Hide and run. Arginine-based endoplasmic-reticulum-sorting motifs in the assembly of heteromultimeric membrane proteins

Arginine-based endoplasmic reticulum (ER)-localization signals are sorting motifs that are involved in the biosynthetic transport of multimeric membrane proteins. After their discovery in the invariant chain of the major histocompatibility complex class II, several hallmarks of these signals have emerged. They occur in polytopic membrane proteins that are subunits of membrane protein complexes; the presence of the signal maintains improperly assembled subunits in the ER by retention or retrieval until it is masked as a result of heteromultimeric assembly. A distinct consensus sequence and their position independence with respect to the distal termini of the protein distinguish them from other ER-sorting motifs. Recognition by the coatomer (COPI) vesicle coat explains ER retrieval. Often, di-leucine endocytic signals occur close to arginine-based signals. Recruitment of 14-3-3 family or PDZ-domain proteins can counteract ER-localization activity, as can phosphorylation. This, and the occurrence of arginine-based signals in alternatively spliced regions, implicates them in the regulated surface expression of multimeric membrane proteins in addition to their function in quality control.     

14-3-3 proteins in membrane protein transport

14-3-3 proteins affect the cell surface expression of several unrelated cargo membrane proteins, e.g., MHC II invariant chain, the two-pore potassium channels KCNK3 and KCNK9, and a number of different reporter proteins exposing Arg-based endoplasmic reticulum localization signals in mammalian and yeast cells. These multimeric membrane proteins have a common feature in that they all expose coatomer protein complex I (COPI)- and 14-3-3-binding motifs. 14-3-3 binding depends on phosphorylation of the membrane protein in some and on multimerization of the membrane protein in other cases. Evidence from mutant proteins that are unable to interact with either COPI or 14-3-3 and from yeast cells with an altered 14-3-3 content suggests that 14-3-3 proteins affect forward transport in the secretory pathway. Mechanistically, this could be explained by clamping, masking, or scaffolding. In the clamping mechanism, 14-3-3 binding alters the conformation of the signal-exposing tail of the membrane protein, whereas masking or scaffolding would abolish or allow the interaction of the membrane protein with other proteins or complexes. Interaction partners identified as putative 14-3-3 binding partners in affinity purification approaches constitute a pool of candidate proteins for downstream effectors, such as coat components, coat recruitment GTPases, Rab GTPases, GTPase-activating proteins (GAPs), guanine-nucleotide exchange factors (GEFs) and motor proteins.     

Novel cargo-binding site in the beta and delta subunits of coatomer

Arginine (R)-based ER localization signals are sorting motifs that confer transient ER localization to unassembled subunits of multimeric membrane proteins. The COPI vesicle coat binds R-based signals but the molecular details remain unknown. Here, we use reporter membrane proteins based on the proteolipid Pmp2 fused to GFP and allele swapping of COPI subunits to map the recognition site for R-based signals. We show that two highly conserved stretches--in the beta- and delta-COPI subunits--are required to maintain Pmp2GFP reporters exposing R-based signals in the ER. Combining a deletion of 21 residues in delta-COP together with the mutation of three residues in beta-COP gave rise to a COPI coat that had lost its ability to recognize R-based signals, whilst the recognition of C-terminal di-lysine signals remained unimpaired. A homology model of the COPI trunk domain illustrates the recognition of R-based signals by COPI.     

Forward Transport of K2p3.1: mediation by 14-3-3 and COPI, modulation by p11.

Surface expression of the K(2P)3.1 two-pore domain potassium channel is regulated by phosphorylation-dependent binding of 14-3-3, leading to suppression of coatomer coat protein I (COPI)-mediated retention in endoplasmic reticulum (ER). Here, we investigate the nature of the macromolecular regulatory complexes that mediate forward and retrograde transport. We demonstrate that (i) the channel employs two separate but interacting COPI binding sites on the N- and C-termini; (ii) disrupting COPI binding to either site interferes with the ER retention; (iii) p11 and 14-3-3 do not interact on their own; (iv) p11 binding to the C-terminal retention motif is dependent on 14-3-3; and (v) p11 is coexpressed in only a subset of tissues with K(2P)3.1, while 14-3-3 expression is ubiquitous. We conclude that K(2P)3.1 forward transport requires 14-3-3 suppression of COPI binding, whereas p11 serves a modulatory role.     

Intracellular trafficking of KA2 kainate receptors mediated by interactions with coatomer protein complex I (COPI) and 14-3-3 chaperone systems

Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the coatomer protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and beta-COP protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 proteins, which mediate forward trafficking of other integral signaling proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs.     

Phosphorylation-dependent C-terminal binding of 14-3-3 proteins promotes cell surface expression of HIV co-receptor GPR15

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.     

A multimeric membrane protein reveals 14-3-3 isoform specificity in forward transport in yeast

Arginine (Arg)-based endoplasmic reticulum (ER) localization signals are sorting motifs involved in the quality control of multimeric membrane proteins. They are distinct from other ER localization signals like the C-terminal di-lysine [-K(X)KXX] signal. The Pmp2p isoproteolipid, a type I yeast membrane protein, reports faithfully on the activity of sorting signals when fused to a tail containing either an Arg-based motif or a -KKXX signal. This reporter reveals that the Arg-based ER localization signals from mammalian Kir6.2 and GB1 proteins are functional in yeast. Thus, the machinery involved in recognition of Arg-based signals is evolutionarily conserved. Multimeric presentation of the Arg-based signal from Kir6.2 on Pmp2p results in forward transport, which requires 14-3-3 proteins encoded in yeast by BMH1 and BMH2 in two isoforms. Comparison of a strain without any 14-3-3 proteins (Deltabmh2) and the individual Deltabmh1 or Deltabmh2 shows that the role of 14-3-3 in the trafficking of this multimeric Pmp2p reporter is isoform-specific. Efficient forward transport requires the presence of Bmh1p. The specific role of Bmh1p is not due to differences in abundance or affinity between the isoforms. Our results imply that 14-3-3 proteins mediate forward transport by a mechanism distinct from simple masking of the Arg-based signal.     

Forward Transport of K2P3.1: Mediation by 14-3-3 and COPI, Modulation by p11

Surface expression of the K_2P3.1 two-pore domain potassium channel is regulated by phosphorylation-dependent binding of 14-3-3, leading to suppression of coatomer coat protein I (COPI)-mediated retention in endoplasmic reticulum (ER). Here, we investigate the nature of the macromolecular regulatory complexes that mediate forward and retrograde transport. We demonstrate that (i) the channel employs two separate but interacting COPI binding sites on the N- and C-termini; (ii) disrupting COPI binding to either site interferes with the ER retention; (iii) p11 and 14-3-3 do not interact on their own; (iv) p11 binding to the C-terminal retention motif is dependent on 14-3-3; and (v) p11 is coexpressed in only a subset of tissues with K_2P3.1, while 14-3-3 expression is ubiquitous. We conclude that K_2P3.1 forward transport requires 14-3-3 suppression of COPI binding, whereas p11 serves a modulatory role.     

Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer

In mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins. Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat. The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells. Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells. We show that a dilysine motif in the -3,-4 position (relative to the cytosolic C-terminus) recruits in a very specific manner all the subunits of the plant coatomer complex. Lysines cannot be replaced by arginines or histidines to bind plant coatomer. A diphenylalanine motif in the -7,-8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif. Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells. The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs. ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgammaS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form.     

Assembly-dependent surface targeting of the heterodimeric GABAB Receptor is controlled by COPI but not 14-3-3

Cell surface expression of transmembrane proteins is strictly regulated. Mutually exclusive interaction with COPI or 14-3-3 proteins has been proposed as a mechanism underlying such trafficking control of various proteins. In particular, 14-3-3 dimers have been proposed to "sense" correctly assembled oligomers, allowing their surface targeting by preventing COPI-mediated intracellular retention. Here we examined whether such a mechanism is involved in the quality control of the heterodimeric G protein-coupled GABAB receptor. Its GB1 subunit, carrying the retention signal RSR, only reaches the cell surface when associated with the GB2 subunit. We show that COPI and 14-3-3 specifically bind to the GB1 RSR sequence and that COPI is involved in its intracellular retention. However, we demonstrate that the interaction with 14-3-3 is not required for proper function of the GABAB receptor quality control. Accordingly, competition between 14-3-3 and COPI cannot be considered as a general trafficking control mechanism. A possible other role for competition between COPI and 14-3-3 binding is discussed.     

Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex

Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K(d) approximately 100 microm), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER.     

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking

COPI‐coated vesicles mediate retrograde membrane traffic from the cis‐Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER‐resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V‐dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI?Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.     

Interaction of the parathyroid hormone receptor with the 14-3-3 protein

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) regulates calcium homeostasis, bone remodeling and skeletal development. 14-3-3 proteins bind to signaling proteins and act as molecular scaffolds and regulators of subcellular localization. We show that the parathyroid hormone receptor (PTHR) interacts with 14-3-3 and the proteins colocalize within the cell. 14-3-3 interacts with the C-terminal tail of the receptor containing a consensus 14-3-3 binding motif, but additional binding sites are also used. Protein kinase-A treatment of the receptor and especially the C-terminal tail reduces 14-3-3 binding. The expressed C-terminal tail is primarily localized in the nucleus, supporting the function of a putative nuclear localization signal that could be involved in the previously described nuclear localization of PTHR. The observed interaction between PTHR and the 14-3-3 protein implies that 14-3-3 could contribute to regulation of PTHR signaling.     

The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER

The reticular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions and undergoes constant remodeling through formation and loss of the three-way junctions. Transmembrane and coiled-coil domain family 3 (TMCC3), an ER membrane protein localizing at three-way junctions, has been shown to positively regulate formation of the reticular ER network. However, elements that negatively regulate TMCC3 localization have not been characterized. In this study, we report that 14-3-3γ, a phospho-serine/phospho-threonine-binding protein involved in various signal transduction pathways, is a negative regulator of TMCC3. We demonstrate that overexpression of 14-3-3γ reduced localization of TMCC3 to three-way junctions and decreased the number of three-way junctions. TMCC3 bound to 14-3-3γ through the N terminus and had deduced 14-3-3 binding motifs. Additionally, we determined that a TMCC3 mutant substituting alanine for serine to be phosphorylated in the binding motif reduced binding to 14-3-3γ. The TMCC3 mutant was more prone than wildtype TMCC3 to localize at three-way junctions in the cells overexpressing 14-3-3γ. Furthermore, the TMCC3 mutant rescued the ER sheet expansion caused by TMCC3 knockdown less than wild-type TMCC3. Taken together, these results indicate that 14-3-3γ binding negatively regulates localization of TMCC3 to the three-way junctions for the proper reticular ER network, implying that the negative regulation of TMCC3 by 14-3-3γ would underlie remodeling of the reticular network of the ER.     

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5′-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.     

Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission

The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271-277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.     

Physical aspects of COPI vesicle formation

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Physical aspects of COPI vesicle formation

Abstract

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Suppression of coatomer mutants by a new protein family with COPI and COPII binding motifs in Saccharomyces cerevisiae

Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell. COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat. Inactivation of COPI vesicle formation in conditional sec21 (gamma-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway. We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability. Mst27p and its homolog Yar033w (Mst28p) are part of the same complex. Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes. Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants. Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation. As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.     

Coatomer, the coat protein of COPI transport vesicles, discriminates endoplasmic reticulum residents from p24 proteins

In the formation of COPI vesicles, interactions take place between the coat protein coatomer and membrane proteins: either cargo proteins for retrieval to the endoplasmic reticulum (ER) or proteins that cycle between the ER and the Golgi. While the binding sites on coatomer for ER residents have been characterized, how cycling proteins bind to the COPI coat is still not clear. In order to understand at a molecular level the mechanism of uptake of such proteins, we have investigated the binding to coatomer of p24 proteins as examples of cycling proteins as well as that of ER-resident cargos. The p24 proteins required dimerization to interact with coatomer at two independent binding sites in gamma-COP. In contrast, ER-resident cargos bind to coatomer as monomers and to sites other than gamma-COP. The COPI coat therefore discriminates between p24 proteins and ER-resident proteins by differential binding involving distinct subunits.