Release from endoplasmic reticulum matrix proteins controls cell surface transport of MHC class I molecules Susanne Fritzsche1, Esam Tolba Abualrous1, Britta Borchert1, Frank Momburg2, and Sebastian Springer1,* 1

Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany; Translational Immunology, German Cancer Research Center/NCT, Heidelberg, Germany

*

2

Department of

To whom correspondence should be addressed: Sebastian Springer Department of Life Sciences and Chemistry Jacobs University Bremen Campus Ring 1 D - 28759 Bremen, Germany E-mail: [email protected] Tel.: +49-421-200-3243 Fax: +49-421-200-4333

Keywords Secretory pathway, anterograde protein transport, MHC class I, Endoplasmic Reticulum (ER), protein folding, ER export Synopsis We show that the rate-limiting step of cell surface transport of the MHC class I peptide receptor H-2Db lies prior to ER exit and delays the access of H-2Db to ER exit sites by acting on the folded and peptide-bound lumenal part of the protein. Our data suggest that cell surface transport of class I and other type I transmembrane glycoproteins is governed by the affinity of all folding and maturation states to the proteins of the ER matrix.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/tra.12279

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Abstract The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi-step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES), and retrieval from the ERGolgi intermediate compartment (ERGIC) or the cisGolgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine MHC class I peptide b b receptor, H-2D and H-2K . We find that a novel pre-ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix. Introduction The intracellular transport of proteins in eukaryotic cells has been intensely investigated for decades. Proteins destined for the secretory pathway, such as cell surface proteins, are co-translationally inserted into the membrane or the lumen of the 1 endoplasmic reticulum (ER ), where molecular chaperones and folding enzymes provide an ideal environment for protein folding and assembly. The progress of protein folding is monitored by ER quality control mechanisms that release only correctly folded proteins into the secretory pathway [1]. Anterograde transport from the ER to the Golgi apparatus is mediated by coat protein complex II (COPII) vesicles that can select their cargo proteins [2]. These transport carriers originate from subdomains of the rough ER called the transitional ER (tER) or ER exit sites (ERES) [3]. In the Golgi, proteins may be retrieved from the cis side to the ER or sorted in the trans-Golgi network for transport to their destination, e.g., the plasma membrane [4]. The transport rate from the ER to the cell surface differs considerably between proteins, but how and where this is determined is barely known,

1

Abbreviations: COPII, coat protein complex II; Db, b H-2D ; EndoF1, endoglycosidase F1 (identical in specificity to EndoH); EndoH, endoglycosidase H; ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERES, ER exit sites; ERGIC, ERGolgi intermediate compartment; HC, heavy chain; b b K , H-2K ; MD, molecular dynamics; MHC, major histocompatibility complex; MEF, mouse ear fibroblasts; TAP, transporter associated with antigen processing; PLC, peptide loading complex; VSV-G, Vesicular stomatitis virus glycoprotein, WT, wild type.

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despite extensive efforts [5,6]. Events that may influence the rate of cell surface transport include protein folding and subunit association in the ER, access to ERES, recruitment into COPII vesicles, retrieval from the ER-Golgi intermediate compartment (ERGIC) as well as the cis-Golgi, and finally transport from the Golgi apparatus to the plasma membrane. Major Histocompatibility Complex (MHC) class I molecules are an ideal system for differential transport studies since the many naturally occurring allotypes – despite their similar protein sequences – differ significantly in their ER-to-Golgi transport rates [6–8]. Since three gene loci exist in mice and humans, up to six endogenously expressed allotypes can be investigated simultaneously in wild type cells, which may avoid artifacts due to protein misfolding, protein overexpression, or variation between cell lines. Most importantly, class I molecules allow the separation of the rates of protein folding and transport, since peptide binding and subunit assembly can be detected with conformation-dependent antibodies. Class I molecules present intracellular peptides to cytotoxic T lymphocytes at the plasma membrane, enabling detection of virus-infected or malignantly transformed cells by the adaptive immune system [9]. In the ER, the glycosylated class I heavy chain (HC) binds transiently to the lectin chaperone calnexin before it assembles with the smaller subunit beta-2 microglobulin (β2m). Peptide binding to the HC/β2m dimer is mediated by the so-called peptide loading complex (PLC), which consists of common chaperones (calreticulin, ERp57), the class I-specific chaperone tapasin, and the peptide transporter TAP (transporter associated with antigen processing), which moves cytosolic peptides into the ER lumen [10]. The members of the PLC act in concert to achieve peptide optimization (the binding of high-affinity peptides) to the HC/β2m dimer. Subsequently, the trimeric HC/β2m/peptide complex dissociates from the PLC and travels to the cell surface via the secretory pathway. Low-affinity peptide complexes may be retained in the ER or in a cycle between the ER and the cis-Golgi [4,11,12]. We have investigated the potential causes of the differential surface transport of the murine class I allotypes H-2Db (Db) and H-2Kb (Kb) with pulse-chase, thermostability, and in vitro COPII vesicle formation assays. We find that the major cause of the difference is a previously undescribed rate-limiting step in the ER that differentiates the lumenal parts, that occurs after assembly with β2m but prior to packaging of the proteins into COPII vesicles, and that is independent of peptide binding and at least partially independent of the PLC. Based on the data, we propose a model in which the differential affinity of all folding states of a membrane protein to the proteins of the ER matrix governs its transport rate to the cell surface.

b

Results Two murine MHC class I allotypes become EndoF1-resistant at different initial rates. The difference between the cell surface transport b b rates of D and K has been described previously. We first examined this phenomenon in RMA cells, where both allotypes are endogenously expressed (Figure 1). We performed pulse-chase experiments that measure the arrival of a newly synthesized glycoprotein in the medial Golgi, where the Nglycans are processed from endoglycosidase F1 (EndoF1)-sensitive to the EndoF1-resistant type (see Materials and Methods) [13]. To ensure that only properly folded class I molecules were observed, we used the conformation-dependent b b antibodies B22.249 (D ) and Y3 (K ), which bind only those forms of class I that are associated with β2m [14–16]. At 37 ºC, Kb acquired EndoF1 resistance much faster than Db (t1/2 = 8 and 30 minutes, respectively; Figure 1B, Rt). Exact quantification of the gels (using absolute band densities normalized to the start of the chase) [17] revealed that the rates of EndoF1 resistance acquisition differed most at the very beginning of b the chase, with D showing a distinct lag phase (Figure 1B; arrow), but were rather similar at times greater than 20 minutes, when EndoF1 resistance acquisition was fast for both allotypes. b

b

Differential access of D and K to ER exit sites and COPII transport vesicles is controlled by a rate-limiting maturation step in the ER. The differential EndoF1 resistance acquisition of Db and Kb may be caused by differential transport from ER to medial Golgi, as we originally assumed, or by differential processing of the glycans. The latter is b conceivable since D carries three glycans, which may be processed more slowly in the medial Golgi than the two glycans of Kb. We therefore developed a second, independent, assay (the COPII vesicle formation assay) to measure the rates of ER export of the two proteins through the efficiencies of "budding", i.e., of the packaging into COPII transport vesicles at the point of ER exit. In this assay, microsomal membranes are isolated from cells, and COPII vesicles are generated in an in vitro "budding reaction" [18]. We first performed in vitro COPII budding reactions with microsomal membranes from [35S]-methionine-labeled wild type fibroblast cells as membrane donors, cytosol as a source of COPII proteins, and recombinant Sar1A protein (the small GTPase that drives COPII coat formation). We controlled for the origin of generated vesicles by replacing Sar1A wild type (WT) with its dominant negative forms, Sar1A T39N or Sar1A H79G [19], which specifically inhibit COPII vesicle formation. Following the in vitro reaction, we isolated the vesicles by differential centrifugation, lysed them with detergent, and detected vesicle proteins. When the budding experiment was carried out following ten minutes of radioactive labeling, the apparent COPII budding efficiency for Kb (defined as the fraction of protein in the donor membranes that is packaged into COPII vesicles during the budding reaction) was significantly greater than that

of D (Figure 2A, C). This result demonstrates that b b K indeed exits the ER faster than D , and that differential glycan processing is not the cause of faster EndoF1 resistance acquisition of Kb in Figure 1B. b The slower ER exit of D may be due either to its inefficient selection by COPII coat proteins (i.e., a lower affinity of Db to COPII coat proteins or cargo receptors) [20,21] or, alternatively, to a maturation b step that delays D prior to the ERES and makes it unavailable for COPII packaging. To distinguish between these alternatives, we used the same experimental system. We reasoned that if an uncompleted pre-ERES step was limiting, then over b time, more D of a given labeled cohort should move into the ERES, and COPII budding efficiency b for D should increase. In contrast, a low affinity of b D to COPII proteins would result in a constant COPII budding efficiency over time. We therefore followed the ten-minute radioactive label with a thirty-minute chase and found that then, strikingly, b D was packaged into the COPII vesicles with a much higher apparent budding efficiency, similar to that of Kb (Figure 2B, C, and S1). Taken together, our data show that the - or at least, one - difference b b in the rate of cell surface transport of D and K is localized in the ER, prior to the packaging into COPII vesicles. The rate-limiting step is independent of peptide binding and optimization. We next sought to further characterize this ratelimiting step. The best-described process between the completion of folding (marked by the association with β2m) and ER export of class I is the binding of high-affinity peptide, an iterative process that occurs – for most class I molecules – with the help of the PLC. We first tested the hypothesis that Db suffers a delay in the progress to the ERES because at a b given time point after synthesis, complexes of D with peptide are less stable (resistant to dissociation) than those of Kb and that therefore, Db remains bound to the PLC or other chaperones for a longer time. To this end, we performed a thermostability experiment (Figure 3A, B). We radiolabeled cells, lysed them immediately (without a chase), incubated the lysates at different temperatures, and then immunoprecipitated Db and Kb molecules with B22.249 and Y3 to test whether their conformation remained native. In this experiment, the higher the temperature required for complete denaturation, the more thermostable the complex is considered to be [22,23]. Db and Kb showed strikingly similar thermostabilities. This suggests that the slower ER b export of D is not caused by a conformational instability. If the rate-limiting step is not connected with peptide binding, as this result suggests, then b b the export rates of D and K should be different even in the absence of high-affinity peptides. To test this prediction, we next performed experiments in RMA-S lymphoblastoma cells, which are syngeneic with RMA but have no functional TAP; thus, their class I molecules have no or few highaffinity peptides available [24] and are mostly empty or bound to low-affinity peptides, i.e., suboptimally loaded [25]. Despite ER and Golgi quality control,

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b

b

some of these suboptimally loaded D and K complexes proceed to the cell surface but then rapidly lose peptide and β2m, followed by endocytosis and lysosomal destruction [26,27]. In a pulse-chase experiment with these TAPdeficient cells, an inspection of the EndoF1-resistant band at the early time points of b the chase shows that K is still transported to the b medial Golgi significantly faster than D , which shows a distinct lag (Figure 3C, D). In a pulsechase done at 25 °C, where the dissociation of the b low-affinity peptide complexes is reduced [27], K b still acquired EndoF1 resistance faster than D (Figure S2A, B). These results show that the ratelimiting step is also present in the cell surface b b transport of suboptimally loaded D and K . In keeping with this result, a COPII vesicle formation assay done with microsomes from TAP-deficient fibroblasts [28] showed that suboptimally loaded Kb was more efficiently recruited into COPII vesicles b then D (Figure 3E, F). As a further control, we b confirmed in a thermostability experiment that K b and D were forming complexes of the same thermodynamic stability in the TAP-deficient cells (Figure S2C, D). Taken together, these data demonstrate that the differentially rate-limiting step for the ER b b exit of D and K is independent of the binding of high-affinity peptide.

The lumenal part of H-2Db confers prolonged residence in the ER. b b To find out which domains of D and K are responsible for the difference in their transport rates, we next generated a series of swap mutants. Keeping the lumenal parts intact, we changed the transmembrane domain and/or the cytosolic tail, b and we also introduced into K the third glycosylation site that is found in the α3 domain of Db (resulting in Kb Y256N, Figure 5A). We then stably expressed the swap mutants in MHCmismatched L cells and performed pulse-chase experiments (Figure S4). Strikingly, the reduced b EndoF1 acquisition observed earlier for D at ten minutes of chase (Figure 1B) was only seen with those constructs that contained the lumenal part of b D (Figure 5B and S4). Thus, the dominant ratelimiting step of ER export depends on a feature of the class I lumenal part. In the same system, we investigated the role of the different numbers of glycans. The b K Y256N mutant, which carries three glycans like b b D , traveled more slowly than K but was not b reduced to D rates (Figure 5B and S4). Thus, the greater number of glycans of Db is not the major cause of the delay of its ER exit.

Interaction with the PLC reduces the rates of b b EndoF1 resistance acquisition of D and K . During their maturation in the ER, class I molecules interact directly with two component proteins of the PLC, namely calreticulin and tapasin [29,30]. Since these two proteins are efficiently retained in the ER, we wondered whether a peptide-independent differential association with them determines the rate of EndoF1 acquisition of class I molecules. We hypothesized that if this was the case, lack of calreticulin or tapasin should make the cell surface b b transport rates of D and K more similar, and we performed pulse-chase experiments as in Figure 1, but with tapasin-deficient [31] and calreticulindeficient fibroblasts [29]. In both deficient cell lines, b b acquisition of EndoF1 resistance of D and K was dramatically accelerated (as observed before [29]), and the difference between Db and Kb was almost abolished (Figure 4A, B, E, F). Still, for both calreticulin- and tapasin-deficient cells, the COPII vesicle formation assay clearly demonstrated b preferential ER export of K (Figure 4C, D, G, H). In pulse-chase experiments performed at 25 °C, which increase the resolution of initial transport events because of their lower overall rate, the difference in b acquisition of EndoF1 resistance between D and b K became more visible (Figure S3A, B, C, D). Again, thermostability was equal for Db and Kb, demonstrating that differential peptide binding was not the reason for the difference (Figure S3E, F, G, H). In summary, these data confirm previous observations that interaction with calreticulin and tapasin critically influences the ER exit and cell surface transport of class I molecules, but they also show clearly that the difference between the ER b b export rates of D and K persists in the absence of

Discussion The class I allotype Kb travels to the medial Golgi faster than Db, as shown by faster acquisition of resistance to EndoF1 (Figure 1) and EndoH [28,32]. We have shown here that this difference is mostly b determined by their lumenal parts. Interestingly, D and Kb are packaged into COPII vesicles with the same efficiency (Figure 2B, C). The rate-limiting step thus lies in the ER itself; theoretically, it might be differential folding (including assembly with β2m), peptide binding, or interactions with the PLC and other chaperones. First: folding. The antibodies B22.249 and Y3 or 20-8-4s only bind if the class I HC is associated with β2m [14,15]. Both Db and Kb acquire these epitopes rapidly, after three to ten minutes of pulse b b (Figure 1A, 4A), and the folded forms of D and K have the same stability against thermal denaturation (Figure 3B, S2D, and S3F, H). This strongly suggests, but does not formally demonstrate, that the antibodies recognize b b equivalent conformations of D and K . We have previously shown that Kb folds faster than Db [17], and in Figure 1C, a plot of the amount of total folded protein (Rt+S) again shows this differential post-pulse folding. Slower acquisition of the conformational epitope may, in principle, contribute to the slower transport of Db but it cannot explain the characteristic lag phase observed for Db at the beginning of the chase (Figure 1B, Rt), where b radiolabeled D , though folded, is not exported. In a match with this conclusion, Degen and Williams have shown that in cell lines with identical Db and Kb folding rates, the difference in their cell surface transport persists [32]. At the early time points, the b b EndoF1-resistant populations of D and K are also not likely to be influenced by differential loss from

either protein. Thus, the rate-limiting step is at least partially dependent on the proteins of the PLC.

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the cell surface. Thus, differential transport is not primarily caused by differential folding, as monitored by epitope acquisition. Second: peptide binding. The b b thermostabilities of folded D and K at the start of the chase are equally high (Figure 3A, B), which suggests that both have reached a similar degree of peptide optimization. The difference in the initial transport rates is not only observed when Db and Kb are both bound to these equally high-affinity peptides (Figure 1B and 3B), but also when they are both bound to no or low-affinity peptides (Figure 3D and S2D). Thus, differential transport is not primarily caused by differential peptide optimization. Third: chaperone interaction. Both in tapasin-deficient and in calreticulin-deficient cells, the difference in the transport rates between Db and Kb is diminished (Figure 4 and S3E, F, G, H). The remaining difference in the ER export in tapasindeficient cells (visible especially well in the COPII vesicle formation assay; Figure 4G, H) may be caused by low-level interaction with another PLC member such as calreticulin [33]. Likewise, in calreticulin-deficient cells, some difference in COPII packaging efficiencies at early time points is still seen (Figure 4C, D, S3C, D). Thus, not any single protein on its own, but the ensemble of the chaperone proteins of the PLC, perhaps together with other ER proteins [34], is responsible for the primary difference in the ER exit and the cell surface transport rates of Db and Kb. We think it most likely that Db, whether bound to low- or to b high-affinity peptides, finds it harder than K to reach the ERES (from which COPII vesicles are budded) because of its tighter, or more frequent, interaction with chaperones. This puts it in a group with the vesicular stomatitis virus glycoprotein (VSV-G) and other proteins for which ER retention was proposed to occur through many short-lived interactions with a 'dynamic matrix' of ER proteins [35,36]. Taken together, we propose the following explanation for our and others' data with respect to differential class I exit from the ER: all those forms b b of class I, D or K , that are bound to any 'matrix protein' chaperone (such as calreticulin, calnexin, BIP, or tapasin) can only exit the ER at a very low rate that is determined by the particular matrix protein; that rate might in fact be close to zero, as for calnexin-bound proteins. In contrast, those forms of class I that are not bound to any matrix protein exit the ER at a higher rate; this includes class I molecules loaded with low-affinity peptides and even free HCs, which are then retrieved from the cis-Golgi [4,11,12,37]. We hypothesize that Kb acquires EndoF1 resistance faster than Db because most, if not all, of its forms have a lower affinity to ER matrix proteins, and thus are exported mostly at the higher rate for matrix-unbound proteins. In principle, our data might also be explained by the preferential binding of all forms of Kb to a putative MHC class I export receptor, which would mediate their uptake into COPII vesicles. Such a receptor has not unequivocally been demonstrated, despite hopeful beginnings [38].

To test whether our explanation of differential ER matrix affinity can indeed account for the differences in the EndoF1 acquisition curves of Db and Kb that we observed, we performed an in silico stochastic simulation using the simplest (most b b parsimonious) theoretical model of K and D folding and cell surface transport (Figure 6). Figure 6B shows surprisingly clearly that the differences in the Db and Kb Rt curves of Figure 1B cannot be explained by differential folding of the proteins (i.e., differential acquisition of the conformational epitope, represented in the model by the folding constant kF, which is varied in Figure 6A). Instead, a small variation of the equilibrium of two intra-ER forms (called S1 and S2) readily yields curves that are close to our experimental observation (compare Figure 6C to Figure 1B). One of these two forms, S1 cannot be exported, for example because it is bound to matrix proteins, whereas the other one, S2, can convert to Rt. The simulation results thus strongly support our b above explanation for the differential ER exit of D b and K . Our data suggest that the differential affinities b of D to the ER matrix are determined by a novel, as yet uncharacterized, feature or property of the class I lumenal part. What is this feature, and how is it read by the cell? According to our data, it is unrelated to peptide binding and not monitored by the conformational antibodies. We hypothesize that it is not a static structural feature but rather the greater or smaller conformational flexibility of the protein. To assess the likelihood of this interpretation being correct, we performed in silico b b molecular dynamics (MD) simulations of D and K bound to the same high-affinity pepide, KAVYNFATM. For each allotype, two independent simulations were compared in a free Gibbs energy plot with respect to the starting structure (Figure 7). In these plots, the ∆G value corresponds to the frequency with which a particular RMSD value (which approximately equates with a conformation of the protein) is populated during the simulation (see [39] and the Materials and Methods). The analysis was performed for the entire lumenal part, and also for the α1/α2 superdomain and the α3 b domain separately. In our results, K shows a single conformation of the lumenal part in both simulations, visible as a single tight peak in the top right-hand two-dimensional plot. In contrast, the lumenal part b of D exists in at least two different conformations, visible as two separate peaks in the top left-hand plot. In the more detailed analysis, both the α1/α2 domain (whose contribution to differential ER exit was already shown for Kd and Kk [40]) and also the b α3 domain of D show a certain conformational diversity (Figure 7, left column, center and bottom panels). This flexibility difference in the α3 domains of Db and Kb may indeed be unrelated to peptide binding and not monitored by the conformational antibodies, and thus it is a good candidate for differential interactions with the ER matrix. In ER quality control, partial unfolding or conformational flexibility of a protein causes monoglucosylation of its glycans, which mediates association with calreticulin and calnexin; both are known to act on murine class I within and outside

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b

the PLC [12]. Dynamic retention of D by this mechanism may be synergistically enhanced by its additional glycan, explaining the small retention effect of the third glycan on Kb (Figure 5B and S4). The differential exit of transmembrane proteins from the ER is governed by many factors such as cytosolic export signals [41], oligomerization [42,43], and membrane curvature [44]. For class I, in conclusion, the rate-limiting step appears to be governed by a novel feature of the lumenal part, perhaps including the α3 domain. Its molecular elucidation will require substantial further effort. Materials and methods Antibodies and reagents The monoclonal antibodies B22.249, Y3, and 20-84s were used to immunoprecipitate β2m-bound HCs of H-2Db and H-2Kb [14,15]. Calnexin was detected using an antiserum. An antiserum for immunoblots against murine p58 was produced by immunizing rabbits with peptides corresponding to residues 43 - 63 (HRRFEYKYSFKGPHLVQSDGT) and position 292 - 310 (SEKEKEKYQEEFEHFQQELDKKKEEFQKG) of the lumenal part (UniProtKB/Swiss-Prot accession number P49257). Cell culture b RMA and RMA-S (H-2 ) lymphoblastoid cells (Alain Townsend, Oxford, UK) were grown in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C and 8% CO2. Adherent cell lines listed below were grown in DMEM enriched with 10% FCS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C and 5% CO2. Wild type, tapasin- and TAP-deficient mouse ear fibroblasts (MEF, H-2b) cells were kindly provided by Luc van Kaer (Nashville, US). b Calreticulin-deficient K42 (H-2 ) cells were obtained from Tim Elliot (Southhampton, UK). Tapasindeficient MC4 cells (H-2b) were described previously [31]. Cloning Db and Kb genes were mutated in the pEGFP-N1 plasmid (Invitrogen/Life Technologies, Carlsbad, US) by site-directed mutagenesis with primers listed in b Table S1. All mutant constructs have the K signal sequence to ensure identical cotranslational translocation into the ER. For the exchange of the cytosolic domains, the full sequence was preamplified with primers specific for Db and Kb in pEGFP-N1 (Table S1) and used as a megaprimer in the mutagenization PCR. Buffers and enzymes were purchased from Fermentas (Thermo Scientific, Waltham, US). Generation of stable cell lines Ltk- cells were transfected in 6-well plates with 5 µg plasmid DNA and peqFECT transfection reagent (peqlab, Erlangen, Germany). Transfected cells b were selected using 1.0 mg/mL G418. K transfectants were sorted by flow cytometry with

b

K10-56 antibody and D transfectants with B22.249 antibody. Metabolic labeling Cells were harvested, washed once in PBS, and starved for methionine and cysteine in modified RPMI 1640 supplemented with 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin for 30 to 60 minutes at 37 °C. Metabolic labeling 35 was performed with EasyTag EXPRESS S Protein Labeling Mix (Perkin Elmer) for various times at 37 °C. Pulse-chase assays Metabolically labeled cells were transferred to DMEM or CO2-independent medium (Invitrogen) supplemented with 10% FCS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, and incubated for the times and at the temperatures indicated in the individual experiments. To terminate the chase, cells were transferred on ice and washed two times in ice-cold PBS and then subjected to lysis. Thermostability assay The assay was performed as published previously [22,23]. Cytosol preparation The preparation of mammalian cytosol was adopted from [45]. RMA cells were harvested and washed once in PBS. The cell pellet was resuspended in the same volume of transport buffer (25 mM HEPESKOH pH 7.2, 115 mM KCl, and 2.5 mM MgCl2). The cell suspension was rapidly frozen in liquid nitrogen and thawed at 25 °C in a waterbath. This freezethaw cycle was repeated twice, and a post-nuclear supernatant was prepared by centrifugation at 500 x g for 5 minutes. Subsequently, the supernatant was subjected to centrifugation at 20.000 x g for 1 hour and stored at -80 °C. For the in vitro COPII vesicle formation assay, the cytosol was centrifuged at 100.000 xg for 1 hour, and the supernatant was used for the budding reaction. Preparation of ER-enriched microsomal membranes Microsomal membrane preparation was adopted from earlier published protocols [11,45]. Metabolically labeled cells were washed once in PBS at 4 °C, and then resuspended in ice-cold swelling buffer (10 mM HEPES-KOH pH 7.2, 15 mM KCl) and incubated for 10 to 30 minutes on ice. Subsequently, the cells were pelleted and resuspended in breaking buffer (50 mM HEPESKOH pH 7.2, 90 mM KCl) and passed 20 times through a 23G needle. Nuclei and unbroken cells were separated from the disrupted membranes by centrifugation at 500 x g for 5 minutes. ER-enriched microsomal membranes were pelleted from the supernatant at 6,000 x g for 10 minutes, washed once in transport buffer, and then used for the in vitro COPII vesicle formation assay. In vitro COPII vesicle formation assay The generation of COPII vesicles in vitro was carried out as published [11,45] with the following

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modifications: To 150 µg microsomal membranes (protein), 450 µg RMA cytosol, 1 µg wild type hamster Sar1A or the respective mutant protein, an ATP regeneration system (1 mM ATP, 40 mM creatine phosphate, and 0.2 mg/mL of creatine phosphokinase), and 0.2 mM GTP were added, and the reaction mixture was adjusted to 90 µL with transport buffer. In vitro vesicle formation was carried out for 30 minutes at 25 °C. Donor membranes and vesicles were separated by differential centrifugation at 100.000 x g. The vesicle pellet was washed once in 100 µL transport buffer and lysed. Lysis of cellular material and immunoprecipitation Lysis of cells, microsomal membranes and vesicles was performed in native lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X100) supplemented with protease inhibitors 5 mM iodoacetamide and 1 mM phenylmethylsulfonyl fluoride for 1 hour. Non-lysed components were pelleted 16.000 x g for 5 minutes. Class I complexes were immunoisolated for 1 hour from precleared (1 hour) lysates using antibodies pre-bound to Protein A agarose beads. Subsequent to immunoprecipitation, the beads were washed three times in 50 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM EDTA and 0.1% Triton X-100, and processed as required. Endoglycosidase F1 digest Class I molecules immobilized on protein A agarose beads were denatured at 95 °C for 10 minutes in denaturation buffer (0.5% sodium dodecyl sulfate, 1% 2-mercaptoethanol). The mixture was neutralized with 50 mM sodium citrate (pH 5.5). In the presence of 1 % Triton X-100, samples were treated at 37 °C for 1 hour with endoglycosidase F1 (EndoF1), which has a cleavage specificity almost identical to that of EndoH [13]. The digest was terminated by addition of SDS-PAGE sample buffer and denaturation at 95 °C for 10 minutes. EndoF1 was produced from a pMal-p2 protein expression plasmid generously provided by Patrick van Roey (Albany, NY, US). SDS-PAGE, immunoblotting, and quantitative protein analysis Proteins were separated by SDS-PAGE. Isotopelabeled proteins were visualized using a Phosphorimager FLA-3000 (Fujifilm). Autoradiographs and immunoblots were analyzed densitometrically using ImageJ (NIH). For precise evaluation of the anterograde transport of class I proteins in pulse-chase experiments with EndoF1 digestion, the protein bands of EndoF1resistant, terminally glycosylated class I HCs (Rt, for definition see Endoglycosidase F1 digest) were quantified and evaluated independently from EndoF1-sensitive (S) as follows: signal intensities of S and Rt protein bands at each time point (tx) were normalized to the total signal intensity S+Rt at the beginning of the chase (t0), i.e. relative S = S(tx)/(S(t0)+Rt(t0)) and relative Rt = Rt(tx)/(S(t0)+Rt(t0)). The total signal intensity (as in Figure 1C) is calculated as the sum of relative S and relative Rt for each time point. The transport

rate was estimated by determining the half-time (t1/2), which corresponds to the time point when half of the initially synthesized and folded class I have become EndoF1-resistant during the chase, i.e., Rt = 0.5. For the analysis of the in vitro COPII vesicle formation assays, the band intensities of each budding sample were normalized to a standard curve generated from the signals of 0%, 5%, and 10% input. Sample duplicates were averaged before further data processing. The specific COPII budding [% input] was the difference of the normalized signals of the wild type Sar1A wild type (WT) sample and the background signal from Sar1A H79G sample. MD simulations The crystal structures of Kb and Db in complex with the peptide KAVYNFATM (single-letter amino acid symbols; PDB entries 1S7R and 1S7V, respectively) served as starting structures for MD simulations. Two sets of MD simulations for each HC/β2m/peptide complex were performed using the PMEMD module of Amber 12 [46]. Proteins were initially placed in an octahedral TiP3 water box [47] + together with ten Na and Cl ions and neutralized with eight counter-ions (Cl-). Short-range nonbonded interactions were taken into account up to a cut-off value of 9 Å. Long-range electrostatic interactions were treated with the particle mesh Ewald option using a grid spacing of 0.9 Å. The structures were energy minimized, positionally restrained using a force constant of 25 kcal mol−1 −2 Å , and heated from 100 K to 300 K. The restraints were resolved, and each complex was equilibrated for 1 ns and simulated for 40 ns. The resulting structural ensembles were evaluated with respect to RMSDs compared to starting structures over the course of simulation. The trajectories were treated as Boltzmann ensembles, and the Gibbs free energy landscapes (∆G) were calculated using the equation ∆G = -R T ln [ρ(RMSDx)/ρ(RMSDeq)], where R is the universal gas constant, T is the temperature, ρ(RMSDx) is the density of states of a certain conformation, and ρ(RMSDeq) is the density of states of the equilibrated conformation [39,48]. Visualization of trajectories and preparation of figures were performed using Visual Molecular Dynamics (VMD) [49] and PyMOL [The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC]. Acknowledgements We would like to thank Ted Hansen, Peter van Endert, Tim Elliott, Luc van Kaer, Patrick van Roey, and David Williams for reagent donations, Ursula Wellbrock for excellent technical support, Gesa Hollermann for her cloning work, and Venkat Raman Ramnarayan and Mohammed Al-Balushi for supplementary and initial experiments for this project. We thank Martin Zacharias (Munich, Germany) for his contribution in establishing MD analysis in our laboratory. We are especially grateful to Hubert Kalbacher (Tübingen, Germany) for help with the p58 antiserum production. Our work was funded by Deutsche Forschungsgemeinschaft (SP 583/2-3 and SP 583/6-1 to SSp)

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Figure le egends b

b

s EndoF1 resiistance faster than D . Figure 1: K acquires b b A, pulse--chase analysis of D and K in wild type RMA cells. Cells C were mettabolically labe eled for 10 minutes and aliqouts w were chased at 37 °C for different d times and then lysed. B22.249 (D ( b) and Y3 (Kb) immunoprrecipitates were dig gested with EndoF1, E separated by SDS S-PAGE, and d detected by y autoradiogra aphy. A repre esentative b b experime ent out of two is shown. D and K HCs sensitive s (S) and resistant (R Rt) to EndoF1 digest are ind dicated. A b b proteolytiic K fragmentt is pointed ou ut with an arro owhead. The distance d between S and Rt is larger for D than for b b K due to o the additiona al glycan in the e α3 domain off D . B, norma alized signal in ntensities of (A A). Representation of relativve EndoF1-se ensitive (S) and EndoF1-ressistant (Rt) populatio ons for Db and d Kb normalize ed to the totall signal intenssity of each allotype at the beginning of the t chase b b (see Matterials and Methods). M The e initial differe ence in ER-G Golgi transport between D (lag phase e) and K (immedia ate transport) at a 10 minutes is marked witth an arrow. C, total signal intensitie es (S+Rt) of (A A) normalized to the total am mount at the beginning b of th he chase (0 minutes).

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b

Figure 2: The ER export rate of D is controlled d by a step prrior to COPII packaging. o COPII vesic cle formation was w performed d with microso omes generate ed from wild type t MEF cellss that had A, in vitro been metabolically rad diolabeled for 10 minutes. In ndividual reacctions were eitther carried ou ut under initiating (Sar1 WT) or in nhibitory condiitions (Sar1 T3 39N, Sar1 H79G, -COPII (n no COPII sourrce), -micr. (no o microsomes)). Vesicle fractions and the inpu ut standard were w lysed in the presencce of 10 µM FAPGNYPAL F SIINFEKL and 10 µM S b b peptides.. D and K were immunoprecipitated using monocclonal antibod dies B22.249 and Y3, resspectively, resolved on 10% SDS--PAGE, and detected d by au utoradiographyy. Remaining lysates were separated on 8% SDSPAGE an nd western blotted b for p58 (positive bu udding contro ol) and calnex xin (negative budding con ntrol). The experime ent was performed three tim mes and quantiified. A repressentative gel iss shown. B, in vittro COPII vessicle formation assay perfformed as in (A), but the cells were additionally a chased for 30 minute es before micrrosome prepa aration. C, speciffic COPII budd ding values fo or Db and Kb of o (A) and (B)) were derived d from the diffference of the e average signal intensities of the t Sar1 WT T reactions an nd the Sar1 H79G contro ol reactions and a normalize ed to the microsom mal input for each experiment (see Materrials and Methods). The mean of three ind dependent exxperiments is plotted for each allottype. Error barrs represent th he SEM; *** means m p