Dual Independent Roles of the p24 Complex in Selectivity of Secretory Cargo Export from the Endoplasmic Reticulum

cells Article Dual Independent Roles of the p24 Complex in Selectivity of Secretory Cargo Export from the Endoplasmic Reticulum Sergio Lopez 1,2 , Ana Maria Perez-Linero 1, Javier Manzano-Lopez 1, Susana Sabido-Bozo 1,2, Alejandro Cortes-Gomez 1,2, Sofia Rodriguez-Gallardo 1,2 , Auxiliadora Aguilera-Romero 1,2, Veit Goder 3and Manuel Muñiz 1,2,* 1Department of Cell Biology, University of Seville, 41012 Seville, Spain; [email protected] (S.L.); [email protected] (A.M.P.-L.); [email protected] (J.M.-L.); [email protected] (S.S.-B.); [email protected] (A.C.-G.); [email protected] (S.R.-G.); [email protected] (A.A.-R.) 2Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41012 Seville, Spain 3Department of Genetics, University of Seville, 41012 Seville, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-954556529 Received: 24 February 2020; Accepted: 20 May 2020; Published: 22 May 2020   Abstract: The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors. Here, we show biochemically that the p24 complex associates at the ER with other cargo receptors in a COPII-dependent manner, forming high-molecular weight multireceptor complexes. Furthermore, live cell imaging analysis reveals that the p24 complex is required to retain in the ER secretory cargos when their specific receptors are absent. This requirement does not involve neither the unfolded protein response nor the retrograde transport from the Golgi. Our results suggest that, in addition to its role as a cargo receptor in the specialized GPI-anchored protein pathway, the p24 complex also plays an independent role in secretory cargo selectivity during its exit through the general ER export pathway, preventing the non-selective bulk flow of native secretory cargos. This mechanism would ensure receptor-regulated cargo transport, providing an additional layer of regulation of secretory cargo selectivity during ER export. Keywords: endoplasmic reticulum; cargo receptor; p24 complex; secretory cargo; bulk flow 1. Introduction Nearly a third of all eukaryotic proteins are synthesized in the endoplasmic reticulum (ER) and subsequently transported by the secretory pathway through the Golgi complex to their functional destination, either the cell surface or the endo-lysosomal system [ 1 , 2 ]. Newly synthesized proteins are translocated into the ER where they become folded, assembled and post-translationally modified by glycosylation and disulfide bond formation. Once correctly matured, secretory proteins are then separated from incompletely folded proteins and ER residents to be incorporated as cargo into COPII-coated transport vesicles that transfer them forward to the Golgi apparatus. COPII vesicles are generated by the sequential assembly of the cytosolic COPII coat components, the small GTPase Sar1 and the two protein dimers Sec23/Sec24 and Sec13/Sec31, which locally bend the ER membrane at specific domains called ER exit sites (ERES) [ 3 ]. Secretory proteins can be selectively captured into Cells 2020,9, 1295; doi:10.3390/cells9051295 www.mdpi.com/journal/cells Cells 2020,9, 1295 2 of 17 nascent COPII vesicles by transmembrane receptors or adaptors that physically link each specific cargo with the COPII coat [ 2 , 4 ]. In contrast to this selective and active cargo capture, secretory proteins have been shown also to enter COPII vesicles by non-selective and passive bulk flow. However, to what extent ER export is driven by selective cargo capture versus non-selective bulk flow is still unknown [ 5 ]. The p24 complex is a heteromeric protein complex formed by members of the conserved p24 family protein, which are abundant type I transmembrane proteins that cycle between ER and Golgi apparatus thanks to cytosolic COPII and COPI coat-binding signals [ 6 – 11 ]. The p24 complex has been shown to play several functions during its bidirectional transport between the ER and Golgi at the early secretory pathway. First, the p24 complex facilitates the formation of COPI retrograde transport vesicles from the cis-Golgi [ 12 , 13 ]. Second, the p24 complex functions as a specialized cargo receptor by promoting the efficient ER export of a class of lipid-anchored cell surface proteins, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) [ 14 – 21 ]. Finally, although facilitating GPI-AP export, the p24 complex contributes to the ER retention of resident proteins and some misfolded non-GPI-APs [ 22 , 23 ]. The special structure and composition of the GPI anchor lead GPI-APs to be differentially trafficked along the secretory pathway [ 24 – 26 ]. In yeast, GPI-APs are segregated from other secretory proteins in the ER, and subsequently incorporated into distinct COPII vesicles [ 27 , 28 ]. This implies the existence of at least two different and parallel ER exit pathways, one of which exports GPI-APs and the other, the rest of the secretory proteins [ 24 , 25 ]. According to its role as a cargo receptor, the p24 complex travels together with its cargo, the GPI-APs, into the same specialized COPII vesicles. However, unexpectedly, the p24 complex was also found in the COPII vesicles that carry non-GPI-anchored cargos [ 16 ]. The fact that a subpopulation of the p24 complex exits the ER via the non-GPI-AP pathway may suggest additional functions to its role as a specific cargo receptor. To gain more insights into this issue, we have identified binding partners of the p24 complex member Emp24 by performing a pull-down assay from the ER membrane fraction. We found that the p24 complex interacts with several other cargo receptors that operate in the non-GPI-AP or general ER export pathway forming High-Molecular Weight multireceptor complexes at the level of the ER exit sites. Moreover, we show that the ER retention of non-GPI-anchored cargos caused by the absence of their specific receptors requires the presence of the p24 complex, suggesting its role in preventing the non-selective bulk flow of native secretory cargo in the general ER export pathway. 2. Materials and Methods 2.1. 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 the 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 the carbon source. 2.2. Yeast Strains and Plasmids Strains of Saccharomyces cerevisiae and plasmids used for this work are listed in Tables 1 and 2, respectively. Table 1. Saccharomyces cerevisiae strains. Strain Genotype Reference/Source BY4742 MATαhis3 leu2 lys2 ura3 Euroscarf Y15936 MATαerv29∆::KanMx his3 leu2 lys2 ura3 Euroscarf Y14567 MATαemp24∆::KanMx his3 leu2 lys2 ura3 Euroscarf Y14421 MATαerv14∆::KanMx his3 leu2 lys2 ura3 Euroscarf Y16420 MATαerv26∆::KanMx his3 leu2 lys2 ura3 Euroscarf MMY679 MATa emp24∆::KanMx erv26∆::KanMx his3 leu2 lys2 ura3 This study Cells 2020,9, 1295 3 of 17 Table 1. Cont. Strain Genotype Reference/Source MMY680 MATαemp24∆::KanMx erv29∆::KanMx his3 leu2 lys2 ura3 This study MMY1009 MATαemp24∆::hph erv14∆::KanMx ade2 his3 leu2 lys2 trp1 ura3 This study MMY1271 MATαERV14-mCi::HIS5Sp sec16-2 ura3 leu2 his4 lys2 trp1 This study MMY1464 MATαerv14∆::KanMx ret1-1 his4 leu2 ura3 This study MMY1673 MATαCPY-GFP::HISMx6 his3 leu2 lys2 ura3 This study MMY1675 MATαemp24∆::KanMx CPY-GFP::HISMx6 his3 leu2 lys2 met15 ura3 This study MMY1676 MATαerv29∆::KanMx CPY-GFP::HISMx6 his3 leu2 lys2 met15 ura3 This study MMY1679 MATαemp24∆::KanMx erv29∆::KanMx CPY-GFP::HISMx6 his3 leu2 lys2 ura3 This study RH3042 MATa ret1-1 his4 leu2 trp1 ura3 H. Riezman RH7016 MATαERV14-mCi::HIS5Sp his4 leu2 trp1 ura3 H. Riezman VGY448 MATa EMP24-TAP::HIS3 his3 leu2 met15 ura3 V. Goder Table 2. Plasmids. Plasmid Description Reference/Source pJML01 ori, CEN, ERV29-3xHA, LEU2 C. Barlowe pRH3083 ori, CEN, CWP2-Venus, URA3 H. Riezman pRS416 ori, CEN, GAS1-GFP, URA3 L. Popolo p3079 ori, CEN, MID2-Venus, LEU2 H. Riezman pRS316 ori, CEN, GNT1-GFP, URA3 K. Kurokawa p3055 ori, CEN, GAP1-ub-GFP, URA3 H. Riezman pDN390 ori, CEN, HAC1i, LEU2 A. Spang 2.3. Proteomic Identification of Binding Partners for Emp24-TAP Emp24-TAP interactors were proteomically identified from an enriched ER membrane fraction as described [ 19 ]. In brief, 15 g of yeast cells expressing Emp24-TAP were lysed by grinding in liquid nitrogen, after which, cell debris was removed by centrifugation. The supernatant was then centrifuged at 13,000 × gfor 15 min at 4 ◦ C. The enriched ER membrane pellet was resuspended in buffer containing 1% digitonin (AppliChem). The extract was incubated for 3 h with IgG-coupled magnetic beads (Dynal). After washing and elution, the eluted proteins were analyzed by SDS-PAGE, and different gel regions were excised and prepared for in-gel digestion with trypsin. Peptide samples were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) using a LCQ Deca XP Plus ion trap mass spectrometer (Thermo Electron), [ 29 , 30 ]. Proteins identified by at least two tryptic peptides or more were characterized using Swiss-Prot/TrEMBL/UniProt database (UniProt Consortium, 2019) [ 31 ] and YeastMine (http://www.yeastgenome.org). 2.4. Native Co-Immunoprecipitation Experiments of native co-immunoprecipitation of Erv14-mCi for subsequent immunoblotting analysis were performed on enriched ER fractions as described [ 15 ]. In brief, 100 optical density 600 (OD600) units of yeast cells expressing Erv14-mCi were washed twice with TNE buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 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 × gfor 15 min at 4 ◦ C. The enriched ER membrane pellet was resuspended in TNE buffer, and digitonin was added to a final concentration of 1%. The suspension was incubated for 1 h at 4 ◦ C with rotation, after which insoluble components were removed by centrifugation at 17,000 × gfor 60 min at 4 ◦ C. For immunoprecipitation of Erv14-mCi, the sample was first preincubated with bab agarose beads (ChromoTek) at 4 ◦C for 1 h and subsequently incubated with GFP-Trap_A (ChromoTek) at 4 ◦ C for 3 h. The immunoprecipitated beads were washed five times with TNE buffer containing 0.2% digitonin, eluted with SDS sample buffer, resolved on SDS-PAGE, and analyzed by immunoblot. Cells 2020,9, 1295 4 of 17 2.5. Separation of High-Molecular-Weight Protein Complexes by Density Gradient Centrifugation 100 OD600 units of log-phase cells were disrupted in TNE buffer with glass beads, after which, cell debris and glass beads were discarded by centrifugation. The resultant supernatant was centrifuged at 13,000 × gfor 15 min at 4 ◦ C. The pellet containing the enriched ER membrane fraction was solubilized in TNE buffer with 1% digitonin for 1 h at 4 ◦ C with rotation. The extract was cleared by centrifugation (17,000 × gfor 1 h at 4 ◦ C) and loaded on top of a 5%–30% sucrose step gradient in TNE buffer with 0.2% digitonin. The step size was 5% sucrose. Centrifugation was performed for 4 h at 50,000 rpm and 4 ◦ C in a TLS-55 rotor (Beckman Coulter). The gradient was fractionated from top to bottom, and fractions were subjected to native co-immunoprecipitation or processed for SDS-PAGE and immunoblotting. 2.6. Fluorescence Live Cell Microscopy For fluorescence live cell microscopy of Mid2-Venus, Gnt1-GFP, CPY-GFP and Gap1-ub-GFP, log-phase cells grown in minimal media were observed directly. For fluorescence live cell microscopy of Cwp2-Venus and Gas1-GFP, log-phase cells were also grown in minimal media and incubated for 2 h in rich media (YPD). Then, these cells were collected by centrifugation, washed twice with PBS, and incubated at least 15 min on ice before being examined under the microscope as described previously [ 32 ]. Images were captured using a Leica DMi8 microscope equipped with an objective lens (HCX PL APO 1003/1.40OIL PH3 CS), L5 (GFP) filter, a Hamamatsu camera, and Application Suite X (LAS X) software, as described previously [15]. 2.7. Staining of Bud Scars with Calcofluor White Log-phase yeast cells grown in minimal media were collected by centrifugation, resuspended in calcofluor white stain at a concentration of 0.1 mg/mL [ 14 ] and incubated for 10 min at room temperature. After calcofluor white treatment, cells were washed three times in distilled water and observed directly under a Leica DMi8 fluorescence microscope. Haploid cells having six or more bud scars were examined for an axial or nonaxial budding pattern. 2.8. Statistical Analysis All values in the figures are expressed as the mean ± SD. Data were evaluated with GraphPad Prism Version 5.01 software. Statistical significances were assessed using the analysis of the variance (ANOVA) with Tukey’s post hoc test for multiple comparisons. The level of significance was set at 0.05. 3. Results 3.1. The p24 Complex Associates with Other ER Cargo Receptors into High Molecular Complexes in a COPII-Dependent Manner The yeast p24 protein complex acts as a specific cargo receptor by promoting the efficient transport of GPI-APs from the ER to the Golgi through their specialized COPII-dependent pathway [ 25 ]. However, the p24 complex was also found in the COPII vesicles that export from the ER the rest of secretory proteins together with their respective cargo receptors [ 16 ]. This dual routing from the ER to the Golgi suggests a possible additional role for the p24 complex in the general ER export pathway. To address this possibility, we first identified ER binding partners of the p24 complex member Emp24 by performing a native co-immunoprecipitation assay. Emp24 was chromosomally tagged with the TAP tag and affinity purified with IgG-coupled magnetic beads from detergent-solubilized ER membrane fractions. Co-precipitated proteins were next identified by tandem mass spectrometry (Figure 1and Figure S1, Tables S1 and S2). As a specificity control, we used a wild-type strain without tagged protein. Validity of the pull-down assay was confirmed by the presence of the other p24 complex members Erv25, Erp1 and Erp2 among the binding partners of Emp24-TAP [ 6 , 33 ]. Interestingly, we also found in the eluate other cargo receptors that operate in the non-GPI-AP or general ER export pathway, including Erv14, Erv29, Svp26/Erv26 or Emp47. These cargo receptors are largely heterogeneous in Cells 2020,9, 1295 5 of 17 terms of structure and cargo recognition motifs. Erv14 mediates the ER export of plasma membrane proteins with long transmembrane domains, Erv29 sorts soluble secretory proteins into COPII vesicles, Erv26 promotes the efficient ER exit of Golgi mannosyltransferases and Emp47 is required for the efficient ER export of some glycoproteins [ 2 , 5 , 34 – 37 ]. In addition, Erv41 and Erv46 were also found in the eluate as p24 interactors. They form a protein complex that has been reported to function as a cargo receptor in both anterograde and retrograde transport pathways [ 38 , 39 ]. To confirm the proteomic results, yeast cells expressing the cargo receptor Erv14 tagged with mCitrine (mCi) were analyzed by native co-immunoprecipitation followed by conventional immunoblotting. As seen in Figure 1B, immunoprecipitation in detergent-solubilized ER membrane fractions revealed that the Erv14-mCi co-precipitated with Emp24, Erv29-HA and Emp47. These physical interactions were specific for cargo receptors because the unrelated translocon subunit Sec61 was not recovered after Erv14-mCi immunoprecipitation. Furthermore, Erv14-mCi also co-precipitated with the core p24 complex member Erv25, indicating that the p24 proteins bind other cargo receptors as a complex (Figure S2). Cells 2020, 9, x FOR PEER REVIEW 5 of 17 general ER export pathway, including Erv14, Erv29, Svp26/Erv26 or Emp47. These cargo receptors are largely heterogeneous in terms of structure and cargo recognition motifs. Erv14 mediates the ER export of plasma membrane proteins with long transmembrane domains, Erv29 sorts soluble secretory proteins into COPII vesicles, Erv26 promotes the efficient ER exit of Golgi mannosyltransferases and Emp47 is required for the efficient ER export of some glycoproteins [2,5,34–37]. In addition, Erv41 and Erv46 were also found in the eluate as p24 interactors. They form a protein complex that has been reported to function as a cargo receptor in both anterograde and retrograde transport pathways [38,39]. To confirm the proteomic results, yeast cells expressing the cargo receptor Erv14 tagged with mCitrine (mCi) were analyzed by native co-immunoprecipitation followed by conventional immunoblotting. As seen in Figure 1B, immunoprecipitation in detergentsolubilized ER membrane fractions revealed that the Erv14-mCi co-precipitated with Emp24, Erv29- HA and Emp47. These physical interactions were specific for cargo receptors because the unrelated translocon subunit Sec61 was not recovered after Erv14-mCi immunoprecipitation. Furthermore, Erv14-mCi also co-precipitated with the core p24 complex member Erv25, indicating that the p24 proteins bind other cargo receptors as a complex (Figure S2). Figure 1. The p24 complex interacts with other cargo receptors at the ER membrane. (A) Coomassie staining of the p24 protein Emp24-TAP interactors. Cargo receptors (red) and p24 proteins (blue) that interact with Emp24-TAP (blue) are indicated. Enriched ER membrane fractions of wild-type yeast cells or cells expressing Emp24-TAP were isolated and solubilized with 1% digitonin. The extract was incubated with IgG-coupled magnetic beads and co-precipitated proteins were prepared and resolved by SDS-PAGE. Visualized bands and regions from the control reaction were subjected to ingel digestion with trypsin and were analyzed by LC-MS/MS techniques (Figure S1, Tables S1 and S2). The star indicates the Emp24-TAP. (B) Co-immunoprecipitation assay between Erv14-mCi and other cargo receptors. Enriched ER membrane fractions of wild-type cells expressing Erv29-HA with or without expressing Erv14-mCi were solubilized, and immunoprecipitated (IP) with anti-GFP antibody, followed by immunoblotting with anti-Emp24, anti-HA, anti-Emp47, anti-Sec61 and anti- GFP antibodies. T represents a 0.5% of the solubilized input material. Next, we determined whether these cargo receptors that interact with each other are present in larger protein complexes. For this purpose, ER membrane fractions from yeast cells expressing Erv14- mCi and Erv29-HA were solubilized with 1% digitonin and subjected to sucrose density gradient centrifugation. The resultant gradient fractions were analyzed by SDS-PAGE and immunoblotting with specific antibodies against mCi, HA, Emp24 and Emp47. As observed in Figure 2A, Emp24, Erv14-mCi, Erv29-HA and Emp47 cargo receptors were found in high density fractions. Their sedimentation patterns showed certain heterogeneity, suggesting the formation of heterogeneous receptor complexes of distinct composition and size (higher than 450 kDa). Nevertheless, all four of the receptors peaked around fraction 10. To verify that they were indeed associated with high molecular weight complexes, Erv14-mCi immunoprecipitation in detergent-solubilized fractions 9 and 10 was performed. The subsequent immunoblot analysis of the eluate revealed that Emp24, Erv29 Figure 1. The p24 complex interacts with other cargo receptors at the ER membrane. ( A ) Coomassie staining of the p24 protein Emp24-TAP interactors. Cargo receptors (red) and p24 proteins (blue) that interact with Emp24-TAP (blue) are indicated. Enriched ER membrane fractions of wild-type yeast cells or cells expressing Emp24-TAP were isolated and solubilized with 1% digitonin. The extract was incubated with IgG-coupled magnetic beads and co-precipitated proteins were prepared and resolved by SDS-PAGE. Visualized bands and regions from the control reaction were subjected to in-gel digestion with trypsin and were analyzed by LC-MS/MS techniques (Figure S1, Tables S1 and S2). The star indicates the Emp24-TAP. ( B ) Co-immunoprecipitation assay between Erv14-mCi and other cargo receptors. Enriched ER membrane fractions of wild-type cells expressing Erv29-HA with or without expressing Erv14-mCi were solubilized, and immunoprecipitated (IP) with anti-GFP antibody, followed by immunoblotting with anti-Emp24, anti-HA, anti-Emp47, anti-Sec61 and anti-GFP antibodies. T represents a 0.5% of the solubilized input material. Next, we determined whether these cargo receptors that interact with each other are present in larger protein complexes. For this purpose, ER membrane fractions from yeast cells expressing Erv14-mCi and Erv29-HA were solubilized with 1% digitonin and subjected to sucrose density gradient centrifugation. The resultant gradient fractions were analyzed by SDS-PAGE and immunoblotting with specific antibodies against mCi, HA, Emp24 and Emp47. As observed in Figure 2A, Emp24, Erv14-mCi, Erv29-HA and Emp47 cargo receptors were found in high density fractions. Their sedimentation patterns showed certain heterogeneity, suggesting the formation of heterogeneous receptor complexes of distinct composition and size (higher than 450 kDa). Nevertheless, all four of the receptors peaked around fraction 10. To verify that they were indeed associated with high molecular weight complexes, Erv14-mCi immunoprecipitation in detergent-solubilized fractions 9 and 10 was performed. The subsequent immunoblot analysis of the eluate revealed that Emp24, Erv29 and Emp47 specifically Cells 2020,9, 1295 6 of 17 co-precipitated with Erv14-mCi, although Emp24 was less efficiently recovered (Figure 2B). This result may indicate that only a subpopulation of the p24 complex travels together with the other cargo receptors through the general ER export pathway. Cargo receptors work by linking their cargos with the COPII coat and they exit the ER as major components of COPII vesicles [ 5 ]. To determine whether the formation of the multireceptor complexes requires the COPII machinery, we analyzed the cargo receptor interactions in vivo by native co-immunoprecipitation in the COPII thermosensitive mutant strain sec16-2. Sec16 is a COPII accessory protein peripherally associated with the ER membrane that regulates the GTP cycle of the COPII coat for vesicle biogenesis at ER exit sites [ 40 – 43 ]. As shown in Figure 2C, in sec16-2 mutant cells, the co-immunoprecipitation of Erv14-mCi with Emp24 and Emp47 is greatly reduced at restrictive temperature (37 ◦ C). This result suggests that the multireceptor complexes are assembled at the level of ER exit sites during the COPII vesicle formation. Cells 2020, 9, x FOR PEER REVIEW 6 of 17 and Emp47 specifically co-precipitated with Erv14-mCi, although Emp24 was less efficiently recovered (Figure 2B). This result may indicate that only a subpopulation of the p24 complex travels together with the other cargo receptors through the general ER export pathway. Cargo receptors work by linking their cargos with the COPII coat and they exit the ER as major components of COPII vesicles [5]. To determine whether the formation of the multireceptor complexes requires the COPII machinery, we analyzed the cargo receptor interactions in vivo by native co-immunoprecipitation in the COPII thermosensitive mutant strain sec16-2. Sec16 is a COPII accessory protein peripherally associated with the ER membrane that regulates the GTP cycle of the COPII coat for vesicle biogenesis at ER exit sites [40–43]. As shown in Figure 2C, in sec16-2 mutant cells, the co-immunoprecipitation of Erv14-mCi with Emp24 and Emp47 is greatly reduced at restrictive temperature (37 °C). This result suggests that the multireceptor complexes are assembled at the level of ER exit sites during the COPII vesicle formation. Figure 2. ER cargo receptors associate into high molecular complexes in a COPII-dependent manner. (A) Isolation of high weight molecular complexes. Enriched ER membrane fractions of wild-type cells expressing Erv14-mCi and Erv29-HA were solubilized with 1% digitonin and were fractionated by 5%–30% sucrose density gradient centrifugation. Fractions were collected from each gradient and directly analyzed by SDS-PAGE followed by immunoblotting with different antibodies. The asterisk represents a non-specific protein crossreacting with the anti-Emp47 antibody. (B) Fractions 9 and 10 were mixed and immunoprecipitated (IP) with anti-GFP antibody, followed by immunoblotting with anti-Emp24, anti-HA, anti-Emp47, anti-Sec61 and anti-GFP antibodies. The co-IP efficiency for Emp24, Erv29 and Emp47 was 0.8%, 4.1% and 2.5% respectively. T represents a 0.5% of the solubilized input material. (C) Physical interactions among ER cargo receptors depends on the COPII regulator Sec16. Enriched ER membrane fractions of wild-type and sec16-2 mutant cells expressing Erv14-mCi, which were previously incubated at 37 °C, were solubilized, and immunoprecipitated (IP) with anti- GFP antibody, followed by immunoblotting with anti-Emp24 and anti-Emp47. T represents a 0.5% of the solubilized input material. Figure 2. ER cargo receptors associate into high molecular complexes in a COPII-dependent manner. ( A ) Isolation of high weight molecular complexes. Enriched ER membrane fractions of wild-type cells expressing Erv14-mCi and Erv29-HA were solubilized with 1% digitonin and were fractionated by 5%–30% sucrose density gradient centrifugation. Fractions were collected from each gradient and directly analyzed by SDS-PAGE followed by immunoblotting with different antibodies. The asterisk represents a non-specific protein crossreacting with the anti-Emp47 antibody. ( B ) Fractions 9 and 10 were mixed and immunoprecipitated (IP) with anti-GFP antibody, followed by immunoblotting with anti-Emp24, anti-HA, anti-Emp47, anti-Sec61 and anti-GFP antibodies. The co-IP efficiency for Emp24, Erv29 and Emp47 was 0.8%, 4.1% and 2.5% respectively. T represents a 0.5% of the solubilized input material. ( C ) Physical interactions among ER cargo receptors depends on the COPII regulator Sec16. Enriched ER membrane fractions of wild-type and sec16-2 mutant cells expressing Erv14-mCi, which were previously incubated at 37 ◦ C, were solubilized, and immunoprecipitated (IP) with anti-GFP antibody, followed by immunoblotting with anti-Emp24 and anti-Emp47. T represents a 0.5% of the solubilized input material. Cells 2020,9, 1295 7 of 17 3.2. The p24 Complex Prevents the Non-Selective ER Exit of Secretory Cargos in the Absence of Their Specific Receptors Emp24 is always assembled with the other p24 proteins into the p24 complex. Thus, the fact that Emp24 associates with other cargo receptors in large multiprotein complexes opens the possibility that the p24 complex plays in the general ER export pathway a different role as the specific cargo receptor role played in the GPI-AP pathway. Because previous studies have shown that the p24 complex participates in the ER retention of resident proteins such as chaperones and some misfolded non-GPI-APs [ 22 , 23 ], we tested by live fluorescence microscopy whether the p24 complex contributes to prevent the bulk flow or non-selective exit of secretory cargos from the ER. For this purpose, we used the deletion of EMP24 that destabilizes the other proteins of the p24 complex, leading to a complete loss of p24 complex function [ 6 ]. Figure 3A addresses the effect of the EMP24 deletion in the ER retention of diverse secretory cargos by the absence of their specific receptors. Mid2-GFP, a plasma membrane-localized protein, was accumulated in the ER in the absence of its cargo receptor Erv14, displaying the ER-characteristic nuclear ring staining [ 34 ]. In contrast, when Mid2-GFP was expressed in emp24 ∆ erv14 ∆ double-mutant cells, no ER staining was observed and Mid2-GFP was mainly localized at the cell surface. The same release effect was observed for other secretory cargos. The N-acetylglucosaminyltransferase Gnt1-GFP localizes at the Golgi apparatus, displaying the typical Golgi-dotted pattern for yeast cells [ 44 ]. As expected, Gnt1-GFP was partially retained at the ER when its cargo receptor, Erv26, was deleted. However, in the emp24 ∆ erv26 ∆ double-mutant cells, Gnt1-GFP was mostly detected in the Golgi. Additionally, the vacuolar carboxypeptidase Y enzyme CPY-GFP, was partially accumulated in the ER in the absence of its cargo receptor Erv29, but this ER retention was also reduced in the emp24∆erv29∆double-mutant cells. Cells 2020, 9, x FOR PEER REVIEW 7 of 17 3.2. The p24 Complex Prevents the Non-Selective ER Exit of Secretory Cargos in the Absence of Their Specific Receptors Emp24 is always assembled with the other p24 proteins into the p24 complex. Thus, the fact that Emp24 associates with other cargo receptors in large multiprotein complexes opens the possibility that the p24 complex plays in the general ER export pathway a different role as the specific cargo receptor role played in the GPI-AP pathway. Because previous studies have shown that the p24 complex participates in the ER retention of resident proteins such as chaperones and some misfolded non-GPI-APs [22,23], we tested by live fluorescence microscopy whether the p24 complex contributes to prevent the bulk flow or non-selective exit of secretory cargos from the ER. For this purpose, we used the deletion of EMP24 that destabilizes the other proteins of the p24 complex, leading to a complete loss of p24 complex function [6]. Figure 3A addresses the effect of the EMP24 deletion in the ER retention of diverse secretory cargos by the absence of their specific receptors. Mid2-GFP, a plasma membrane-localized protein, was accumulated in the ER in the absence of its cargo receptor Erv14, displaying the ER-characteristic nuclear ring staining [34]. In contrast, when Mid2-GFP was expressed in emp24Δ erv14Δ double-mutant cells, no ER staining was observed and Mid2-GFP was mainly localized at the cell surface. The same release effect was observed for other secretory cargos. The N-acetylglucosaminyltransferase Gnt1-GFP localizes at the Golgi apparatus, displaying the typical Golgi-dotted pattern for yeast cells [44]. As expected, Gnt1-GFP was partially retained at the ER when its cargo receptor, Erv26, was deleted. However, in the emp24Δ erv26Δ double-mutant cells, Gnt1-GFP was mostly detected in the Golgi. Additionally, the vacuolar carboxypeptidase Y enzyme CPY-GFP, was partially accumulated in the ER in the absence of its cargo receptor Erv29, but this ER retention was also reduced in the emp24Δ erv29Δ double-mutant cells. Figure 3. The p24 complex is required for the ER retention of different secretory cargos in the absence of their receptors. (A–C) Fluorescent live images of wild-type and different deletion strains expressing the secretory cargos Mid2-GFP (A), Gnt1-GFP (B) or CPY-GFP (C). The white arrow points out the perinuclear ER. Scale bar represents 5 μm. (D–F) Quantification of several micrographs described in Figure 3. The p24 complex is required for the ER retention of different secretory cargos in the absence of their receptors. ( A – C ) Fluorescent live images of wild-type and different deletion strains expressing the secretory cargos Mid2-GFP ( A ), Gnt1-GFP ( B ) or CPY-GFP ( C ). The white arrow points out the perinuclear ER. Scale bar represents 5 µ m. ( D – F ) Quantification of several micrographs described in ( A , B ) or ( C ). The graphs plot the average percentage of cells displaying perinuclear ER pattern. n, number of cells plotted ≥ 100. Different letters denote significant differences between strains (one-way ANOVA test, p≤0.05). Cells 2020,9, 1295 8 of 17 Thus, these results indicate that ER retention of a specific cargo in the absence of its selective receptor is compromised by the p24 loss of function. However, this lack of ER retention was not observed for their own specific cargos of the p24 complex, the GPI-APs. As shown in Figure 4, two different GPI-APs, Gas1-GFP and Cwp2-Venus, localize at the cell surface in wild-type and erv14 ∆ , erv26 ∆ ,erv29 ∆ receptor mutant cells while they are detected in the ER in the emp24 ∆ and emp24 ∆ erv14 ∆ ,emp24 ∆ erv26 ∆ or emp24 ∆ erv29 ∆ mutant cells. Therefore, all together, these results strongly suggest that one of the p24 complex functions during the ER export is to prevent the non-selective cargo export from the ER and that this function is independent of its role as a cargo receptor for GPI-APs. Cells 2020, 9, x FOR PEER REVIEW 8 of 17 (A,B) or (C). The graphs plot the average percentage of cells displaying perinuclear ER pattern. n, number of cells plotted ≥ 100. Different letters denote significant differences between strains (one-way ANOVA test, p ≤ 0.05). Thus, these results indicate that ER retention of a specific cargo in the absence of its selective receptor is compromised by the p24 loss of function. However, this lack of ER retention was not observed for their own specific cargos of the p24 complex, the GPI-APs. As shown in Figure 4, two different GPI-APs, Gas1-GFP and Cwp2-Venus, localize at the cell surface in wild-type and erv14Δ, erv26Δ, erv29Δ receptor mutant cells while they are detected in the ER in the emp24Δ and emp24Δ erv14Δ, emp24Δ erv26Δ or emp24Δ erv29Δ mutant cells. Therefore, all together, these results strongly suggest that one of the p24 complex functions during the ER export is to prevent the non-selective cargo export from the ER and that this function is independent of its role as a cargo receptor for GPI- APs. Figure 4. The p24 complex is still required for the ER export of GPI-APs in the absence of other cargo receptors. (A) Fluorescent live images of wild-type and different deletion strains expressing Gas1- GFP or Cwp2-Venus. The white arrow points out the perinuclear ER. Scale bar represents 5 μm. (B,C) Quantification of several micrographs described in (A). The graphs plot the average percentage of cells displaying perinuclear ER pattern. n ≥ 100. Different letters denote significant differences between strains (one-way ANOVA test, p ≤ 0.05). To get more insight into the cargo-receptor independent role of the p24 complex in selectively and fidelity of cargo export from the ER, we tested whether those secretory cargos retained at the ER by the p24 complex in the absence of their respective receptors can reach a functional native status. For this reason, we analyzed the functionality of a specific cargo of the Erv14 receptor, the plasma membrane protein Axl2, in the emp24Δ erv14Δ double mutant strain. Haploid yeast requires Axl2 to bud in an axial budding pattern, where the new bud site occurs adjacent to a structure called the bud scar, leftover from the previous cell division [45]. Therefore, the axial budding pattern results in a continuous chain of bud scars on older mother cells that can be visualized by staining with the fluorescent dye calcofluor (Figure 5). In contrast to wild-type cells, the erv14Δ mutant cells display a non-axial pattern, since Axl2 is retained at the ER and thus, cannot reach the plasma membrane [46]. However, the axial pattern is substantially recovered in emp24Δ erv14Δ double mutant cells, indicating that this secretory cargo retained in the ER by the p24 complex is mostly functional. Figure 4. The p24 complex is still required for the ER export of GPI-APs in the absence of other cargo receptors. ( A ) Fluorescent live images of wild-type and different deletion strains expressing Gas1-GFP or Cwp2-Venus. The white arrow points out the perinuclear ER. Scale bar represents 5 µ m. ( B , C ) Quantification of several micrographs described in ( A ). The graphs plot the average percentage of cells displaying perinuclear ER pattern. n ≥ 100. Different letters denote significant differences between strains (one-way ANOVA test, p≤0.05). To get more insight into the cargo-receptor independent role of the p24 complex in selectively and fidelity of cargo export from the ER, we tested whether those secretory cargos retained at the ER by the p24 complex in the absence of their respective receptors can reach a functional native status. For this reason, we analyzed the functionality of a specific cargo of the Erv14 receptor, the plasma membrane protein Axl2, in the emp24 ∆ erv14 ∆ double mutant strain. Haploid yeast requires Axl2 to bud in an axial budding pattern, where the new bud site occurs adjacent to a structure called the bud scar, leftover from the previous cell division [ 45 ]. Therefore, the axial budding pattern results in a continuous chain of bud scars on older mother cells that can be visualized by staining with the fluorescent dye calcofluor (Figure 5). In contrast to wild-type cells, the erv14 ∆ mutant cells display a non-axial pattern, since Axl2 is retained at the ER and thus, cannot reach the plasma membrane [ 46 ]. However, the axial pattern is substantially recovered in emp24 ∆ erv14 ∆ double mutant cells, indicating that this secretory cargo retained in the ER by the p24 complex is mostly functional. Cells 2020,9, 1295 9 of 17 Cells 2020, 9, x FOR PEER REVIEW 9 of 17 Figure 5. A secretory cargo retained at the ER by the p24 complex in the absence of its receptor is functional. (A) Budding patterns in wild-type (WT) and erv14 strains. Yeast cells were stained with calcofluor to visualize bud scars. Scale bar represents 5 μm. Wild-type and emp24 cells display the typical axial budding pattern whereas erv14 cells exhibit nonaxial budding patterns. However, the axial budding pattern is recovered in the emp24 erv14 double mutant cells. (B) Quantification of several micrographs described in (A). The graph plots the average percentage of cells displaying polarized scars. n ≥ 100. Different letters denote significant differences between strains (one-way ANOVA test, p ≤ 0.05). 3.3. Secretory Cargo ER Retention by the p24 Complex Involves Neither the UPR nor the Retrograde Transport from the ER Deletion of p24 genes in yeast has been previously shown to cause the ER escape of the resident chaperone Kar2 and its subsequent secretion to the extracellular media [23]. However, this phenotype has been attributed to the fact that the p24 deletion mutant strains have chronically activated the unfolded protein response (UPR), which increases Kar2 expression [47]. Therefore, it is possible that a chronic UPR triggered by EMP24 deletion leads also to the ER escape of secretory cargos in the absence of their receptors. To address this possibility, we transformed different strains with a plasmid expressing spliced HAC1 mRNA [48]. HAC1 is a transcriptional activator of UPR response genes whose functional expression is driven by non-conventional cytosolic splicing of its mRNA. Thus, expression of spliced HAC1 causes a constitutively active UPR. Under these conditions, we observed that GAP1-ub-GFP, a plasma membrane protein and specific cargo for Erv14 receptor [28], was still retained at the ER in the erv14Δ cells and localized at the plasma membrane in wild-type cells (Figure 6). These results show that the activated UPR does not cause the ER escape of secretory cargo, and therefore, the function of the p24 complex on ER retention is independent of UPR activation. This is in agreement with previous observations that ER leakage of soluble chaperones in the absence of the p24 function occurred independently of the UPR [22,49]. Figure 5. A secretory cargo retained at the ER by the p24 complex in the absence of its receptor is functional. ( A ) Budding patterns in wild-type (WT) and erv14 ∆ strains. Yeast cells were stained with calcofluor to visualize bud scars. Scale bar represents 5 µ m. Wild-type and emp24 ∆ cells display the typical axial budding pattern whereas erv14 ∆ cells exhibit nonaxial budding patterns. However, the axial budding pattern is recovered in the emp24 ∆ erv14 ∆ double mutant cells. ( B ) Quantification of several micrographs described in ( A ). The graph plots the average percentage of cells displaying polarized scars. n ≥ 100. Different letters denote significant differences between strains (one-way ANOVA test, p≤0.05). 3.3. Secretory Cargo ER Retention by the p24 Complex Involves Neither the UPR nor the Retrograde Transport from the ER Deletion of p24 genes in yeast has been previously shown to cause the ER escape of the resident chaperone Kar2 and its subsequent secretion to the extracellular media [ 23 ]. However, this phenotype has been attributed to the fact that the p24 deletion mutant strains have chronically activated the unfolded protein response (UPR), which increases Kar2 expression [ 47 ]. Therefore, it is possible that a chronic UPR triggered by EMP24 deletion leads also to the ER escape of secretory cargos in the absence of their receptors. To address this possibility, we transformed different strains with a plasmid expressing spliced HAC1 mRNA [ 48 ]. HAC1 is a transcriptional activator of UPR response genes whose functional expression is driven by non-conventional cytosolic splicing of its mRNA. Thus, expression of spliced HAC1 causes a constitutively active UPR. Under these conditions, we observed that GAP1-ub-GFP, a plasma membrane protein and specific cargo for Erv14 receptor [ 28 ], was still retained at the ER in the erv14 ∆ cells and localized at the plasma membrane in wild-type cells (Figure 6). 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