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

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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 dem- onstrate 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 September 26, 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.
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