, which is required for the bidirectional transport endosome-TGN and for the exit of secretory vesicles from the TGN (Alder-Baerens et al,2006; Liu et al,2008;Xuet al,2013). While cells harboring an empty vector do not improve Ape1 processing in drs2Δcells, cells expressing a mutant unable to flip PS (drs2-GA) and cells expressing an ATPase-dead mutant of Drs2 (drs2-D560N) rescue either totally or partially the Cvt pathway (Fig 3C). Cells lacking Cho1, an enzyme essential for the synthesis of PS (Choi et al,2010), show normal processing of Ape1, confirming that flipping of PS is not required for the Cvt pathway (Fig 3D). The cytosolic C-terminal tail of the flippase is also important for the known functions of Drs2. For instance, the Gea2 interacting motif (GIM) formed by the LSSLVMK sequence (Chantalat, 2004), is required for the Drs2-Gea2-Arl1 complex assembly, a mechanism based on protein–protein interactions that regulate a number of trafficking pathways at the TGN and endosomes (Chantalat, 2004; Tsai et al,2013). Adjacent to the GIM, Drs2 presents a conserved motif (CM). Conserved motif is fundamental for Drs2 function and to bind to the cytosolic domains of Drs2 that stabilize the autoinhibitory conformation (Chantalat, 2004; Timcenko et al,2019a). Drs2 has also two NPFXD motifs (hereafter referred to as NPF motif) that constitute some of its multiple endocytosis signals (Liu et al,2007). However, expression of drs2-ΔGIM,drs2-ΔCM or drs2-ΔGIMΔNPF also show more than 50% of Ape1 processing (Fig 3E). Thus, the mutagenesis experiments indicate that known functional motifs of 4of 18 EMBO reports e56134 |2023 Ó2023 The Authors EMBO reports Irene Pazos et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License the Drs2 C-terminal tail are not essential for the role of the flippase in the Cvt pathway. The I(S/R)TTK motif is required for the function of Drs2in the Cvt pathway We then investigated the cytosolic N-terminal tail of Drs2. Although P4-ATPases present low homology in this region, multiple sequence alignment identified a conserved stretch of 15 amino acids (residues 198–212 in Drs2; Fig 4A). The stretch defines a cavity of 18.8  Ain depth located adjacent to the first transmembrane helix in the structure of Drs2 (Fig 4B). Homology modeling suggests that all yeast P4- ATPases present the N-terminal cavity in their structure (Fig EV3), although the function of this cavity is not known. We observed that the bottom of the cavity of those P4-ATPases that bind MTCs (Dnf1, Dnf2, and Drs2) is characterized by an I(S/R)TTK motif that is lacking in the cavity of Dnf3 and Neo1 (Fig 4A). The correlation between this structural feature and the P4-ATPases-MTCs interaction pattern suggested that the I(S/R)TTK motif might be relevant for the function of P4-ATPases as regulatory partners of MTCs. We first generated cells expressing Drs2 with its I(S/R)TTK motif substituted by five alanines (drs2-5A). The expression of drs2-5A rescues the cold-sensitive phenotype of drs2Δcells, confirming that the loss of this motif does not induce general protein unfolding (Fig 4C). In agreement, comparative co-localization of Drs2-GFP and drs2-5A-GFP with Sec7-RFP, a marker of the TGN, shows that drs2- 5A-GFP is properly transported to the Golgi (Fig EV4A). However, the expression of drs2-5A in drs2Δcells cannot rescue Ape1 processing (Fig 4D), which accumulates in large cytosolic Ape1-GFP aggregates similar to those seen in trs85Δand drs2Δcells (Movie EV2). These results prompted us to investigate the molecular bases that control the function of the Drs2 I(S/R)TTK motif. Drs2I(S/R)TTK mediates proximal interactions with MTCs We first designed a competition assay to corroborate that Drs2 function in the Cvt pathway is mediated by interactions with its N- terminal tail. We measured the impact on Ape1 processing of overexpressing amino acids 1–212 of Drs2, which include the I(S/R)TTK motif. Remarkably, overexpression of Drs2 N-terminal tail caused a 30% inhibition of Ape1 processing (Fig 5A). Then, to systematically analyze the mechanism mediated by the Drs2 I(S/R)TTK motif, we carried out cross-linking-mass spectrometry (XL-MS) immunoprecipitation experiments (see Materials and * 1 μm 200 nm D pep4∆ pep4∆ drs2∆ A drs2∆ trs85∆ WT drs2∆ trs85∆ drs2∆ trs85∆ 1 0 0.12 1 0 0.22 + + + - - - 0.18 0.03 0.13 0.02 0.00 0.02 prApe1 mApe1 23ºC 30ºC 37ºC C Trs85-FRB Drs2-GFP Trs85-FRB Bet5-GFP 23ºC 0.4 0.8 1.2 37ºC Interaction score 0 P = 0.03 E Ape1-GFPZoom in EM drs2∆ 1 μm 200 nm Ape1 processing 55 1 0 0.96 + + + - - - 0.04 0.02 0.02 + + + - - - B trs85∆ Ape1-GFP drs2∆ BF WT WT WT Figure 2. Drs2is critical for the biogenesis of the Cvt vesicle. A Ape1processing was analyzed by Western blot in the indicated strains and temperatures and normalized to the processing achieved in wild-type cells SD, n=3. B Aggregation of Ape1. Representative images of Ape1-GFP in wild-type, trs85Δand drs2Δcells. BF, brightfield images. Scale bar, 5μm. 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’st-test (P<0.05). D Representative EM images of pep4Δand pep4Δdrs2Δstrains. Black squares show a zoom-in in the vacuole. White arrowheads point to Cvt bodies. E Representative CLEM images of drs2Δcells. Ape1-GFP (top) correlates with a membrane-free ribosome exclusion area (middle). Black square (bottom) shows a zoomin at the position correlating with Ape1-GFP (white arrowhead). Data information: n=biological replicate. Ó2023 The Authors EMBO reports e56134 |2023 5of 18 Irene Pazos et al EMBO reports 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Methods). We performed a partial cross-linking with disuccinimidyl sulfoxide (DSSO), a cross-linker of 10.1  A in length that allowed us to detect proximal peptides in cell extracts (Kao et al,2011). Upon a GFP-specific pulldown, the comparative analysis between the interactome of Drs2-GFP and drs2-5A-GFP identified 26 proteins that are likely to recognize directly, or in the near vicinity, Drs2-GFP and whose binding requires the I(S/R)TTK motif (Fig EV4B). This set of interactions is enriched with proteins involved in endosomal transport (P=3.9 E-06), Golgi vesicle transport (P=3.5 E-05), and endosome-to-Golgi transport (P=4.9 E-08). In agreement with the cold-sensitivity rescue assay (Fig 4C), known protein–protein interactions required for canonical Drs2 functions were not affected by Drs2 mutation (e.g., binding to Cdc50 or Rcy1). Overall, our results suggest that the mutation of the I(S/R)TTK motif allows us to dissect different mechanisms controlled by Drs2. Although Drs2-GFP pulled down subunits of the GARP, TRAPPII, and TRAPPIII complexes, we could not detect binding of Drs2-GFP to any subunit of the COG complex in the XL-MS experiments, suggesting that Drs2 and COG do not interact directly. Interestingly, drs2-5A-GFP could not recapitulate the interaction with Trs85 (the specific subunit of TRAPPIII), and the interactions with Trs65, Trs120, Trs130 (specific subunits of TRAPPII) and Vps53 (GARP) were also destabilized upon mutation of the I(S/R)TTK motif. Thus, the interactome data suggest that the I(S/R)TTK motif plays a role in the network of interactions between Drs2 and GARP, TRAPPII, and TRAPPIII (Figs 5B and EV4B). We further confirmed this observation using PICT. Although our assay was not sensitive enough to recapitulate the interaction between Drs2-GFP and GARP in Δdrs2 cells expressing Drs2 from a centromeric plasmid, PICT confirmed that the lack of the I(S/R)TTK motif abrogated the interaction between the lipid flippases and TRAPPII and TRAPPIII (Fig 5C and EV4C). These results encouraged us to investigate the general contribution that the I(S/R)TTK motif might have in the network of interactions between P4-ATPases and MTCs. A general mechanism for the interplay between P4-ATPases and MTCs The N-terminal tail of P4-ATPases is composed of a large unstructured region that is not conserved and a stretch of 15 amino acids that define the cavity that links the first transmembrane helix and the unstructured region (Timcenko et al,2019a). We cannot exclude 55 55 rcy1Δ apl4Δ apl5Δ gga1Δ gga2Δ prApe1 mApe1 0.04 0.02 0.02 0.05 1.02 1.06 1.01 0.97 + + + + - - - - 55 prApe1 mApe1 1 1.02 0.17 0.11 + + - - 55 cho1Δ WT drs2Δ + drs2-GA drs2Δ + empty vector drs2Δ + WT DRS2 + + + + - - - - Ape1 processing prApe1 mApe1 drs2Δ + drs2-D560N CD A 0.07 0.08 0.27 0.23 0.80 0.69 0.69 1 drs2Δ + WT DRS2 drs2Δ + drs2-ΔGIM drs2Δ + drs2-ΔCM drs2Δ + drs2-ΔGIM ΔNPF + + + + - - - - prApe1 mApe1 E 1220 13551250 1270 1274 1298 1311 1333 GIM CM NPFXD NPFXD 237 238 aa 560 WT DRS2 drs2-GA drs2-D560N drs2- Δ GIM drs2- Δ CM drs2- Δ GIM Δ NPF B Ape1 processing Ape1 processing Ape1 processing 0.17 0.09 0.08 0.09 0.31 1 1.04 0.54 Figure 3. Drs2role in the Cvt pathway is independent from its known mechanisms. A–E (A, C, E) Ape1processing was analyzed by Western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild-type Drs2SD, n≥3biological 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 Drs2sequence. (D) Ape1processing was analyzed by Western blot in the indicated strains, and normalized to the processing achieved in wild-type cells SD, n=3biological replicates. 6of 18 EMBO reports e56134 |2023 Ó2023 The Authors EMBO reports Irene Pazos et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License that MTCs bind the unstructured region of the P4-ATPases N- terminal tail. However, the lack of sequence homology among the P4-ATPases that bind MTCs hints that this is unlikely to be a universal mechanism. Alternatively, MTCs might bind the N-terminal cavity of P4-ATPases that nest the I(S/R)TTK motif. Although it has no known function, the cavity is conserved in the atomic structure of yeast and human P4-ATPases (Bai et al,2019; Hiraizumi et al, 2019a; Timcenko et al,2019a; Nakanishi et al,2020a,2020b). The presence of the I(S/R)TTK motif in yeast Dnf1 and Dnf2 and the related ISTAK motif in the human ATP8A2, a homolog of Drs2, suggests that this structural element represents a mechanistic feature conserved in other P4-ATPases (Fig EV3). Using PICT, we assessed the general contribution of the I(S/R)TTK motif in the network of interactions between P4-ATPases and MTCs. Haploid cells expressed MTCs (GARP or TRAPPIII) subunits fused to FRB (bait) and P4-ATPases (Dnf1 or Dnf2) fused to GFP (prey), and when required, DNF1 and DNF2 genes were edited with CRISPR/Cas9. In consonance with a conserved mechanism for the interaction between P4-ATPases and MTCs, mutation of the I(S/R)TTK motif to five alanines weakens significantly all the interactions tested (Fig 5B and EV4D). It is particularly interesting the loss of interaction between GARP and dnf1-5A-GFP and dnf2-5A-GFP, whose Interaction score decreased 56.8 and 63.4%, respectively. In agreement with the essential role of GARP in the transport of Dnf1 and Dnf2 to the bud (Takagi et al,2012; Eising et al,2019), cells expressing dnf1-5A-GFP and dnf2-5A-GFP failed to polarize properly the two flippases (Fig EV4E). Thus, our results demonstrate that the I(S/R) TTK motif is required to preserve the overall network, with consequences on the cellular performance of both MTCs and P4-ATPases. To illustrate the biological relevance of such interactions, we further characterized the functional implications of the interaction between Drs2 and TRAPPIII. Binding to the Drs2N-terminal tail enhances the function of TRAPPIII TRAPPIII is in charge of transporting Atg9 from endocytic reservoirs to the Golgi, before Atg9 is delivered to the PAS (Shirahama-Noda et al,2013). We implemented a microscopy-based system with highsensitivity and high-temporal resolution to evaluate the relevance of Drs2 binding for the function of TRAPPIII in the transport of Atg9 vesicles (see Materials and Methods). In growing yeast cells, . . Lumen Cytosol Cdc50p Drs2p y x z I ST T K A drs2Δ + drs2-5A drs2Δ + WT DRS2 drs2Δ + empty vector prApe1 mApe1 55 0.14 1 0.21 + + + - - - 0.21 0.09 0.16 Ape1 processing DRS2 1335 1151 1571 1612 1656 D WT drs2Δ + empty vector drs2Δ + WT DRS2 drs2Δ + drs2-5A 30ºC 18ºC C Lumen Cytosol B Drs2 Cdc50 1 1 1 1 1 198 212 191 205 229 243 138 152 170 184 DNF1 DNF2 DNF3 NEO1 Figure 4. The I(S/R)TTK motif is required for the interaction between Drs2and TRAPPIII and its function in the Cvt pathway. 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, Dnf1and Dnf2. B The I(S/R)TTK motif in the Drs2-Cdc50 structure (PDB ID: 6ROH; Timcenko et al,2019a; 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. C Cold-sensitive assay for the indicated strains. Cells streaked onto YPD plates were incubated at 30 and 18°C for 3days. D Ape1processing was analyzed by Western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild-type Drs2SD, n=3bio- logical replicates. Ó2023 The Authors EMBO reports e56134 |2023 7of 18 Irene Pazos et al EMBO reports 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Atg9-GFP can be observed as highly mobile puncta, the vast majority of which correspond to vesicles within the cytoplasm (Yamamoto et al,2012). In agreement with a previous publication (Yamamoto et al,2012), growing wild-type cells present two populations of Atg9 puncta (Fig 6A and Movie EV3). The larger population comprises 86% of the puncta and it is highly mobile (mean speed of 2.3 0.7 nm/ms). The smaller population comprises 14% of the puncta and it has slower mobility (mean speed of 0.7 0.2 nm/ms). Interestingly, in drs2Δand drs2-5A cells, the low mobility population increases up to 54% (0.6 0.3 nm/ms) and 50% (0.6 0.2 nm/ms) of the puncta, respectively (Fig 6A and Movies EV4 and EV5). We could not detect Drs2-GFP at the PAS (Fig EV5), suggesting that the I(S/R)TTK motif of Drs2 plays a major role in the trafficking of Atg9 prior to reaching the PAS, most likely in the transport of higher mobility vesicles from lower mobility Atg9 reservoirs. This is further supported by the 39.6% decrease of Atg9-GFP detected at the Ape1-mCherry aggregates upon mutation of the Drs2 I(S/R)TTK motif (Fig 6B). Note that the inability of drs2-5A cells of transporting Atg9 to the PAS is likely to be more prominent, because large Ape1 aggregates in mutant cells are more prone to artifactually co-localize with neighboring Atg9 puncta. Mutation of the I(S/R)TTK motif disrupts the interaction of Drs2 with multiple proteins that contribute to Atg9 transport, such as GARP and TRAPPII subunits (Figs 5B and EV4B). This challenges the interpretation of the Atg9 miss-sorting and the specific contribution that the TRAPPIII-Drs2 interplay might have in this process. We confirmed that the interaction with Drs2 is required for TRAPPIII function with the PICT assay, a technique that provides a good read-out to measure the capability of proteins to bind membranes in situ (Picco et al,2017). We inferred TRAPPIII association with Atg9- loaded membranes by measuring the recruitment of Atg9-GFP to the anchoring platform when using Trs85-FRB as bait. Indeed, the Interaction score between TRAPPIII and Atg9-GFP dropped 84.1% in cells where TRAPPIII could not bind Drs2 (Fig 6C). The partial delivery of Atg9 to the PAS and the residual ability of TRAPPIII to bind Atg9-loaded membranes are in agreement with the observation that the Cvt pathway remains slightly active in drs2-5A cells under our experimental conditions (Fig 4D). Vesicle tracking, co-localization and the PICT assay demonstrate that the I(S/R)TTK motif in the Drs2 N-terminal tail strengthens the binding of TRAPIII on Atg9- loaded membranes and facilitates the delivery of Atg9 for the Cvt pathway. These results, together with the competition assay and XL-MS experiments (Figs 5A and B and EV4B), uncovered the mechanism used by Drs2 as a regulatory partner of TRAPPIII: Trs85 proximal binding to the Drs2 N-terminal tail stabilizes TRAPPIII on Atg9- loaded membranes, which in turn is necessary for the normal transport of Atg9 from the reservoirs and for the progression of the early stages of the Cvt pathway (Fig 6D). Discussion Understanding the mode of action of MTCs requires an approach dedicated to compare side-by-side the mechanistic features of these protein machines. The PICT assay is based on live-cell imaging. ** B Interaction score Trs85-FRB P = 0.00005 MTCs P4-ATPases Requires I(S/R)TTK to bind MTCs Yes Yes No No AC 55 prApe1 mApe1 WT + empty vector WT + N-terminal Drs2 + + - - 0.01 0.01 Anchor- RFP-FKBP Prey-GFP Merge Trs85-FRB + RAP Drs2-GFP drs2-5A-GFP NeoI GARP TRAPPII TRAPPIII Dnf2 Dnf3 DSL1 Exocyst CORVET HOPS Drs2 COG Dnf1 Neo1 Ape1 processing 1 0.7 Yes 1 0 0.2 0.4 0.6 0.8 1.2 Drs2 -GFP drs2- 5A-GFP Figure 5. The I(S/R)TTK motif and the interaction between MTCs and P4-ATPases. A Ape1processing 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. 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). 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’st-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, 0.9μm. Scale bar, 5μm. Data information: n=biological replicate. 8of 18 EMBO reports e56134 |2023 Ó2023 The Authors EMBO reports Irene Pazos et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Trs85 Cytosol Drs2 I(S/R)TTK ** B Atg9-GFPApe1-mCherryMerge drs2Δ + WT DRS2 drs2Δ + drs2-5A Colocalization score Atg9-GFP Ape1- mCherry 0 0.2 0.4 0.6 0.8 1.2 1 Interaction score Trs85-FRB Atg9-GFP ** C P = 0.005 0 0.2 0.4 0.6 0.8 1.2 1 drs2Δ + WT DRS2 drs2Δ + drs2-5A Anchor- RFP-FKBP Atg9-GFP Merge WT DRS2 drs2-5A Trs85-FRB drs2Δ + RAP 4 drs2Δ + WT DRS2 drs2Δ + drs2-5A A P = 0.0009 Atg9 vesicles Atg9 reservoir Cvt pathway D Atg9 Drs2 TRAPPIII Trs85 0.8 1.6 2.4 3.20 54% puncta 0.6 nm/ms 46% puncta 1.5 nm/ms 150 100 50 0 40.8 1.6 2.4 3.20 14% puncta 0.7 nm/ms 86% puncta 2.3 nm/ms 40 30 20 10 0 drs2Δ Atg9 puncta WT Atg9 puncta 0.8 1.6 2.4 3.2 40 50% puncta 0.6 nm/ms 50% puncta 1.1 nm/ms 160 120 80 40 0 200 drs2Δ + drs2-5A Atg9 puncta speed (nm/ms) Figure 6. 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J Mol Biol 430:2237–2243 Zou S, Chen Y, Liu Y, Segev N, Yu S, Liu Y, Min G, Ye M, Zeng Y, Zhu X et al (2013) Trs130 participates in autophagy through GTPases Ypt31/32 in Saccharomyces cerevisiae.Traffic 14:233–246 Zuo X, Guo W, Lipschutz JH (2009) The exocyst protein Sec10 is necessary for primary ciliogenesis and cystogenesis in vitro. Mol Biol Cell 20: 2522–2529 License: This is an open access article under the terms of the Creative Commons Attribution-NonCom- mercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 18 of 18 EMBO reports e56134 |2023 Ó2023 The Authors EMBO reports Irene Pazos et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Expanded View Figures Sec7-GFP Trs85-FRB + RAP Vps10-GFP Prey-GFP Anchor- RFP-FKBP Merge 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, 0.9μm. Scale bar, 5μm. Source data are available online for this figure. 55 Ape1 processing D 1 0 0.3 0 0.3 prApe1 mApe1 drs2Δ trs85Δ drs2Δ atg19Δ drs2Δ atg17Δ WT CBA prApe1 mApe1 55 drs2Δ 16º 18º 23º 30º 37º (C) 0.1 0.8 1 1.7 6.7 Ape1 processing Ape1 processing 1.1 1 1.2 1.1 drs2Δ 55 1 2 3 4 (h) Ape1 processing prApe1 mApe1 WT 55 drs2Δ trs85Δ mApe1 1 0 0.05 prApe1 55 1 0.78 0.73 prApe1 mApe1 + + + - - - 0.01 0.01 0.02 drs2∆ trs85∆ WT -N 4h Ape1 processing E 16ºC Figure EV2. Drs2is essential for the Cvt pathway as the temperature decreases. Ape1processing was analyzed by Western blot in the indicated strains. ? Adrs2Δcells were grown at 30°C and then cultured for 2 h at the indicated temperatures. BWild-type, trs85Δand drs2Δcells were grown at 30°C and then cultured for 2 h at 16°C. CAnalysis of Ape1 processing in drs2Δcells where either the Cvt pathway (atg19Δ) or nonselective autophagy (atg17Δ) were blocked. Ddrs2Δcells were grown at 30°C and then cultured for 1, 2 or 4 h at 23°C. EWild-type, trs85Δand drs2Δcells 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, Ape1processing was normalized to the processing achieved after 2hat23°C. (B, C, E) Below, Ape1processing was normalized to the processing achieved in wild-type cells. Source data are available online for this figure. Irene Pazos et al EMBO reports Ó2023 The Authors EMBO reports e56134 |2023 EV1 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 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 A), Dnf2(11.6×9.5 A), Dnf3(13.5×15.5 A) and Neo1(18.1×13.8 A). Drs2was used as a template PDB ID: 6ROH:A (Timcenko et al,2019a; Data ref: Timcenko et al,2019b). The I(S/R)TTK motif present in Dnf1and Dnf2is highlighted in yellow. Structural representation of the 15 aa cavity (purple) in the N-terminal tail of the human ATP8A2(17.7×20.5 A). ATP8A1was used as a template (PDB ID: 6K7N; Hiraizumi et al,2019a; 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. 0.8 Co-localization score Sec7-RFP GFPSec7-RFPMerge Drs2-GFP drs2-5A-GFP drs2∆ + 0 0.2 0.4 0.6 1 drs2∆ + Drs2-GFP drs2∆ + drs2-5A-GFP 1.2 GARP TRAPPII TRAPPIII CAB ***** TRAPPIII-FRB 0 0.2 0.4 0.6 0.8 1 1.2 GARP-FRB Dnf1-GFP dnf1-5A-GFP Dnf2-GFP dnf2-5A-GFP P = 0.0002 P = 0.048 P = 0.019 Interaction score P = 0.014 D E Vps13 Vti1 Emp70 Gnp1 Akr1 Kex1 Trs65 Pep1 Pdr12 Ste13 Sho1 Vps5Ste4 Mon2 Vps45 Trs85 Lcb4 Dop1 Trs120 Cct2 Trs130 Ypr148c Vps17 Vph1 Gcs1 Vps53 Drs2 I(S/R)TTK BFDR < 0.05 BFDR < 0.1 Interacts with Atg9 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Interaction score TRAPPII-FRB drs2∆ + Drs2-GFP drs2∆ + drs2-5A-GFP P = 0.005 ** Dnf1-GFP dnf1-5A-GFP Dnf2-GFP dnf2-5A-GFP Figure EV4. EMBO reports Irene Pazos et al EV2EMBO reports e56134 |2023 Ó2023 The Authors 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure EV4. The role of the I(S/R)TTK motif. ARepresentative images (top) and quantification (bottom) of the co-localization between Sec7-RFP and Drs2-GFP or drs2-5A-GFP in drs2Δcells. 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. BGraphic 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. CInteraction between Trs130-FRB and Drs2-GFP or drs2-5A-GFP analyzed by PICT. The Interaction score was normalized to the measurement of Drs2-GFP. DInteraction 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. ERepresentative images of Dnf1-GFP, dnf1-5A-GFP, Dnf2-GFP or dnf2-5A-GFP. Data information: (A, C, E) Error bars: mean SD, n=3biological replicates. Asterisk indicates significant difference as determined by a two-tailed Student’st-test (*P<0.05;**P<0.01). Source data are available online for this figure. atg1Δ Drs2-GFPAtg11-mCherry Merge Figure EV5. GFP-tagged Drs2do 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, 5μm. Source data are available online for this figure. Irene Pazos et al EMBO reports Ó2023 The Authors EMBO reports e56134 |2023 EV3 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256134 by Universidad De Sevilla, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License