The P24 Complex Contributes to Specify Arf1 for COPI Coat Selection

International Journal of Molecular Sciences

Article The p24 Complex Contributes to Specify Arf1 for COPI Coat Selection

Susana Sabido-Bozo 1,2,† , Ana Maria Perez-Linero 1,2,†, Javier Manzano-Lopez 1,3,†, Soﬁa Rodriguez-Gallardo 1,2, Auxiliadora Aguilera-Romero 1,2, Alejandro Cortes-Gomez 1,2, Sergio Lopez 1,2 , Ralf Erik Wellinger 3,4 and Manuel Muñiz 1,2,*

1 Department of Cell Biology, Faculty of Biology, University of Seville, 41012 Seville, Spain; [email protected] (S.S.-B.); [email protected] (A.M.P.-L.); [email protected] (J.M.-L.); [email protected] (S.R.-G.); [email protected] (A.A.-R.); [email protected] (A.C.-G.); [email protected] (S.L.) 2 Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41012 Seville, Spain 3 Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, 41012 Sevilla, Spain; [email protected] 4 Department of Genetics, University of Seville, 41012 Seville, Spain * Correspondence: [email protected] † These authors contributed equally.

Abstract: Golgi trafﬁcking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis-Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI-coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor-coated vesicles at the trans-Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the Citation: Sabido-Bozo, S.; interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, Perez-Linero, A.M.; Manzano-Lopez, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI J.; Rodriguez-Gallardo, S.; by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, Aguilera-Romero, A.; Cortes-Gomez, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study A.; Lopez, S.; Wellinger, R.E.; Muñiz, supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for M. The p24 Complex Contributes to selective COPI coat assembly at the cis-Golgi compartment. Specify Arf1 for COPI Coat Selection. Int. J. Mol. Sci. 2021, 22, 423. Keywords: p24 complex; COPI; Golgi; ArfGEFs; Arf1 https://doi.org/10.3390/ijms22010423

Received: 30 October 2020 Accepted: 30 December 2020 Published: 3 January 2021 1. Introduction COPI vesicle-mediated retrograde transport along the early secretory pathway is an Publisher’s Note: MDPI stays neu- essential process that recycles resident proteins and anterograde transport factors within tral with regard to jurisdictional clai- the Golgi compartments, and from the Golgi apparatus back to the endoplasmic reticulum ms in published maps and institutio- (ER). Thereby it contributes to maintain ER homeostasis, to initiate new rounds of ER-to- nal afﬁliations. Golgi vesicle transport and to drive Golgi cisternal maturation [1–4]. In yeast, COPI vesicles are primarily formed at the earlier Golgi compartments by the small GTPase Arf1 which, upon activation by the functionally redundant exchange factors or ArfGEFs Gea1 and Gea2, is thought to directly recruit and assemble the cytosolic heptameric protein complex COPI Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. for membrane deformation and cargo capture [5–12]. However, the presence of Arf1 is not This article is an open access article limited to the early compartments within the Golgi. Indeed, Arf1 also functions at the late distributed under the terms and con- Golgi where, upon activation by the ArfGEF Sec7, it recruits other types of coat effectors ditions of the Creative Commons At- such as clathrin coat adaptors to form vesicles that export fully processed secretory cargo to tribution (CC BY) license (https:// the endosomal system, or to the plasma membrane [13–15]. The fact that Arf1 is activated creativecommons.org/licenses/by/ in early as well as late Golgi compartments raises the fundamental question of how the 4.0/). active form of Arf1 can preferentially produce COPI vesicles at the early Golgi.

Int. J. Mol. Sci. 2021, 22, 423. https://doi.org/10.3390/ijms22010423 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 423 2 of 13

A previous study described an Arf1 activation-independent, direct interaction of Gea1 with the COPI coat [16]. This finding led to postulate a feed-forward mechanism by which the formation of Gea1/COPI coat complexes would channel Arf1 into specifically binding to the COPI effector by triggering Arf1 activation in proximity to the COPI coat. Thus, selective COPI coat assembly by Arf1 at the early Golgi compartments could be then determined by the previous formation or stabilization of Gea1/COPI coat complexes [16,17]. However, since ArfGEF/coat interaction itself has been shown to be insufficient to maintain the coat bound to membranes, additional factors may contribute to specifically stabilize Gea1/COPI coat complexes at the early Golgi membrane [17]. One of these factors could be transmembrane cargos of COPI vesicles such as the p24 proteins, which are abundant and conserved type I transmembrane proteins harboring COPI binding signals on their cytoplasmic tails [18–21]. They assemble in a heteromeric complex that continuously cycles between the ER and Golgi, being itself a major cargo of COPI retrograde transport vesicles [22–24]. Besides acting as a specific cargo receptor during the ER export [25,26], the p24 complex also participates in COPI vesicle formation from the Golgi [5,27,28]. This later role has been mainly attributed to the potential ability of the p24 proteins to stabilize the COPI coat onto the Golgi membrane after Arf1-GTP hydrolysis [5,27,28]. Here, we provide evidence suggesting an even earlier role of the p24 complex in establishing COPI vesicle budding sites. We find that the p24 complex is required to direct Arf1 for specific COPI binding as it promotes the local association of COPI with Gea1. Our results suggest that COPI vesicle cargos, such as the abundant p24 complex, act to spatially delimit the Golgi territory where Arf1 will form COPI retrograde transport vesicles.

2. Results 2.1. The p24 Complex Specifically Interacts with the ArfGEF Gea1 Through COPI Coat Binding The previous finding, that the COPI coat associates specifically with the ArfGEF Gea1, has led to postulate a feed-forward mechanism by which the COPI coat and Gea1 would form a complex to channel Arf1 activation for COPI vesicle budding [16,17]. We therefore wondered if ArfGEF/coat complexes could be selectively stabilized at the early Golgi by transmembrane COPI vesicle cargos, such as the p24 complex, in order to establish COPI vesicle budding sites. As a first step to evaluate this hypothesis, we tested whether the interaction between Gea1 and COPI coats occurs at the Golgi membrane or in the cytoplasm. Therefore, we examined the subcellular distribution of Gea1/COPI coat complexes by differential fractionation, followed by specific immunoprecipitation of each fraction for the COPI coat subunit Sec21 (yeast γ-COP) tagged with GFP and subsequent immunoblotting for HA-Gea1. As shown in Figure1, Gea1/COPI coat complexes were mainly found in the membrane fraction, which agrees with the idea that transmembrane proteins might contribute to their formation on the Golgi membrane. As the p24 proteins efficiently bind the COPI coat [19], we next assessed whether the p24 complex is also able to specifically associate with Gea1. For this purpose, yeast cells expressing HA-Gea1 were analyzed by native coimmunoprecipitation. As seen in Figure2A, immunoprecipitation in detergent-solubilized extracts revealed that the p24 protein Emp24-CFP coprecipitated with HA-Gea1. As a positive control, binding of Emp24-CFP with the COPI coat subunit Cop1 (yeast α-COP) was also observed. These physical interactions were specific because the unrelated cytosolic protein Pgk1 was not recovered after Emp24-CFP coimmunoprecipitation. If the p24 complex is involved in the selective formation of COPI vesicles from the early Golgi by stabilizing specific complexes containing Gea1 and COPI, it should not interact with Sec7, the other class of ArfGEF that forms clathrin-coated vesicles at the late Golgi [29]. Cells expressing Sec7-dsRed were also analyzed by native coimmunoprecipitation (Figure2A). As expected, Emp24-CFP was not coprecipitated with Sec7-dsRed, indicating that, specifically, the p24 proteins form a complex only with Gea1 and COPI. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 15

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Figure 1. Gea1 and COPI coat specifically interact at the Golgi membrane. Total (T) lysate prepared Figurefrom 1. wild-type Gea1 cellsand expressing COPI coat HA-Gea1 specifically and the COPI interact coat subunit at Sec21 the tagged Golgi with membrane. GFP was Total (T) lysate pre- separated into low-speed supernatant (S13) and pellet (P13) fractions by a 13,000× g centrifugation paredstep. from The S13 wild-type enriched-Golgi cells fraction expressing was further separated HA-Gea1 into high-speed and the supernatant COPI coat (S100) subunit and Sec21 tagged with GFP was pelletseparated (P100) fractions into low-speed by centrifugation supernatant at 100,000× g. The (S13) collected and fractions pellet were (P13) subjected fractions to by a 13,000 ×g centrifu- gationspecific step. immunoprecipitation The S13 enriched-Golgi for Sec21-GFP and fraction subsequent wa immunoblottings further usingseparated antisera to into HA, high-speed supernatant Pgk1, Bos1 and GFP. (S100) and pellet (P100) fractions by centrifugation at 100,000 ×g. The collected fractions were subjected toDue specific to the direct, immunoprecipitation specific interaction of Gea1 for withSec21-GFP the COPI coat,and wesubsequent next assessed immunoblotting using anti- whether the p24 complex binds Gea1 indirectly through the COPI coat. The Gea1 residues sera 434–521to HA, are Pgk1, part of Bos1 the conserved and GFP. HUS (Homology Upstream Sec7) domain that directly binds the γ subunit of the COPI coat [16]. Thus, we analyzed the interaction of p24 with a HA-Gea1 N-terminal deletion mutant lacking residues 434–521 (HA-Gea1∆COPI) by coimmunoprecipitation.As the p24 proteins (Figure efficiently2B). HA-Gea1 bind∆COPI wasthe stablyCOPI expressed coat [19], to wild-type we next assessed whether the p24 levelscomplex and showed is also reduced able binding to specifically to Sec21-GFP indicating associate that COPI with binds Gea1. to the 434–For this purpose, yeast cells 521 region of Gea1 as previously shown by Deng et al. (Figure2C,D) [ 16]. Furthermore, expressingas seen in FigureHA-Gea12D, the full-lengthwere analyzed wild-type HA-Gea1by native was efficientlycoimmunoprecipitation. recovered by As seen in Figure 2A, Emp24-CFPimmunoprecipitation coimmunoprecipitation. in However,detergent-solubilized HA-Gea1∆COPI failed extracts to efficiently revealed bind that the p24 protein Emp24-CFP. The reduced capacity of HA-Gea1∆COPI to bind Sec21-GFP and Emp24- Emp24-CFPCFP suggests coprecipitated that the p24 complex with binds Gea1HA-Gea1. through the As COPI a positive coat. To confirm control, this binding of Emp24-CFP withobservation, the COPI we performedcoat subunit an in vitro Cop1binding (yeast assay with α-COP) immobilized was p24 also tail peptidesobserved. These physical inter- and cytosol (Figure2C). Synthetic peptides corresponding to the C-terminal 10 amino actionsacids were of the p24 specific protein Erv25because were coupledthe unrelated to Sepharose cy beadstosolic and incubated protein with Pgk1 a was not recovered after Emp24-CFPcytosolic protein coimmunoprecipitation. extract prepared from wild-type If cells the expressing p24 complex HA-Gea1. Afteris involved the in the selective for- pulldown, both HA-Gea1 and the COPI subunit Cop1 were detected bound to the Erv25 tail mationsequence. of COPI Nevertheless, vesicles the binding from of thesethe twoearly proteins Golgi was by significantly stabilizing reduced specific when complexes containing Gea1the and aromatic COPI, residues it at should−7 and −8 werenot convertedinteract to alanineswith (YFSec7, to AA) the in theother Erv25 tailclass of ArfGEF that forms sequence. The di-aromatic motif of Erv25 has been shown to be necessary for efficient clathrin-coatedand direct binding vesicles of purified at COPI the [19 late]. Therefore, Golgi the [29]. reduced Cells binding expressing of HA-Gea1 toSec7-dsRed were also ana- the mutated Erv25 peptide suggests that Gea1 binds to the p24 cytosolic tails indirectly lyzedthrough by native COPI. coimmunoprecipitation (Figure 2A). As expected, Emp24-CFP was not coprecipitated with Sec7-dsRed, indicating that, specifically, the p24 proteins form a complex only with Gea1 and COPI.

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Figure 2. The p24 complex specifically associates with the ArfGEF Gea1 through the COPI coat. (A) Emp24-CFP specifically Figure 2. The p24 complex specifically associates with the ArfGEF Gea1 through the COPI coat. (A) Emp24-CFP specifi- binds thecally early binds Golgi-localized the early Golgi-localized ArfGEF HA-Gea1 ArfGEF HA-Gea1 and the COPIand the coat COPI subunit coat subunit Cop1 Cop1 but not but the not latethe late Golgi-localized Golgi-localized ArfGEF Sec7-dsRed.ArfGEF Native Sec7-dsRed. coimmunoprecipitation Native coimmunoprecipitation assay between assay the between p24 complex the p24 subunit complex Emp24-CFP subunit Emp24-CFP and HA-Gea1, and HA-Gea1, Cop1 or Sec7- DsRed. S13Cop1 enriched-Golgi or Sec7-DsRed. fractionsS13 enriched-Golgi of wild-type fractions cells of expressing wild-type cells the indicatedexpressing taggedthe indicated proteins tagged were proteins immunoprecipitated were im- with anti-GFPmunoprecipitated antibody, followedwith anti-GFP by immunoblotting.antibody, followed by Total immunobl (T) representsotting. Total a fraction (T) represents of the a solubilized fraction of the input solubilized material. (B) input material. (B) Scheme of full-length HA-Gea1 and deletion construct of HA-Gea1p (HA-Gea1ΔCOPI) lacking residues Scheme of434–521, full-length a region HA-Gea1 involvedand in direct deletion binding construct to the COPI of HA-Gea1p coat [16]. DCB, (HA-Gea1 dimerization∆COPI) an lackingd cyclophilin residues binding 434–521, domain; a region involvedHUS, in direct homology binding upstream to the of Sec7 COPI domain; coat [ Sec7,16]. DCB,guanine dimerization nucleotide exchange and cyclophilin factor domain; binding HDS1, domain; HDS2, HDS3, HUS, homol- homology upstreamogy of downstream Sec7 domain; of Sec7 Sec7, domains. guanine (C nucleotide) HA-Gea1Δ exchangeCOPI is expressed factor domain; to the level HDS1, of the HDS2, full-length HDS3, HA-Gea1. homology Protein downstream expression levels of each construct were analyzed by Western blot of whole cell extracts. Pkg1 serves as a loading control. of Sec7 domains. (C) HA-Gea1∆COPI is expressed to the level of the full-length HA-Gea1. Protein expression levels (D) The lack of the COPI coat binding region (434–521) [16] in HA-Gea1ΔCOPI prevents its efficient association to the p24 of each constructcomplex. S13 were enriched-Golgi analyzed byfractions Western of wild-type blot ofwhole cells ex cellpressing extracts. indicated Pkg1 tagged serves proteins as a loadingwere immunoprecipitated control. (D) The lack of the COPIwith coatanti-GFP binding antibody, region followed (434–521) by immunoblotting. [16] in HA-Gea1 Total∆ (T)COPI represents prevents a fraction its efficient of the solubilized association input to material. the p24 complex.(E) S13 enriched-Golgi fractions of wild-type cells expressing indicated tagged proteins were immunoprecipitated with anti-GFP antibody, followed by immunoblotting. Total (T) represents a fraction of the solubilized input material. (E) The p24 proteins interact with Gea1 through COPI binding. Synthetic peptides corresponding to the cytoplasmic tail of the p24 protein Erv25 (KNYFKTKHII) or to Erv25-AA with aromatic residues replaced by alanines (KNAAKTKHII) were coupled to thiopropyl-Sepharose beads. These peptide-bound beads and empty beads (Control) were incubated with cytosol from a wild-type strain expressing HA-Gea1. Bound material was resolved by SDS-PAGE and analyzed by immunoblot. Int. J. Mol. Sci. 2021, 22, 423 5 of 13

2.2. The Specific Association of the Arf1GEF Gea1 with the COPI Coat is Stabilized by the p24 Complex Since we found that the p24 complex indirectly interacts with Gea1 through the COPI coat, it was conceivable that the presence of the p24 complex increases the stability of the COPI coat on the early Golgi membrane and thus, promotes the association of Gea1 with COPI coat to initiate COPI vesicle formation. To assess this possibility, we first examined, in vivo, if the yeast p24 complex enhances COPI coat association to the membrane as previously reported for mammalian p24 proteins [30]. For this purpose, we used the deletion of EMP24 that destabilizes the other proteins of the complex, leading to a complete loss of p24 complex function, and analyzed the subcellular distribution of COPI coat in the absence of the p24 complex by differential fractionation [22]. We thereby observed that Cop1 was less associated to the membrane fraction in the emp24∆ mutant compared to the wild-type strain (Figure3). Thus, we next assessed if the p24 complex is also required for the efficient recruitment of HA-Gea1 to the membrane (Figure3). In contrast to Cop1, the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 15 binding of HA-Gea1 to the membrane was not affected by the lack of the p24 complex, suggesting that recruitment of HA-Gea1 is independent of the presence of the COPI coat onto the Golgi membrane.

Figure 3. The absence of Emp24 reduces the Golgi membrane association for the COPI coat but not for HA-Gea1. (A) Golgi Figure 3. Themembrane absence association of Emp24 of reduces Cop1 and the HA-Gea1Golgi membrane in wild-type asso andciationemp24 for∆ strains.the COPI Total coat (T) but lysates not for prepared HA-Gea1. from ( cellsA) Golgi membraneexpressing association HA-Gea1 of Cop1 were and differentially HA-Gea1 centrifuged in wild-type as in Figureand emp241. TheΔ collected strains. soluble Total S100 (T) andlysates membrane prepared P100 from fractions cells expressing HA-Gea1were analyzed were bydifferentially Western blot centrifuged using antisera as toin Cop1,Figure HA 1. The and collected the SNARE soluble Bos1, usedS100 asand Golgi membrane membrane P100 marker. fractions were analyzed(B) Quantification by Western ofblot three using independent antisera experiments.to Cop1, HA The and graph the plotsSNARE the averageBos1, used percentage as Golgi of themembrane Golgi membrane marker. (B) Quantificationassociation of three of independent Cop1 and HA-Gea1 experiments. normalized The to graph the association plots the in average the wild-type percentage strain. of Membrane the Golgi association membrane was association of Cop1determined and HA-Gea1 by quantification normalized of the to amountthe association of protein in present the wild-type in the membrane strain. Membrane (P100) fraction association divided by was the amountdetermined by quantificationpresent inof boththe amount the membrane of protein and soluble present fractions in the (P100membrane + S100). (P100) Error barsfrac indicatetion divided the SD. by Statistical the amount significance present was in both the membranedetermined and soluble by using fractions the Student (P100t test. + **S100).p < 0.01. Error bars indicate the SD. Statistical significance was determined by using the Student t test. ** p < 0.01. Our finding, that the yeast p24 complex contributes to have more COPI coat associated to the membrane, prompted us to study whether the p24 complex also contributes to the Ourefficient finding, interaction that the between yeast Gea1p24 andcomplex COPI coat.contributes To address to thishave possibility, more COPI we assessed coat associated tothe the fraction membrane, of HA-Gea1 prompted bound byus Sec21-GFPto study whether in the absence the orp24 presence complex of the also p24 contributes complex. to the efficientThereby, interaction we observed between significant Gea1 reduction and COPI in the coat. coprecipitation To address of HA-Gea1 this possibility, with Sec21- we as- sessedGFP the fraction in emp24 ∆ofmutant HA-Gea1 extracts bound as compared by Sec21-GFP to wild-type in the extracts absence (Figure or presence4A,B). These of the p24 results suggest that the efficient association of Gea1 with the COPI coat depends on the complex. Thereby, we observed significant reduction in the coprecipitation of HA-Gea1 Golgi presence of the p24 complex. with Sec21-GFP in emp24∆ mutant extracts as compared to wild-type extracts (Figure 4A,B). These results suggest that the efficient association of Gea1 with the COPI coat depends on the Golgi presence of the p24 complex.

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FigureFigure 4. 4.TheThe Gea1/COPI Gea1/COPI coat coat complex complex is is stabilized stabilized by the p24p24 complex.complex. ( A(A)) The The p24 p24 complex complex is requiredis required for for the the efficient efficient interactioninteraction between between Gea1 Gea1 and and the the COPI COPI coat.coat. Detergent-solubilizedDetergent-solubilized S13 S13 fractions fractions of of wild-type wild-type and andemp24 emp24∆ mutantΔ mutant cells cells expressingexpressing HA-Gea1 HA-Gea1 and and the COPICOPI coat coat subunit subunit Sec21-GFP Sec21-GFP were were subjected subjected to native to native immunoprecipitation immunoprecipitation (IP) with (IP) anti-GFP with anti- GFPantibody, antibody, followed followed by immunoblotting. by immunoblotting. Total (T)Total represents (T) repres a fractionents a fraction of the solubilized of the solubilized input material. input ( Bmaterial.) Quantification (B) Quantifi- of cationthree of independent three independent experiments. experiments. The graph The plots graph the plots average the percentageaverage percentage of the recovery of the recovery of HA-Gea1 of HA-Gea1 normalized normalized to the to recoverythe recovery in the in wild-type the wild-type strain. Errorstrain. bars Error indicate bars indicate the SD. Statistical the SD. Statistical significance significance was determined was determined by using the by Student usingt the Studenttest. * pt test.< 0.05. * p( C< )0.05. The ( lossC) The of the loss p24 of complexthe p24 complex intensifies intensifies the thermosensitive the thermosensitive growth defect growth in the defectgea1-6 in gea2the ∆gea1-6double gea2Δ doublemutant mutant but not but in thenot sec7-4in themutant sec7-4 mutant strain.Cells strain. were Cells tested were for tested growth for at growth 24, 30 and at 24, 37 30◦C and on YPUAD. 37 °C on YPUAD.

WeWe next strove toto seesee ifif the the p24 p24 complex complex may ma participatey participate in in the the initial initial stage stage of COPIof COPI vesiclevesicle formation formation by stabilizing thethe Gea1/COPIGea1/COPI coat complex.complex. UnderUnder physiologicalphysiological con- condi- tions,ditions, the the existence existence of ofmany many other other transmembr transmembraneane cargos cargos of of COPI COPI vesicles vesicles can likely at- tenuateattenuate the the defect defect caused caused by by the the absence absence ofof thethe p24p24 proteins. proteins. However, However, when when the the ArfGEF ArfGEF activity of partially redundant Gea1 and Gea2 proteins is compromised, p24 could become activity of partially redundant Gea1 and Gea2 proteins is compromised, p24 could become indispensable for COPI vesicle generation. To investigate this possibility, we assessed the indispensable for COPI vesicle generation. To investigate this possibility, we assessed the viability of gea1-6 gea2∆ double mutant strains and a gea1-6 gea2∆ emp24∆ triple mutant viabilitystrains2 byof dropgea1-6 test gea2 analysis∆ double (Figure mutant4C). In strains line with and our a gea1-6 idea, the gea2 growth∆ emp24 of cells∆ triple carrying mutant strainsthe temperature2 by drop sensitivetest analysisgea1-6 (Figureallele lacking4C). In Gea2line with and our Emp24 idea, became the growth displayed of cells as a carrying slow thegrowth temperature phenotype sensitive and increased gea1-6 temperatureallele lacking sensitivity Gea2 and at 30Em◦C.p24 As became control, displayed we took ad- as a slowvantage growth of a mutantphenotype impaired and inincreased Sec7 function temperature because Sec7sensitivity is the other at 30 ArfGEF °C. As present control, at we tookthe late advantage Golgi, where of a itmutant activates impaired Arf1 to producein Sec7 function clathrin-coated because vesicles. Sec7 is Interestingly, the other ArfGEF the presentemp24∆ atmutant the late did Golgi, not show where any it synthetic activates interaction Arf1 to produce with sec7-4 clathrin-coated, a temperature-sensitive vesicles. Inter- estingly,mutant allelethe emp24 of SEC7Δ mutant. These did results not show strengthen any synthetic the idea interaction that the functional with sec7-4 interaction, a temperature-sensitivebetween the EMP24 mutantand alleleGEA1 ofis SEC7 speciﬁc. These for COPIresults vesicle strengthen formation. the idea that the functional interaction between the EMP24 and GEA1 is specific for COPI vesicle formation.

2.3. The p24 Complex Contributes to Specify Arf1 Activation for COPI Binding Gea1 was previously shown to interact with the COPI coat subunit Sec21 in the presence of the ArfGEF inhibitor Brefeldin A (BFA) [16], which blocks the GDP/GTP exchange

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2.3. The p24 Complex Contributes to Specify Arf1 Activation for COPI Binding reactionGea1 by wastrapping previously the Arf1-GDP/GEF shown to interact comp withlex the [31–33]. COPI coat This subunit finding Sec21 indicates in the that presence of the ArfGEF inhibitor Brefeldin A (BFA) [16], which blocks the GDP/GTP Gea1/COPI complex formation does not depend on Arf1 activation. Likewise, since the exchange reaction by trapping the Arf1-GDP/GEF complex [31–33]. This ﬁnding indicates p24that complex Gea1/COPI facilitates complex the formationformation does of the not Gea1/COPI depend on Arf1 complex activation. by increasing Likewise, sincethe COPI associationthe p24 complex to the facilitates membrane, the formation this initial of the COPI-p24 Gea1/COPI binding complex byshould increasing also the occur COPI inde- pendentlyassociation of toArf1 the membrane,activation. this As initial shown COPI-p24 in Figure binding 5A, shouldcoimmunoprecipitation also occur independently confirms theof interaction Arf1 activation. of Sec21-GFP As shown with in Figure Emp245A, coimmunoprecipitation and HA-Gea1, even in conﬁrms the presence the interaction of BFA. No- tably,of Sec21-GFP since the BFA with Emp24treatment and was HA-Gea1, effective even because in the presenceArf1 was of trapped BFA. Notably, in complex since with HA-Gea1the BFA (Figure treatment 5B), was this effective result suggests because Arf1that wasCOPI trapped can be in recruited complex by with the HA-Gea1 p24 complex to the(Figure early5B) Golgi, this resultmembrane suggests before that COPI Arf1 canactivation. be recruited by the p24 complex to the early Golgi membrane before Arf1 activation.

FigureFigure 5. The 5. Thep24 p24complex complex can can interact interact with with COPI COPI independently independently ofof Arf1 Arf1 activation. activation. (A )(Aerg6) erg6∆ mutantΔ mutant cells cells expressing expressing HA-Gea1HA-Gea1 and the and COPI the COPI coat coat subunit subunit Sec Sec21-GFP21-GFP were were incubated incubated withwith or or without without 100 100µg/mL µg/mL BFA BFA (Brefeldin (Brefeldin A) for A) 20 for min. 20 min. erg6Δerg6 mutation∆ mutation was wasused used to increase to increase the the plasma plasma membrane membrane permeability to to BFA. BFA. Detergent-solubilized Detergent-solubilized S13 fractionsS13 fractions from from these thesecells cellswere were subjected subjected to tonative native immunoprecipitation immunoprecipitation (IP) (IP) with with anti-GFP anti-GFP antibody, antibody, followed follo bywed immunoblotting. by immunoblotting. Total (T) Total (T) representsrepresents a a fraction fraction ofof thethe solubilized solubilized input input material. material. (B) BFA(B) BFA treatment treatment is effective is effective because because it traps Arf1 it traps in complex Arf1 in with complex with HA-Gea1.erg6 erg6∆Δmutant mutant cells cells expressing expressing HA-Gea1 HA-Gea1 were were incubated incubated with or with without or without BFA for 20BFA min. for Detergent-solubilized 20 min. Detergent-solu- bilizedS13 S13 fractions fractions from from these these cells cells were were subjected subjected to native to nati immunoprecipitationve immunoprecipitation (IP) with (IP) anti-HA with anti-HA antibody, antibody, followed followed by by immunoblotting.immunoblotting. Total Total (T) representsrepresents a a fraction fraction of theof the solubilized solubilized input input material. material. The subsequent association of COPI to Gea1 might then ensure that COPI is al- readyThe insubsequent place when association Arf1-GTP is of generated COPI to by Gea1 Gea1. might Accordingly, then ensure the stabilization that COPI of is the already in Gea1/COPIplace when complex Arf1-GTP by the is p24generated complex by should Gea1. direct Accordingly, Arf1 to bind the the stabilization proximal COPI of the Gea1/COPIeffector. We complex addressed by thisthe prediction p24 complex by determining should direct the amount Arf1 to of bind Cop1 the bound proximal to Arf1- COPI effector.GFP in We the addressed absence or this the presenceprediction of theby dete p24 complex.rmining the We observedamount of that Cop1 in the boundemp24 to∆ Arf1- GFPmutant, in the Cop1absence was or less the precipitated presence byof the Arf1-GFP p24 complex. than in the We wild-type observed strain that (Figurein the 6emp24). ∆ mutant,This result Cop1 further was less conﬁrms precipitated that the p24 by complexArf1-GFP contributes than in tothe specify wild-type Arf1 forstrain COPI (Figure coat 6). Thisselection result further at the early confirms Golgi. that the p24 complex contributes to specify Arf1 for COPI coat selection at the early Golgi.

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Figure 6. The p24 complex facilitates the interaction of Arf1-GFP with COPI. (A) Detergent-solubilized S13 fractions of Figure 6. The p24 complex facilitates the interaction of Arf1-GFP with COPI. (A) Detergent-solubilized S13 fractions of wild-type and emp24∆ mutant cells expressing Arf1-GFP were subjected to native immunoprecipitation (IP) with anti-GFP wild-type and emp24Δ mutant cells expressing Arf1-GFP were subjected to native immunoprecipitation (IP) with anti-GFP antibody, followed by immunoblotting. Total (T) represents a fraction of the solubilized input material. (B) Quantiﬁcation antibody, followed by immunoblotting. Total (T) represents a fraction of the solubilized input material. (B) Quantification of three independent experiments. The graph plots the average percentage of the recovery of Cop1 normalized to the of three independent experiments. The graph plots the average percentage of the recovery of Cop1 normalized to the recovery in the wild-type strain. Error bars indicate the SD. Statistical signiﬁcance was determined by using the Student t recovery in the wild-type strain. Error bars indicate the SD. Statistical significance was determined by using the Student t test. *** p < 0.001. test. *** p < 0.001.

3.3. Discussion Discussion HowHow the the same Arf1Arf1 GTPase, GTPase, upon upon activation, activation, manages manages to generate to generate different different types of types of coatedcoated vesicles, vesicles, depending on on the the donor donor Golgi Golgi compartment, compartment, remains remains a central a central question question inin Golgi Golgi trafficking. trafficking. Here, Here, we we investigated investigated how how Arf1 selectively contributescontributes to to COPI COPI vesi- vesicle formation at the cis-Golgi. According to the classical model, different cytosolic cle formation at the cis-Golgi. According to the classical model, different cytosolic coats coats can be potentially targeted by the activated Arf1-GTP to the cis-Golgi membrane but canonly be COPI potentially would betargeted selectively by the assembled activated upon Arf1-GTP additional to binding the cis- withGolgi transmembrane membrane but only COPIretrograde would cargo be selectively such as the p24assembled proteins upon to form additional COPI vesicles binding [5]. Deng with et transmembrane al. suggested ret- rogradean alternative cargo mechanismsuch as the based p24 proteins on their observationto form COPI that vesicles Gea1, the [5]. Arf1-GEF Deng et specifically al. suggested an alternativelocalized at mechanism the cis-Golgi, based directly on interactstheir observation with the COPI that coatGea1, in thethe presenceArf1-GEF of specifically BFA, localizedthereby, independentat the cis-Golgi, of Arf1 directly activation interacts [16]. This with finding the COPI led to postulatecoat in the a feed-forward presence of BFA, thereby,mechanism independent by which Gea1/COPI of Arf1 activation complexes [16] would. Thischannel finding Arf1 led to activation postulate to selecta feed-forward the mechanismadjacent COPI by coatwhich effector Gea1/COPI for COPI complexes vesicle budding would [17 ].channel Our study Arf1 supports activation this modelto select the and suggests that the p24 complex, a major cargo of COPI vesicles, contributes to specify adjacent COPI coat effector for COPI vesicle budding [17]. Our study supports this model Arf1 for COPI selection by stabilizing the Gea1/COPI complexes at the cis-Golgi membrane and(Figure suggests7). We that show the that p24 association complex, of COPIa major to thecargciso-Golgi of COPI membrane vesicles, is promotedcontributes by theto specify Arf1p24 complex,for COPI while selection Gea1 isby separately stabilizing recruited the Gea1/COPI by the Rab complexes GTPase Ypt1 at to the the cis-cis-GolgiGolgi mem- branelacking (Figure exposed 7). We anionic show lipids that such association as phosphatidylserine of COPI to the (PS) cis-Golgi or phosphatidylinositol- membrane is promoted by4 phosphatethe p24 complex, (PI4P) [9 while]. The Gea1 p24-dependent is separately COPI recruited recruitment by tothe the Rabcis -GolgiGTPase membrane Ypt1 to the cis- Golgifavors lacking the local exposed encounter anionic of COPI lipids with such Gea1, as leadingphosphatidylserine to the formation (PS) of or the phosphatidylin- transient ositol-4ternary phosphate complex Gea1/COPI/p24. (PI4P) [9]. The Accordingly,p24-dependent we found COPI that recruitment the p24 complex to the facilitatescis-Golgi mem- braneGea1/COPI favors associationthe local encounter and indirectly of COPI interacts with with Gea1, Gea1 leading through COPI.to the Most formation importantly, of the tran- we observed that COPI binds to the p24 complex in the presence of the ArfGEF inhibitor sient ternary complex Gea1/COPI/p24. Accordingly, we found that the p24 complex facil- BFA. This finding suggests that the COPI coat can be recruited to the cis-Golgi membrane via itatesthe p24 Gea1/COPI complex and association thereby will and interact indirectly with Gea1 interacts before with Gea1-dependent Gea1 through Arf1 activation.COPI. Most importantly,Consistently, we COPIobserved was that also COPI shown binds to bind to Gea1the p24 in thecomplex presence in the of BFA presence [16]. Theof the fact ArfGEF inhibitorthat Gea1 BFA. can activate This finding Arf1 while suggests simultaneously that the interactingCOPI coat with can COPIbe recruited should imply to the that cis-Golgi membranethe generated via Arf1-GTPthe p24 complex binds COPI and coatthereby rather wi thanll interact to other with coat Gea1 effectors. before Coherent Gea1-depend- entwith Arf1 this activation. prediction, Consistently, we show that Arf1 COPI binding was al toso COPI shown is reduced to bind in Gea1 the absence in the ofpresence the of BFAp24 [16]. complex. The fact In addition, that Gea1 to can facilitate activate the Arf1 formation while of simultaneously the Gea1/COPI interacting complex before with COPI should imply that the generated Arf1-GTP binds COPI coat rather than to other coat effectors. Coherent with this prediction, we show that Arf1 binding to COPI is reduced in the absence of the p24 complex. In addition, to facilitate the formation of the Gea1/COPI complex before Arf1 activation, the p24 complex could also play other roles that contribute to the local formation of COPI vesicles at the cis-Golgi membrane. First, p24 proteins

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Arf1 activation, the p24 complex could also play other roles that contribute to the local could initially recruit the deactivated form of Arf1 (Arf1-GDP) [23], facilitating its interac- formation of COPI vesicles at the cis-Golgi membrane. First, p24 proteins could initially tionrecruit with thethe deactivated pre-assembled form ofGea1/COPI Arf1 (Arf1-GDP) complex [23 ],to facilitating be subsequently its interaction activated with theand di- rectedpre-assembled for COPI binding. Gea1/COPI Furthermore, complex to the be subsequently direct interaction activated of p24 and proteins directed forwith COPI the Arf- GAPbinding. Glo3 [27], Furthermore, which is thestably direct associated interaction to of COPI, p24 proteins could contribute with the ArfGAP to regulate Glo3 [its27], func- tionswhich in COPI is stably vesicle associated biogenesis to COPI, and could uncoat contributeing [34–36]. to regulate Therefore, its functions we in COPIsuggest vesicle that the abilitybiogenesis of p24 andproteins uncoating to oligomerize [34–36]. Therefore, could wecontribute suggest thatto establish the ability multiple, of p24 proteins transient to in- teractionsoligomerize with could the different contribute regulatory to establish elemen multiple,ts transientof the COPI interactions vesicle withmachinery, the different favoring theirregulatory local encounter elements on of thethe COPI cis-Golgi vesicle membrane machinery, for favoring productive their local vesicle encounter budding on the(Figure cis-Golgi membrane for productive vesicle budding (Figure7). 7).

Figure 7. Schematic representation of the proposed role of the p24 complex in programming Arf1 activation by Gea1 for Figureselective 7 Schematic COPI coatrepresentation assembly. COPI of the can proposed be initially recruitedrole of the by p24 the p24complex complex, in aprogramming major retrograde Arf1 cargo activation of COPI vesicles,by Gea1 for selectiveto the COPIcis-Golgi coat assembly. membrane COPI independently can be initially of Arf1 activation.recruited by There, the thep24 p24 complex, complex a andmajor COPI retrograde form a transient cargo of complex COPI vesicles, towith the thecis- ArfGEFGolgi membrane Gea1 through independently direct binding of to Arf1 COPI. activation. Gea1 is separately There, recruitedthe p24 complex by the Rab and GTPase COPI Ypt1 form to thea transientcis-Golgi complex withmembrane the ArfGEF lacking Gea1 anionic through lipids. direct The p24 binding complex to hasCOPI. been Gea1 also is proposed separately to facilitate recruited the by recruitment the Rab GTPase of the inactive Ypt1 to the cis-Golgiform membrane of Arf1 (Arf1-GDP). lacking anionic Arf1-GDP lipids. could The be alsop24 recruited complex by has Gea1 been as itsalso substrate. proposed The to local facilitate formation the of recruitment this transient of the inactivecomplex form ensuresof Arf1that (Arf1-GDP). Arf1 activation Arf1-GDP by Gea1 could occurs be in also the proximity recruited of by COPI, Gea1 which as its leads substrate. to the COPI The coat local assembly formation by the of this transientgenerated complex Arf1-GTP ensures and that the p24Arf1 complex. activation Arf1-GTP by Gea1 can occurs be deactivated in the byproximity the speciﬁc of COPI-associatedCOPI, which leads ArfGTPase to the Glo3 COPI to coat assemblyinitiate by athe new generated activation Arf1-GTP cycle by Gea1. and Thus,the p24 through complex. this feed-forwardArf1-GTP can mechanism, be deactivated the p24 by complex the specific could contributeCOPI-associated to ArfGTPaseestablish Glo3 COPI to initiate vesicle buddinga new activation sites by facilitating cycle by theGea1. local Thus, encounter through of COPI, this feed-forward Gea1 and Arf1. mechanism, the p24 complex could contribute to establish COPI vesicle budding sites by facilitating the local encounter of COPI, Gea1 and Arf1. Our study is consistent with a mechanism that could contribute to the spatial organi- zation of vesicle budding in the maturing Golgi [14,37]. Transmembrane retrograde cargos, Our study is consistent with a mechanism that could contribute to the spatial organ- such as the abundant p24 proteins, would contribute, through their ability to bind COPI, to izationthe formation of vesicle of budding Gea1/COPI in the complexes maturing on the Golgicis-Golgi [14,37]. membrane, Transmembrane which should retrograde activate cargos,Arf1 such for as speciﬁc the abundant COPI binding p24 proteins, and thus, woul to establishd contribute, COPI vesiclethrough budding their ability sites. Anto bind COPI,analogous to the mechanism formation should of Gea1/COPI operate for complexes Gea2, which on has the been cis- alsoGolgi shown membrane, to bind COPI which shouldand isactivate present Arf1 in later for Golgi specific compartments COPI bindin thang Gea1and thus, [16,38 to]. Byestablish using this COPI mechanism, vesicle budding the sites.COPI An coat analogous might be mechanism able to outcompete should with operate other Arf1for Gea2, coat effectors which forhas Arf1-GTP been also binding shown to bindat COPI the early and Golgi is present compartments in later where Golgi retrograde compartments cargos arethan mostly Gea1 present.[16,38]. Therefore,By using this mechanism,the collective the presence COPI coat of retrograde might be cargosable to could outcompete contribute with to spatially other Arf1 delimit coat the effectors Golgi for territory where Arf1 will form COPI retrograde transport vesicles. In the maturing Golgi, Arf1-GTP binding at the early Golgi compartments where retrograde cargos are mostly the progressive depletion of retrograde cargo from late Golgi compartments by COPI- present.dependent Therefore, recycling the could collective facilitate presence the Arf1 of switch retrograde to generate cargos clathrin/adaptor-coated could contribute to spa- tiallyvesicles delimit at the TGNGolgi for territory Golgi export where of Arf1 anterograde will form secretory COPI retrograde cargo. In thetransport absence vesicles. of In theretrograde maturing cargo, Golgi, the the recruitment progressive of the deplet Arf1GEFion of Sec7 retrograde to the TGN cargo to from activate late Arf1 Golgi for com- partmentsselective by clathrin COPI-dependent vesicle budding recycling involves could several facilitate regulatory the mechanismsArf1 switch includingto generate the clathrin/adaptor-coatedpresence of anionic vesicles lipids atat the the membrane TGN for Golgi surface, export autoinhibition, of anterograde the positive secretory feedback cargo. In the loopabsence between of retrograde Arf1 and Sec7, cargo, the Rabthe GTPaserecruitmen Ypt1t andof the the Arf1GEF Arf-like GTPase Sec7 to Arl1 the [ 14TGN,39,40 to]. acti- vateInterestingly, Arf1 for selective the anterograde clathrin cargo vesicle has beenbudding also implicated involves inseveral the establishment regulatory ofmechanisms vesicle including the presence of anionic lipids at the membrane surface, autoinhibition, the positive feedback loop between Arf1 and Sec7, the Rab GTPase Ypt1 and the Arf-like GTPase Arl1 [14,39,40]. Interestingly, the anterograde cargo has been also implicated in the establishment of vesicle budding sites at the TGN [41,42]. Therefore, in addition to the extensive

Int. J. Mol. Sci. 2021, 22, 423 10 of 13

budding sites at the TGN [41,42]. Therefore, in addition to the extensive crosstalk and feedback between different Arf and Rab GTPases, their GEF and GAP regulatory proteins, effectors and speciﬁc membrane lipids [43,44], cargo proteins could also contribute to the directionality of Golgi trafﬁcking. Further work, also including in vitro reconstitution and live cell microscopy techniques, will be necessary to determine the collective role of cargo in the complex regulatory circuitry that controls Golgi maturation.

4. Materials and Methods 4.1. Media and Growth Conditions Yeast cultures were grown at 24 ◦C in rich YP medium (1% yeast extract, 2% peptone) supplemented with 0.2% adenine and containing 2% glucose (YPD) as carbon source or in synthetic minimal medium (0.15% yeast nitrogen base, 0.5% ammonium sulfate) supplemented with the appropriate amino acids and bases as nutritional requirements, and containing 2% glucose (SD) as carbon source.

4.2. Yeast Strains and Plasmids Strains of Saccharomyces cerevisiae and plasmids used for this work are listed in Tables1 and2 respectively.

Table 1. Saccharomyces cerevisiae strains.

Strain Genotype Source MMY1156 MATa SEC21-GFP::HIS3 ura3 leu2 his3 lys2 ade2 trp1 This study BY4742 MATα ura3 leu2 his3 lys2 Euroscarf Y06829 MATa gea1∆::kanMx ura3 leu2 his3 met15 Euroscarf MMY835 MATa gea1∆::kanMx emp24∆::HmBx ura3 leu2 his3 met15 This study MMY1157 MATa SEC21-GFP::HIS3 emp24∆::kanMx ura3 leu2 his3 lys2 trp1 This study RSY1818 MATα ura3 leu2 his3 lys2 ade2 A. Spang MMY914 MATα emp24∆::kanMx leu2.3,112 ura3-52 his3∆200 This study APY022 MATα gea2∆::HIS3 gea1-6 leu2 his3 lys2 ade2 ura3 A. Spang MMY816 MATα gea1-6 gea2∆::HIS3 emp24∆::HmBx ura3 leu2 his3 ade2 lys2 This study MMY1320 MATa sec7-4 trp1 ura3 leu2 his3 ade2 This study MMY1321 MATα emp24∆::kanMx sec7-4 ura3 leu2 his3 ade2 This study INV-ARFGFP MATa ARF1-GFP::HIS3 ura3∆0 leu2∆0 his3∆1 met15∆0 Invitrogen MMY562 MATα emp24∆::KanMx4 ARF1-GFP::HIS3 ura3 leu2 his3 This study MMY1668 MATa erg6∆::hph SEC21-GFP::HIS3 This study

Table 2. Plasmids.

Number Yeast Marker Yeast Replication Plasmid Source pRC4 URA3 2µ 5xHA-GEA1 C.L. Jackson pRC4-GEA1-∆COPI URA3 2µ pRC4-5xHA-GEA1-∆COPI This study RH3127 URA3 CEN EMP24-CFP (YCPlac33) G. Castillón pRS415 LEU2 CEN SEC7-dsRED A. Spang pYD111 URA3 2µ YEp352-5xHA GEA1 (5-611) C.L. Jackson

4.3. Plasmid Construction Plasmid pRC4-GEA1-∆COPI contains a GEA1 allele expressing a truncated Gea1 protein lacking amino acids 434 to 521. To generate plasmid pRC4-GEA1-∆COPI, DNA fragments up and downstream of nucleotides 1302 and 1563 of the GEA1 ORF were ampli- ﬁed from genomic DNA using overlapping nucleotides. The PCR products were mixed and ampliﬁed using external oligonucleotides, digested with XbaI/NheI and inserted into XbaI/NheI site of pRC4, thereby replacing a 1777 kb fragment with a 1516 bp fragment. Int. J. Mol. Sci. 2021, 22, 423 11 of 13

4.4. Differential Fractionation Differential fractionation was performed as described by Chen et al. [45] with some modiﬁcations. A total of 100 mL of cells was harvested at 5 × 106 cell/mL, washed with 10 mM sodium azide, spheroplasted, lysed, layered on a 1 M sorbitol cushion (1.7 M sorbitol and 50 mM potassium phosphate [pH 7.5]), and centrifuged at 6500× g for 3 min. The pellet was lysed in 1 mL of lysis buffer (20 mM HEPES (pH 7.4) with protease inhibitors) and centrifuged at 2500× g for 2 min in a microfuge. The supernatant was removed and spun at 13,000× g for 30 min. The resultant low-speed supernatant (S13 enriched-Golgi fraction) was further separated into high-speed supernatant (S100) and pellet (P100) fractions by centrifugation at 100,000× g. The S100 supernatant was saved, and the P100 pellet was resuspended in lysis buffer.

4.5. Native Coimmunoprecipitation Native coimmunoprecipitation experiments were performed on enriched-Golgi fractions as follows. 100 OD600 units of yeast cells were washed twice with HEPES buffer (20 mM HEPES (pH 7.2), 100 mM KCl, 5 mM MgCl2, 1 mM phenylmethylsulfonylfluoride, and protease inhibitor cocktail; Roche Diagnostics) and disrupted with glass beads, after which cell debris and glass beads were removed by centrifugation. The supernatant was then centrifuged at 13,000× g for 15 min at 4 ◦C. The resultant supernatant was diluted with HEPES buffer, and Triton X-100 was added to a final concentration of 1%. The suspension was incubated for 60 min at 4 ◦C with rotation, after which insoluble components were removed by centrifugation at 13,000× g for 60 min at 4 ◦C. For immunoprecipitation of GFP-tagged proteins, the sample was first preincubated with Bab agarose beads (Chro- moTek) at 4 ◦C for 1 h and subsequently incubated with GFP-Trap_A (ChromoTek, Planegg, Germany) at 4 ◦C for 3 h. The immunoprecipitated beads were washed five times with HEPES buffer containing 0.1% Triton X-100, eluted with SDS sample buffer, resolved on SDS-PAGE, and analyzed by immunoblot.

4.6. BFA Treatment erg6∆ mutation was used to increase the plasma membrane permeability to BFA. For BFA treatment, erg6∆ cells were incubated at 24 ◦C in the presence of 100 µg/mL BFA for 20 min. This concentration of BFA was maintained during the subsequent native coimmunoprecipitation [16].

4.7. In Vitro Pull-Down Assay Synthetic peptides corresponding to the 10 C-terminal amino acids of Erv25 (KNYFK- TKHII) and Erv25-AA (KNAAKTKHII) with an N-terminal cysteine residue were generated (JPT Peptide Technologies) and linked to thiopropyl-Sepharose 6B (GE Healthcare) as described by Belden and Barlowe [19]. Cytosol from a strain expressing HA-Gea1 was obtained as described by Muñiz et al. [26]. In vitro binding reactions were performed as described by Belden and Barlowe [19].

Author Contributions: Conceptualization, J.M.-L. and M.M.; methodology and investigation, J.M.-L., A.M.P.-L., S.S.-B., A.A.-R., S.R.-G., S.L., A.C.-G. and R.E.W.; writing—original draft preparation, J.M.- L. and M.M.; writing—review and editing, S.S.-B., A.A-R., J.M.-L., A.M.P.-L., S.R.-G., S.L., A.C.-G., R.E.W. and M.M.; supervision, M.M.; project administration, M.M.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the FEDER/Ministerio de Ciencia, Innovación y Universi- dades—Agencia Estatal de Investigación/BFU2017-89700-P to Manuel Muñiz and to and BFU2015- 69183-P (MINECO/FEDER, UE) to Ralf Erik Wellinger, and by the “VI Own Research Plan” of the University of Seville VIPPIT-2020-I.5 to Manuel Muñiz. University of Seville fellowships to Soﬁa Rodriguez-Gallardo, Ana Maria Perez-Linero and Javier Manzano-Lopez. Ministry of Education, Cul- ture and Sport (MECD) fellowship to Susana Sabido-Bozo. ‘V Own Research Plan’ of the University of Seville (VPPI-US) contract (cofounded by the European Social Fund) to Sergio Lopez. Int. J. Mol. Sci. 2021, 22, 423 12 of 13

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments: We are grateful to Catherine L. Jackson and Anne Spang for material and advice. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations GEF Guanosine Exchange Factor GAP GTPase Activating Protein COPI Coat Protein Complex I ARF ADP-Ribosylation Factor BFA Brefeldin A

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