14-3-3 dimers probe the assembly status of multimeric membrane proteins

BACKGROUND: Arginine-based endoplasmic reticulum (ER) localization signals are involved in the heteromultimeric assembly of membrane protein complexes like ATP-sensitive potassium channels (K(ATP)) or GABA(B) G protein-coupled receptors. They constitute a trafficking checkpoint that prevents ER exit of unassembled subunits or partially assembled complexes. For K(ATP) channels, the mechanism that leads to masking of the ER localization signals in the fully assembled octameric complex is unknown. RESULTS: By employing a tetrameric affinity construct of the C terminus of the K(ATP) channel alpha subunit, Kir6.2, we found that 14-3-3 isoforms epsilon and zeta specifically recognize the arginine-based ER localization signal present in this cytosolic tail. The interaction was reconstituted by using purified 14-3-3 proteins. Competition with a nonphosphorylated 14-3-3 high-affinity binding peptide implies that the canonical substrate binding groove of 14-3-3 is involved. Comparison of monomeric CD4, dimeric CD8, and artificially tetramerized CD4 fusions correlates the copy number of the tail containing the arginine-based signal with 14-3-3 binding, resulting in the surface expression of the membrane protein. Binding experiments revealed that the COPI vesicle coat can specifically recognize the arginine-based ER localization signal and competes with 14-3-3 for the binding site. CONCLUSIONS: The COPI vesicle coat and proteins of the 14-3-3 family recognize arginine-based ER localization signals on multimeric membrane proteins. The equilibrium between these two competing reactions depends on the valency and spatial arrangement of the signal-containing tails. We propose a mechanism in which 14-3-3 bound to the correctly assembled multimer mediates release of the complex from the ER.     

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.     

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.     

Diacidic motifs influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.     

Assembly-dependent trafficking assays in the detection of receptor-receptor interactions

Assembly-dependent trafficking is a property of many multimeric membrane protein complexes; this coupling of assembly and trafficking processes provides an important cellular quality control mechanism, ensuring that only properly folded and assembled complexes are expressed on the cell surface. In all membrane protein complexes whose trafficking is known to be assembly-dependent, at least one of the subunits contains an endoplasmic reticulum (ER) retention/retrieval signal that is shielded on subunit assembly, allowing the assembled protein complex to traffic to the plasma membrane. Under these conditions, presence of the normally retained subunit on the cell surface can be used as an indirect index of protein assembly in the ER. In this article, I describe the design of two complementary approaches (trafficking enhancement and trap assays) that can be used separately or in combination to determine whether two (or more) proteins assemble in the ER, i.e., whether they constitutively oligomerize. Both of the approaches are based on the measurement of plasma membrane-expressed proteins using antibody-mediated detection of extracellularly expressed epitopes and subsequent luminometric quantification. These methods provide a straightforward and relatively inexpensive way to assess protein-protein interactions early in the synthetic pathway.     

A novel function for the ER retention signals in the C-terminus of kainate receptor subunit,GluK5

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons.SignificanceWe present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways.     

Endoplasmic reticulum quality control of oligomeric membrane proteins: topogenic determinants involved in the degradation of the unassembled Na,K-ATPase alpha subunit and in its stabilization by beta subunit assembly

The molecular nature of determinants that mediate degradation of unassembled, polytopic subunits of oligomeric membrane proteins and their stabilization after partner subunit assembly is largely unknown. Expressing truncated Na,K-ATPase alpha subunits alone or together with beta subunits, we find that in unassembled alpha subunits neither the four N-terminal transmembrane segments acting as efficient alternating signal anchor-stop transfer sequences nor the large, central cytoplasmic loop exposes any degradation signal, whereas poor membrane insertion efficiency of C-terminal membrane domains M5, M7, and M9 coincides with the transient exposure of degradation signals to the cytoplasmic side. beta assembly with an alpha domain comprising at least D902 up to Y910 in the extracytoplasmic M7/M8 loop is necessary to stabilize Na,K-ATPase alpha subunits by favoring M7/M8 membrane pair formation and by protecting a degradation signal recognized from the endoplasmic reticulum (ER) lumenal side. Thus our results suggest that ER degradation of Na,K-ATPase alpha subunits is 1) mainly mediated by folding defects caused by inefficient membrane insertion of certain membrane domains, 2) a multistep process, which involves proteolytic and/or chaperone components acting from the ER lumenal side in addition to cytosolic, proteasome-related factors, and 3) prevented by partner subunit assembly because of direct protection and retrieval of degradation signals from the cytoplasm to the ER lumenal side. These results likely represent a paradigm for the ER quality control of unassembled, polytopic subunits of oligomeric membrane proteins.     

Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval.

Two new thermosensitive yeast mutants defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2-1 and ret3-1 were defective for ER retrieval, only ret2-1 exhibited a defect in forward ER-to-Golgi transport at the non-permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro. The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively. Both proteins show significant homology to clathrin adaptor subunits. These results emphasize the role of coatomer in retrieval of dilysine-tagged proteins back to the ER, and the similarity between clathrin and coatomer coats.     

Masking of Transmembrane‐Based Retention Signals Controls ER Export of γ‐Secretase

γ‐Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of γ‐secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER‐retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of γ‐secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER‐retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER‐retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent γ‐secretase complex could mask TMD‐based ER‐retention/retrieval signals to allow plasma membrane transport of fully assembled γ‐secretase.     

Surface recognition elements of membrane protein oligomerization

Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts.     

Endoplasmic reticulum quality control of unassembled iron transporter depends on Rer1p-mediated retrieval from the golgi

Endoplasmic reticulum (ER) quality control is a conserved process by which misfolded or unassembled proteins are selectively retained in the endoplasmic reticulum (ER). Failure in oligomerization of multisubunit membrane proteins is one of the events that triggers ER quality control. The transmembrane domains (TMDs) of unassembled subunits are determinants of ER retention in many cases, although the mechanism of the TMD-mediated sorting of unassembled subunits remains elusive. We studied a yeast iron transporter complex on the cell surface as a new model system for ER quality control. When Fet3p, a transmembrane subunit, is not assembled with the other membrane subunit, Ftr1p, unassembled Fet3p is exclusively localized to the ER at steady state. The TMD of Fet3p contains a determinant for this process. However, pulse-chase analysis and in vitro budding assays indicate that unassembled Fet3p rapidly escapes from the ER. Furthermore, Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi, is responsible for the TMD-dependent ER retrieval of unassembled Fet3p. These findings provide clear evidence that the ER quality control of unassembled membrane proteins can be achieved by retrieval from the Golgi and that Rer1p serves as a specific sorting receptor in this process.     

Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER

Background  

In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails.     

New COP1-binding motifs involved in ER retrieval.

Coatomer-mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins. For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER). Making use of the two-hybrid system, we characterized a new sequence (deltaL) which interacts specifically with the delta-COP subunit of the COP1 complex. Transfer of deltaL to the cytoplasmic domain of a reporter membrane protein resulted in its localization in the ER, in yeast and mammalian cells. This was due to continuous retrieval of tagged proteins from the Golgi back to the ER, in a manner similar to the ER retrieval of KKXX-tagged proteins. Extensive mutagenesis of deltaL identified an aromatic residue as a critical determinant of the interaction with COP1. Similar COP1-binding motifs containing an essential aromatic residue were identified in the cytoplasmic domain of an ER-resident protein, Sec71p, and in an ER retention motif previously characterized in the CD3epsilon chain of the T-cell receptor. These results emphasize the role of the COP1 complex in retrograde Golgi-to-ER transport and highlight its functional similarity with clathrin-adaptor complexes.     

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.     

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.     

Endoplasmic reticulum localization of DHHC palmitoyltransferases mediated by lysine-based sorting signals

Intracellular palmitoylation dynamics are regulated by a family of 24 DHHC (aspartate-histidine-histidine-cysteine) palmitoyltransferases, which are localized in a compartment-specific manner. The majority of DHHC proteins localize to endoplasmic reticulum (ER) and Golgi membranes, and a small number target to post-Golgi membranes. To date, there are no reports of the fine mapping of sorting signals in mammalian DHHC proteins; thus, it is unclear how spatial distribution of the DHHC family is achieved. Here, we have identified and characterized lysine-based sorting signals that determine the restricted localization of DHHC4 and DHHC6 to ER membranes. The ER targeting signal in DHHC6 conforms to a KKXX motif, whereas the signal in DHHC4 is a distinct KXX motif. The identified dilysine signals are sufficient to specify ER localization as adding the C-terminal pentapeptide sequences from DHHC4 or DHHC6, which contain these KXX and KKXX motifs, to the C terminus of DHHC3, redistributes this palmitoyltransferase from Golgi to ER membranes. Recent work proposed that palmitoylation of newly synthesized peripheral membrane proteins occurs predominantly at the Golgi. Indeed, previous analyses of the peripheral membrane proteins, SNAP25 and cysteine string protein, are fully consistent with their initial palmitoylation being mediated by Golgi-localized DHHC proteins. Interestingly, ER-localized DHHC3 is able to palmitoylate SNAP25 and cysteine string protein to a similar level as wild-type Golgi-localized DHHC3 in co-expression studies. These results suggest that targeting of intrinsically active DHHC proteins to defined membrane compartments is an important factor contributing to spatially restricted patterns of substrate palmitoylation.     

Signals for COPII-dependent export from the ER: what's the ticket out?

Export of many secretory proteins from the endoplasmic reticulum (ER) relies on signal-mediated sorting into ER-derived transport vesicles. Recent work on the coat protein complex II (COPII) provides new insight into the mechanisms and signals that govern this selective export process. Conserved di-acidic and di-hydrophobic motifs found in specific transmembrane cargo proteins are required for their selection into COPII-coated vesicles. These signaling elements are cytoplasmically exposed and recognized by subunits of the COPII coat. Certain soluble cargo molecules depend on receptor-like proteins for efficient ER export, although signals that direct soluble cargo into ER-derived vesicles are less defined.     

ERp29 Restricts Connexin43 Oligomerization in the Endoplasmic Reticulum

Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus.     

Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval.

Dilysine signals confer localization of type I membrane proteins to the endoplasmic reticulum (ER). According to the prevailing model these signals target proteins to the ER by COP I-mediated retrieval from post-ER compartments, whereas the actual retention mechanism in the ER is unknown. We expressed chimeric membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF constructs, which had access to post-ER compartments, the KKAA chimeras were localized to the ER by confocal microscopy and were neither processed by cis-Golgi-specific enzymes in vivo nor included into ER-derived transport vesicles in an in vitro budding assay, suggesting that KKAA-bearing proteins are permanently retained in the ER. The ER localization was nonsaturable and exclusively mediated by the dilysine signal because mutating the lysines to alanines led to cell surface expression of the chimeras. Although the KKAA signal avidly binds COP I in vitro, the ER retention by this signal does not depend on intact COP I in vivo because it was not affected in an epsilon-COP-deficient cell line. We propose that dilysine ER targeting signals can mediate ER retention in addition to retrieval.