Δ-COP Contains a Helix C-Terminal to Its Longin Domain Key to COPI Dynamics and Function

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

Eric C. Arakela, Kora P. Richtera, Anne Clancya, and Blanche Schwappacha,b,1

aDepartment of Molecular Biology, Universitätsmedizin Göttingen, 37073 Goettingen, Germany; and bMax Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany

Edited by David J. Owen, University of Cambridge, Cambridge, United Kingdom, and accepted by Editorial Board Member Pietro De Camilli May 11, 2016 (received for review March 3, 2016)

Membrane recruitment of coatomer and formation of coat protein I Results (COPI)-coated vesicles is crucial to homeostasis in the early secretory Functional Dissection of δ-COP. There are numerous examples where pathway. The conformational dynamics of COPI during cargo capture genes from higher eukaryotes, including plants, can functionally and vesicle formation is incompletely understood. By scanning the replace their counterparts in yeast. Functional complementation length of δ-COP via functional complementation in yeast, we dissect may entail a partial, but not total, loss of function in yeast because the domains of the δ-COP subunit. We show that the μ-homology evolutionary divergence in amino acid sequence can cause minor domain is dispensable for COPI function in the early secretory path- but significant variations in intermolecular contacts within a large way, whereas the N-terminal longin domain is essential. We map a complex. Such discrepancies provide a useful means to dissect the previously uncharacterized helix, C-terminal to the longin domain, functional role of a target gene, especially when analyzed reciprocally that is specifically required for the retrieval of HDEL-bearing endo- with chimaeras of the two genes that display different degrees of plasmic reticulum-luminal residents. It is positionally analogous to an functional complementation. We used such a complementation assay unstructured linker that becomes helical and membrane-facing in the to characterize the molecular features of δ-COP. open form of the AP2 clathrin adaptor complex. Based on the am- The RET2 gene of Saccharomyces cerevisiae encoding the phipathic nature of the critical helix it may probe the membrane for δ-subunit of COPI is essential for viability. The Bos taurus δ-COP lipid packing defects or mediate interaction with cargo and thus gene ARCN1 sustained survival in S. cerevisiae following the contribute to stabilizing membrane-associated coatomer. deletion of RET2, despite a sequence similarity of only 34% (Fig. S1A). Binding experiments and reporter-based localization assays COPI | coatomer | ARCN1 | HDEL | KDEL confirmed that many canonical functions of coatomer, such as the recognition of Arg-based and di-lysine–based ER retrieval signals, oat protein I (COPI) is a heptameric complex that is recruited were unaffected in the yeast strain with ARCN1 replacing RET2 Cen bloc to the Golgi by GTP-bound Arf1. Coatomer is evolu- (δc*) (Fig. S1 B and C). The overall gross morphology of the ER tionarily related to the adaptin family and can be conceptually was also unchanged because the reporter was localized at both the C divided into an F-subcomplex or adaptor (β-, γ-, δ-, and ζ-COP typical perinuclear and cortical ER (Fig. S1 ). Affinity purification α β′ e of coatomer from the δc* strain revealed that its coatomer is intact subunits) and a B-subcomplex or cage ( -, -, and -COP subunits) D α β′ (1, 2). COPI vesicles mediate the retrograde transport of proteins (Fig. S1 ). Subunits of the B-subcomplex of COPI ( and )were isolated at similar levels from both wild-type and δc* strains. and lipids from the Golgi apparatus to the endoplasmic reticulum However, the levels of isolated β-andγ-COP (subunits of the (ER). F-subcomplex) were lower, possibly owing to partial dissociation of Several studies have culminated in a medium-resolution (13 Å) molecular model of the COPI vesicle (3). Partial crystal structures Significance for six of the seven subunits are available [i.e., a segment of the αβ′-core, the C-terminal domain of α-ande-COP, the μ-homology δ domain (μHD) of δ-COP, the γ-COP appendage domain, and Arf1- We systematically dissect the structural elements of the γ-ζ-COP in complex] (4–9). However, no structural information is subunit of the coat protein I (COPI) vesicle coat. We demonstrate that the μ-homology domain is dispensable for available for the β-subunit and the N-terminal half of δ-COP, essential COPI functions. We map a key helix, positionally containing a predicted longin domain (Fig. S1A). Studies addressing analogous to a helix observed in clathrin adaptor (AP) struc- the evolution of the ancestral coat theorize that the large and small tures but without assigned function in AP, let alone COPI. This subunits of the F-subcomplex arose as a result of gene duplication helix shows all features of an amphipathic helix that can enable μ with only one of the two small subunits attaining a HD (present in interactions of a protein with the membrane. Indeed, the resi- μ δ -adaptin in AP and -COP in COPI) (10). The flexible C-terminal dues on its hydrophobic surface were specifically required for domain of μ-adaptin (μHD) serves both as a cargo-binding domain its function. Both findings substantially refine current models and as a membrane anchor (11). The μHD of the closely related of coat formation and restrict the mechanisms of how the coat δ-COP has likewise been ascribed a role in the binding of ArfGAP1/ polymer is constructed or how HDEL cargo is recognized Gcs1, the vesicle-tether Dsl1, Arg-based ER retrieval signals, and in by COPI. interactions mediating the linkage of COPI subunits in the assem- bled coat (3, 7, 12–14). The μHD is also found in the cargo-binding Author contributions: E.C.A. and B.S. designed research; E.C.A., K.P.R., and A.C. per- stonins and the muniscin family (Syp1 in yeast, and FCHo1/2 in formed research; E.C.A. contributed new reagents/analytic tools; E.C.A., K.P.R., and B.S. analyzed data; and E.C.A. and B.S. wrote the paper. mammals), which serve as endocytic adaptors (15, 16). Using yeast as a genetically tractable model system for a structure- The authors declare no conflict of interest. δ This article is a PNAS Direct Submission. D.J.O. is a guest editor invited by the Editorial guided functional analysis of the -COP subunit, we identify the Board. minimal fraction of this subunit required to sustain COPI function. μ Freely available online through the PNAS open access option. In doing so, we reassess the functional importance of the HD of 1 δ To whom correspondence should be addressed. Email: [email protected] -COP and highlight the role of a previously unidentified helical goettingen.de. μ region, positionally analogous to the linker-helix in 2ofAP2,thatis This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. central to the function of coatomer at the membrane. 1073/pnas.1603544113/-/DCSupplemental.

6916–6921 | PNAS | June 21, 2016 | vol. 113 | no. 25 www.pnas.org/cgi/doi/10.1073/pnas.1603544113 Downloaded by guest on October 2, 2021 the F-subcomplex. This suggests that a segment of Ret2 is crucial in The inverse mutant, chimaera 6 containing the N-terminal maintaining the ensemble of the coatomer complex. equivalent of bovine δ-COP and its adjacent helical region secreted Soluble and luminal ER residents such as the chaperones Kar2 maximal levels of Pdi1, suggesting that the longin domain and the and Pdi1 are efficiently retrieved following erroneous ER exit. These two adjoining helices of Ret2 were essential for HDEL-mediated proteins contain an HDEL (or KDEL in higher eukaryotes) se- COPI function (Fig. 1 A and C).Thiscouldalsoindicatethatthe quence in their C termini. When mislocalized to the lumen of the intermolecular contacts forging the β–δ interface are affected pos- Golgi, HDEL-exposing proteins are recognized by the HDEL re- sibly due to differences between the amino acid sequence of yeast ceptor (encoded by the ERD2 gene) in a pH-dependent man- and bovine δ and β-COP. ner and subsequently retrieved back to the ER in a COPI- dependent mechanism (17, 18). Deletion of, or mutations in, Functional Relevance of the δ-COP μHD. The predicted secondary retrograde trafficking-related genes results in the perturbation of structure of δ-COP is evolutionarily conserved (Fig. S2), permit- ret2 the HDEL-retrieval pathway, causing secretion of ER resident ting the complementation of a deletion strain by bovine δ proteins with an HDEL signal into the culture medium (19, 20). -COP despite low sequence similarity. This similarity in predicted The secretion of such HDEL-bearing proteins has been inter- secondary structure is also found in the cognate subunit of the adaptin complex, μ-adaptin. The C-terminal domain of μ2-adaptin preted to occur either as a result of the failure to retrieve the Φ HDEL receptor or due to the activation of the unfolded protein binds endocytic cargo via the YXX motif and also binds PtdIns4,5P2 (11). Likewise, the δ-COP μHD binds di-tryptophan (WXn(1–6)[WF]) response where the capacity of the HDEL-retrieval pathway is δ exceeded (20, 21). The secretion assay examining HDEL-bearing -containing cargo [previously characterized as L motif by Cosson et al. (23)]. The mammalian ArfGAP1, its yeast homolog Gcs1, proteins therefore presents a highly efficient and sensitive read- δ μ δ out of the steady-state status of the retrograde transport and the vesicle-tether Dsl1 bind the -COP HD via such a L motif (7, 12, 13). pathway. The erd1 deletion strain was used as a positive control Schizosaccharomyces pombe is unique in that it possesses only one in this assay because ERD2 is an essential gene. Erd1 was RET2-homologous gene encoding a truncated variant of the δ-COP identified as being required for the efficient retrieval of HDEL- subunit that entirely lacks the C-terminal μHD (Fig. 2A). Curiously, bearing proteins and has been proposed to facilitate the S. pombe also lacks a DSL1-like gene and its Gcs1 homolog lacks a interaction of the HDEL receptor Erd2 and its ligands in the δL sequence, suggesting that this whole functional network has been Golgi (22). deleted in S. pombe. The complete lack of the δ-COP μHD in this Despite compensating for the loss of RET2, replacement of RET2 ARCN1 particular yeast species and the ensuing evolutionary adaptation with resulted in the secretion of Kar2 and Pdi1 into seems remarkable given the recently proposed role of the μHD in the growth medium, implying that COPI function was not wholly A δ a set of interactions that contribute to coat formation via a triad restored (Fig. 1 ). Overexpression of Ret2 in the c* strain rescued linkage (3). RET2 ARCN1 the observed secretion phenotype. Chimaeras of and We deleted the μHD of Ret2 to ascertain the relevance of the were constructed in an effort to identify the minimal region of Ret2 μHD in S. cerevisiae (Fig. 2A). Surprisingly, deletion of the μHD did required to salvage the retrieval of HDEL-bearing proteins. Chi- not result in any discernible differences in the viability of yeast (Fig. maeras were constructed around four well-conserved sequence 2B). The strain containing a truncated Ret2 missing the μHD elements of the genes (Fig. 1B and Fig. S1E). Chimaera 2, con- (Ret2LD2α) did not exhibit temperature sensitivity up to 39 °C taining an N-terminal span of Ret2, comprising the predicted longin despite RET2’s being an essential gene. The indispensability of domain and two adjoining predicted helices, secreted negligible RET2 is presumably due to the requirement of its longin domain to levels of Kar2 and Pdi1, comparable to the wild-type strain (Fig. 1 A maintain structural integrity of the F-subcomplex. Consistently, we and C). Chimaera 3 and chimaera 4 secreted elevated levels of Kar2 observed a loss of viability in the ret2 mutant complemented with despite containing the same N-terminal segment of Ret2 as chi- bovine δ-COP at elevated temperatures. There was no elevation in maera 2, possibly owing to defective maneuverability of the flexible the levels of secreted HDEL-bearing Pdi1 into the growth medium μHD as a result of improper protein folding. for yeast lacking the Ret2 μHD, implying that the retrograde

Fig. 1. Expansion on an incomplete COPI structure via CELL BIOLOGY functional dissection of a highly flexible coatomer subunit. (A) Secretion assay of a ret2 deletion strain harboring a functional B. taurus ARCN1 gene (δc*) was BCtransformed with plasmids containing Ret2-Archain chimaeras (CHI 1–8). Proteins secreted into the culture medium (i.e., extracellular) were analyzed by SDS/PAGE and immunoblot analysis using antibodies specific for Kar2 and Pdi1. Cell pellets from the same cultures were also similarly analyzed using antibodies specific for Pgk1. (B) Schematic representation of the Ret2-Archain chimaeras used in A. Boundaries at which the chimaeras were constructed and alignments of their sequences are indicated. (C) Chimaera 2, containing the longin domain and two predicted adjoining helices of Ret2 rescues the Pdi1 secretion phenotype of δc*. Bar graph of the relative amounts of Pdi1 secreted into the culture medium as quantified by the densitometric analysis of immunoblots. Quantification of five independent experiments. Error bars depict SEM.

Arakel et al. PNAS | June 21, 2016 | vol. 113 | no. 25 | 6917 Downloaded by guest on October 2, 2021 pathway and thus COPI was fully functional regardless of the loss of strain incorporating the truncated variant of ARCN1 (δCLD2α,with the μHD (Fig. 2C). In this strain, S. cerevisiae possibly buffers the loss the longin domain and the two adjoining helices) is conceivably in- of the μHD by relying on redundant mechanisms of regulating coat capable of facilitating unaltered retrograde transport owing to its dynamics. The vesicle tether Dsl1 can interact with COPI via both inability to form the proper intermolecular contacts with the yeast the α-andδ-COP subunits (13). Likewise, the pertinent GTPase- β-subunit of COPI (as seen with the dissociation of the F-subcomplex activating proteins are thought to be redundant. Therefore, Glo3, in Fig. S1D). which was shown to interact with the β′-andγ-subunit of coatomer in a yeast-2-hybrid assay (24), can potentially compensate Characterization of the α-Helix Central to δ-COP Function. We de- for the loss of Gcs1 recruitment by the μHD. signed additional chimaeras of RET2 and ARCN1 to comprehen- The observation that the μHD is dispensable for the essential sively dissect the role of individual structural elements and their role functionsofCOPIinS. cerevisiae is in line with the notion that the within the longin domain (Fig. 4A). Swapping the first α-helix ad- μHD was acquired only recently in evolutionary terms (10) and that it joining the longin domain (CHI9) was of no consequence (Fig. 4 B expanded its functional scope fairly late in evolution. The lack of any and C). The two ends of the second α-helix are conserved between acute phenotype in the strain lacking the μHD impinges on a recent Ret2 and bovine δ-COP (Fig. S3A). Swapping the less-conserved structural model of the linkages within the COPI coat (3) where the intervening 17 residues of Ret2 for those of bovine δ-COP (CHI10) δ-COP subunits are central to one type of linkage. Our data func- caused secretion of the HDEL-bearing Pdi1 (Fig. 4). Interestingly, a tionally refines this model and implies that the relevant set of inter- strain containing a chimaera of the N-terminal longin domain of actions cannot occur via the μHD of δ-COP. Ret2, but the two adjoining α-helices of bovine δ-COP, with its equivalent 17 residues replaced by yeast Ret2 (CHI13), resulted in a Identification of the Minimal Functional Element of δ-COP Enabling rescue of the secretion phenotype (Fig. 4, and compare Ret2LD+δC HDEL-Dependent Retrieval. Longin domains occur in a number of 2α in Fig. 3). This pinpoints these 17 residues as being central to the membrane trafficking proteins. The fold is based on a unique role of the second predicted α-helix, C-terminal to the longin domain, arrangement of a ββαβββαα(α) sandwich (25). Both COPI and in supporting retrograde trafficking of HDEL-bearing proteins. the adaptin family contain two subunits with longin domains at the Successful affinity purification of intact coatomer from a wild- core of the trunk-like complex (σ and μ in AP, and δ and ζ in the type strain, the Ret2LD2α (lacking the μHD) and CHI10 (identical case of COPI). Comparison of the prediction-based structural to Ret2LD2α but with the critical 17 residues of the Ret2 helix alignment of δ-COP and the μ-adaptins revealed a distinct varia- swapped with its bovine equivalent) strains excluded defects in tion in its underlying architecture in that the longin domain of protein folding and coatomer assembly (Fig. 4D). Binding assays δ-COP is followed by an additional α-helix (Fig. S2). using coatomer purified from wild-type (BY4741) or Ret2 variant We constructed additional truncations of Ret2 and Archain to strains (Ret2LD2α, CHI10, and Ret2LD) revealed that all coat- further delineate the minimal region of δ-COP required to sustain omer variants recognized both di-lysine (KKXX and KXKXX) and unimpaired COPI function (Fig. 3A). Deletion of the two helices Arg-based ER retrieval signals with equal efficacy (Figs. S4 A–C adjoining the longin domain of Ret2 caused elevated secretion of and S5). As previously reported, we confirmed that oligomerization HDEL-bearing Pdi1 (Fig. 3 B and C), suggesting that the presence of of the p24 C termini (via the coiled-coil forming region of Gcn4) solely the longin domain was insufficient to maintain efficient re- greatly increased its affinity for COPI (26). Reporter-based locali- trieval from the Golgi (Fig. 3 B and C). Likewise, strains expressing zation assays using the same signals [in addition to a COPI-recog- truncated variants of Archain, lacking the μHD and the two adjoining nition motif present in the C-terminus of the TASK-3 ion channel helices, were viable but did not support Pdi1 retrieval. Furthermore, (27)] confirmed that the reporter constructs exposing the respective we assessed chimaeras between Ret2 and bovine δ-COP where the sorting motifs were efficiently retrieved even in the Ret2LD and helices (contiguous with the longin domain) were interchanged be- CHI10 strains (Fig. S4D). Collectively, these data demonstrate that tween the two species (Ret2LD+δC2α and δCLD+Ret2 2α;Fig. the distal α-helix adjoining the longin domain of Ret2 is crucial for 3A). The presence of the Archain helices resulted in the secretion of the efficient ER-retrieval of HDEL-bearing cargo, but not for the Pdi1 into the growth medium (Fig. 3 B and C), indicating that spe- retrieval of cargo containing other canonical COPI-recognition cifically the two adjoining helices of Ret2 were indispensable. The signals, such as KKXX or KXKXX motifs on the cytoplasmic

Fig. 2. The μHD of Ret2 is dispensable. (A) Sche- matic representation of JPred secondary structure predictions of the δ-COP subunit from different species. Note that S. pombe lacks a functional μHD. BCThe secondary structure of μ-adaptin has been illus- trated for comparison. The UniProt accession numbers have been provided within parentheses. (B) Growth assay demonstrating that deletion of the Ret2 μHD does not cause temperature sensitivity up to 39 °C. (C) Secretion analysis of ret2 deletion strains expressing ARCN1, RET2, or truncated RET2 (i.e., lacking the μHD; Ret2LD2α). Proteins secreted into the culture medium were analyzed by SDS/PAGE and immunoblot analysis using antibodies specific for Pdi1 and Kar2. Cell pellets from the same cultures were similarly analyzed using antibodies specific for Pgk1, coatomer, and a His-tag present on Ret2LD2α. Note that the coat antibody detects the μHD of Ret2.

6918 | www.pnas.org/cgi/doi/10.1073/pnas.1603544113 Arakel et al. Downloaded by guest on October 2, 2021 AB C

Fig. 3. A predicted helix C-terminal to the Ret2 longin domain plays a pivotal role in the HDEL-dependent retrieval system. (A) Schematic representation of the JPred secondary structure predictions of the truncated and chimeric constructs of Ret2 and Archain used in B. α-Helices are colored red and purple and β-sheets are green and brown (for Ret2 and Archain, respectively). (B) ret2 deletion strains containing either truncated genes or chimaeras of RET2 and ARCN1 (i.e., lacking the μHD, the μHD and the two helices C-terminal to the longin domain and chimaeras with swapped helices). Proteins secreted into the culture medium were analyzed by SDS/PAGE and immunoblot analysis using antibodies specific for Pdi1. Cell pellets from the same cultures were also similarly analyzed using antibodies specific for Ret2, Archain, coatomer, and Pgk1. (C) Bar graph of the relative amounts of Pdi1 secreted into the culture medium as quantified by the densitometric analysis of immunoblots. Quantification of three independent experiments. Error bars depict SEM.

domains of transmembrane proteins that are ensnared by the Additional chimaeras swapping the αx–αy helices of the longin β-propeller domains of α-andβ′-COP (28, 29) or Arg-based signals domain (CHI-14) did not result in the secretion of Pdi1 from that were proposed to bind β-COP and the μHD of δ-COP (14). cells (Fig. 4 A–C). Likewise, CHI-12 containing the αp and β(1–5)

AB E

CD CELL BIOLOGY

Fig. 4. Seventeen residues at the core of the identified α-helix are key to the retrieval of HDEL-bearing cargo. (A) Schematic representation of JPred secondary structure predictions. α-Helices are colored red and purple and β-sheets are green and brown (for Ret2 and Archain, respectively). Nomenclature of longin domain secondary structural elements corresponds to ref. 25. (B) ret2 deletion strains containing chimeric constructs of Ret2 and Archain in the two helices C-terminal to the longin domain were analyzed for the secretion of HDEL-bearing proteins. Proteins secreted into the culture medium were analyzed by SDS/PAGE and immunoblot analysis using antibodies specific for Pdi1. Cell pellets from the same cultures were also similarly analyzed using antibodies specific for coat, Pgk1, and a His-tag present in Ret2LD2α.(C) Bar graph of the relative amounts of Pdi1 secreted into the culture medium as quantified by the densitometric analysis of immunoblots. Quantification of four independent experiments. Error bars depict SEM. (D) TAP-tagged coatomer purified from the three indicated strains. (E) Schematic illustration of the conformational change in COPI following membrane recruitment. Like AP2, this potentially results in displacement of the μHD of δ-COP, the opening of the coatomer core via extension of the F-subcomplex, and the binding of the newly identified helix in δ-COP (colored red) onto β-COP. The interaction of individual COPI subunits with cargo (through ER retrieval signals) further stabilizes the open conformation of coatomer. ArfGAP1 (colored purple) with a δL-motif interacts with the μHD of δ-COP and Arf1.

Arakel et al. PNAS | June 21, 2016 | vol. 113 | no. 25 | 6919 Downloaded by guest on October 2, 2021 of bovine δ-COP, the αx–αy of Ret2, and its two adjoining helices between the μHD of δ-COP are thought to give rise to linkage type resulted in a limited, yet minor, secretion of Pdi1 (compared with III. However, our data excludes a model in which the δ-COP μHD δCOPLD2α), suggesting that the identified second predicted α-helix is required for an essential linkage within the COPI coat. Curiously, C-terminal to the longin domain was indeed vital to coatomer e-COP (encoded by SEC28) is the only nonessential subunit of the function (Fig. 4 A–C). COPI coat. In sec28 deletion cells linkage type I involving a set of Plotting the N-terminal half of the helix as a helical wheel (Fig. interactions between e-COP and the C-terminus of α-COP cannot S3C) reveals a segregation of charged and hydrophobic residues for form as proposed. However, sec28 deletion yeasts are viable up to Ret2 and δ-COP. The putative hydrophobic surface was most ex- 34 °C (32). This suggests that linkages involving e-COP are also not tensive for Ret2, featuring four isoleucines, interrupted by an as- essential. Furthermore, we found that combined deletion of the paragine. We engineered single-residue substitutions for all four μHD and e-COP did not affect the viability of yeast up to 34 °C isoleucines (I145, I148, I149, and I155) and found three of them (Fig. S7 C–E). In conclusion and in line with the fact that the re- affected the role of this α-helix (Fig. S6 A, B,andD). Conversely, constructions of the linkages were of lower resolution (18–23 Å) in changing any of the nonhydrophobic residues to the equivalent side the EM tomogram (3), further verification of the structural model chain found in bovine δ-COP had no effect (Fig. S6 C and D). We will be required to assess the precise set of interactions underlying conclude that the hydrophobic surface of the predicted α-helix in the different types of coatomer triads and their relevance to the Ret2 is key to its function. physiological formation of COPI vesicles. We also provide additional insight into the function of coatomer Discussion at the membrane by demonstrating that a predicted helix immedi- Coatomer is recruited to membranes by GTP-Arf1 through a bi- ately C-terminal to the longin domain of δ-COP is required for valent interaction (9). This binding results in the opening of the efficient retrieval of HDEL proteins. This process was clearly per- coatomer core through an extension of the α-solenoid domains of turbed in the absence of this helix or mutations within its N-terminal β-COP and γ-COP [the hyperopen form (3)] and presumably dis- portion. Here, we discuss two mechanistic interpretations of this placement of the μHD from its inactive (cytosolic) seat. The finding, which will require validation once high-resolution structures β-propeller domains of α and β′-COP bind di-lysine-based ER re- of COPI are available. trieval signals (28, 29) and might stabilize the open conformation of The first model focuses on the evolutionary relatedness of AP2 COPI in a cooperative manner. Binding of additional signals such as and COPI, which suggests that this helix may bind back onto the Arg-based ER retrieval signals to the trunk may further stabilize the coatomer trunk and occupy a trough formed between δ-COP and open conformation and thus make coatomer increasingly resistant β-COP [similar to the μ2–β2 trough in AP2, PDB ID code 2XA7 to dissociation (Fig. 4E). Using structure-guided manipulation of (11)]. By secondary structure prediction the identified segment in endogenous yeast coatomer, we now provide additional insight into yeast Ret2 and mammalian δ-COP is expected to form an α-helix; the role of coatomer at the membrane. however, as observed for the μ2-adaptin subunit (11), this segment First, we reassessed the functional relevance of the δ-COP μHD could potentially transition between an unstructured linker in the by demonstrating that it is dispensable for maintaining COPI func- closed state and an α-helix in the open, membrane-associated state tion in the early secretory pathway. Unlike the open conformation (Fig. S8). The loss of function following replacement of the core 17 of AP2, where the C-terminal μHD of μ2-adaptinisbelievedtobe residues of the Ret2 helix by bovine δ-COP in the CHI10 strain can closely apposed to the membrane surface, in the recent structure of potentially be explained by a loss of interaction with the β-COP Dodonova et al. the δ-COP μHD is displaced far from the mem- subunit (i.e., failure to occupy the furrow). We speculate that the brane (3, 11). Most cargo recognition domains are closely apposed function of this helix is similarly conserved in higher organisms, and to the membrane surface, as is the case with the β-propellers do- that the packing into this trough may prevent the destabilization of mains of α and β′ COPI subunits that recognize di-lysine signals the distended α-solenoid or heat-repeat domains of β-COP. (28, 29). Deletion of the μHD (in the Ret2LD2α, CHI10, and The identified helix in Ret2 and δ-COP is significantly longer Ret2LD) did not affect the recognition of Arg-based ER retrieval than the back-binding helix identified in μ-adaptin (Fig. S3B). This signals, indicating that either the μHD does not bind Arg-based raises the question of how the helix in coatomer is packed in a signals or that COPI contains more than one recognition site (a manner akin to AP2. It is conceivable that the helix, classified as second region important for recognizing Arg-based signals was in- such by structure prediction, could exist as two smaller helices ad- deed identified in the β-subunit) (14). Mutations in the μHD of the jacent to each other, with the anterior helix forming the back- δ-COP subunit such as those in the ret2-1 yeast strain or the nur17 binding helix. Likewise, the linker coupling the helix and the μHD mouse model (with an I422T substitution in the μHD) have been in COPI is significantly longer than in AP2 (Fig. S3B), possibly reportedtoresultinaberrantCOPIfunction(30).Inlightofthe explaining the divergence in membrane proximity of the μHD in dispensability of this domain, the phenotypes caused may be due AP2 and coatomer (3, 11). to the presence of a misfolded domain that may interfere with Interestingly, the linker that transforms into a helix in μ-adaptin of coatomer dynamics. AP2 is phosphorylated at Thr156 in the open conformation (33, 34), To account for the surprising difference in membrane proximity either inducing or stabilizing a conformational change in AP2, which between the μ2andδ-COP C-terminal μHD domains, Suckling augments the binding of the YXXΦ endocytic motif. AAK1, which et al. (7) propose a theory that the highly negative surface of δ-COP copurifies with AP2, has been implicated in the phosphorylation of prevents inaccurate vesicle fusion with negatively charged mem- μ2Thr156 (33). The α-helix that we mapped to be functionally crucial branes such as those of the late Golgi. Such a model is consistent in δ-COP contains only one phosphorylatable residue in both with the concept of membrane territories (31) where characteristic S. cerevisiae (Thr158) and B. taurus (Thr150), within the span of the features of the ER and the early secretory pathway stem from lipid- central 17 residues. Thr150 in δ-COPisconservedinallhigher-order packing defects, whereas electrostatics play a larger role in mem- eukaryotes and is predicted to be phosphorylated, unlike the Thr158 brane identity and dynamics in the late secretory and endocytic of S. cerevisiae (35, 36). A putative phosphorylation at this site could pathway and at the plasma membrane. potentially function in a manner akin to the μ2Thr156. The nature of Dodonova et al. (3) determined that flexible, membrane-distal the bovine δ-COP helix possibly necessitates phosphorylation to sta- domains of COPI, such as the μHD of δ-COP, the C-terminus of blybindbacktomammalianβ-COP, offering an explanation for the α-COP and e-COP, mediate the linkage between three COPI com- loss of function in the CHI10 strain. Besides binding back onto plexes on the membrane (coatomer triads) and thereby the COPI β-COP, the α-helix C-terminal to the longin domain of δ-COP may lattice. To discuss our finding that the μHD is dispensable in the also directly bind to sorting machinery or cargo. Ret2LD2α strain we have extrapolated a schematic illustration The second model of the mechanism builds on the amphipathic from the structural data provided for each proposed linkage type nature of the identified helix. Amphipathic helices have been shown (Fig. S7 A and B). Linkage type I relies on a set of interactions to enable lipid binding and support membrane remodeling in sev- between e-COP and C-terminus of α-COP, whereas interactions eral proteins involved in the dynamics of the endomembrane system.

6920 | www.pnas.org/cgi/doi/10.1073/pnas.1603544113 Arakel et al. Downloaded by guest on October 2, 2021 Both the Ret2 and δ-COP helices are amphipathic in nature (Fig. Antibodies. Primary antibodies used in this study include the following: mouse S3C), as they present an array of hydrophobic residues on one surface monoclonal anti-His (34660, Penta·His; Qiagen), mouse monoclonal anti- interrupted by a single polar residue (N in Ret2 and Q in δ-COP). PGK1 (459250, clone 22C5D8; Life technologies), and rabbit polyclonal anti- This rift is reminiscent of data by Ford et al. (37), who demonstrated Pdi1, Ret2, Kar2, COPI, and δ-COP (877) antisera. that substitution of a hydrophobic residue by a polar residue (L6Q) in Secretion Assay. Secretion assays were performed as previously described the amphipathic helix (α0) of the epsin ENTH domain (Fig. S3D) results in a loss of its ability to tubulate membranes but not in a loss of by Belden et al. (21) with minor modifications as specified in Supporting Information. its affinity to lipids. Additionally, the α0 becomes ordered only upon lipid binding. We propose that the helix in Ret2 is unable to remodel membranes but instead serves as a membrane-adsorbing surface. By Fluorescence Microscopy. Stationary phase yeast cultures grown in minimal doing so, the helix may position coatomer in close apposition with the SC medium were imaged at room temperature. For details see Supporting membrane surface and hence enable it to probe for ER-retrieval Information. signals on short cytoplasmic loops or tails. In consequence, stable recruitment of coatomer to the membrane would rely on the co- Purification of GST Fusion Proteins. Recombinant proteins were purified as incident detection of activated Arf1, lipid-packing defects, and cargo previously described by Michelsen et al. (14) with minor modifications as signals and thus be restricted to membranes of correct identity with a specified in Supporting Information. need for cargo sorting (Fig. 4E). Purification of Yeast Coatomer. Coatomer was purified as previously de- Methods scribed by Yip and Walz (38) with minor modifications as specified in Supporting Information. Yeast Strains. A spore derived from the heterozygous RET2/ret2 strain (marked with KanMX4), transformed with an URA3-containing CEN plasmid carrying the ACKNOWLEDGMENTS. We thank H. D. Schmitt for the gift of the anti-Ret2, temperature-sensitive ret2-1 allele, with a stop codon at position 385 [by selec- M. Schuldiner for the anti-Pdi1; M. Seedorf for the anti-Kar2; A. Spang for tion for growth on geneticin and 5-fluoroorotic acid (FOA) sensitivity] (14), was anti-yeast-COPI; F. Wieland for the anti–δ-COP antiserum; and R. Ernst, used as the antecedent to all strains created in this study. Strains with LEU2- R. Jahn, H. D. Schmitt, M. Schuldiner, and A. Spang for helpful comments containing CEN plasmids (p415) carrying truncations or mutants of RET2 were on the project. This work was supported by the Jacob-Henle-Programm, selected on 5-FOA for the loss of the ret2-1 carrying URA3 plasmid. University Medical Center Göttingen (K.P.R.).

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Arakel et al. PNAS | June 21, 2016 | vol. 113 | no. 25 | 6921 Downloaded by guest on October 2, 2021