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.     

Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum.

Several families of transmembrane endoplasmic reticulum (ER) proteins contain retention motifs in their cytoplasmically exposed tails. Mutational analyses demonstrated that two lysines positioned three and four or five residues from the C-terminus represent the retention motif. The introduction of a lysine preceding the lysine that occurs three residues from the terminus of Lyt2 renders this cell surface protein a resident of the ER. Likewise, the appropriate positioning of two lysine residues in a poly-serine sequence confines marker proteins to the ER. Arginines or histidines cannot replace lysines, suggesting that simple charge interactions are not sufficient to explain the retention. The identified consensus motif may serve as a retrieval signal that brings proteins back from a sorting compartment adjacent to the ER.     

Retrieval of transmembrane proteins to the endoplasmic reticulum

A COOH-terminal double lysine motif maintains type I transmembrane proteins in the ER. Proteins tagged with this motif, eg., CD8/E19 and CD4/E19, rapidly receive post-translational modifications characteristic of the intermediate compartment and partially colocalized to this organelle. These proteins also received modifications characteristic of the Golgi but much more slowly. Lectin staining localized these Golgi modified proteins to ER indicating that this motif is a retrieval signal. Differences in the subcellular distribution and rate of post-translational modification of CD8 maintained in the ER by sequences derived from a variety of ER resident proteins suggested that the efficiency of retrieval was dependent on the sequence context of the double lysine motif and that retrieval may be initiated from multiple positions along the exocytotic pathway.     

The double lysine motif of tapasin is a retrieval signal for retention of unstable MHC class I molecules in the endoplasmic reticulum

Tapasin (tpn), an essential component of the MHC class I (MHC I) loading complex, has a canonical double lysine motif acting as a retrieval signal, which mediates retrograde transport of escaped endoplasmic reticulum (ER) proteins from the Golgi back to the ER. In this study, we mutated tpn with a substitution of the double lysine motif to double alanine (GFP-tpn-aa). This mutation abolished interaction with the coatomer protein complex I coatomer and resulted in accumulation of GFP-tpn-aa in the Golgi compartment, suggesting that the double lysine is important for the retrograde transport of tpn from late secretory compartments to the ER. In association with the increased Golgi distribution, the amount of MHC I exported from the ER to the surface was increased in 721.220 cells transfected with GFP-tpn-aa. However, the expressed MHC I were less stable and had increased turnover rate. Our results suggest that tpn with intact double lysine retrieval signal regulates retrograde transport of unstable MHC I molecules from the Golgi back to the ER to control the quality of MHC I Ag presentation.     

Endoplasmic Reticulum Protein Targeting of Phospholamban: A Common Role for an N-Terminal Di-Arginine Motif in ER Retention?

Background

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase, which transports Ca²⁺ into the SR lumen, leading to muscle relaxation. A mutation of PLN in which one of the di-arginine residues at positions 13 and 14 was deleted led to a severe, early onset dilated cardiomyopathy. Here we were interested in determining the cellular mechanisms involved in this disease-causing mutation.

Methodology/Principal Finding

Mutations deleting codons for either or both Arg13 or Arg14 resulted in the mislocalization of PLN from the ER. Our data show that PLN is recycled via the retrograde Golgi to ER membrane traffic pathway involving COP-I vesicles, since co-immunoprecipitation assays determined that COP I interactions are dependent on an intact di-arginine motif as PLN RΔ14 did not co-precipitate with COP I containing vesicles. Bioinformatic analysis determined that the di-arginine motif is present in the first 25 residues in a large number of all ER/SR Gene Ontology (GO) annotated proteins. Mutations in the di-arginine motif of the Sigma 1-type opioid receptor, the β-subunit of the signal recognition particle receptor, and Sterol-O-acyltransferase, three proteins identified in our bioinformatic screen also caused mislocalization of these known ER-resident proteins.

Conclusion

We conclude that PLN is enriched in the ER due to COP I-mediated transport that is dependent on its intact di-arginine motif and that the N-terminal di-arginine motif may act as a general ER retrieval sequence.     

Arginine/lysine residues in the cytoplasmic tail promote ER export of plant glycosylation enzymes

Plant N -glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study signals and mechanisms, promoting their localization and retention in the endoplasmic reticulum (ER) and Golgi apparatus. Herein, we focused on a detailed investigation of amino acid sequence motifs present in their short cytoplasmic tails in respect to ER export. Using site-directed mutagenesis, we determined that single arginine/lysine residues within the cytoplasmic tail are sufficient to promote rapid Golgi targeting of Golgi-resident N -acetylglucosaminyltransferase I (GnTI) and α-mannosidase II (GMII). Furthermore, we reveal that an intact ER export motif is essential for proper in vivo function of GnTI. Coexpression studies with Sar1p provided evidence for COPII-dependent transport of GnTI to the Golgi. Our data provide evidence that efficient ER export of Golgi-resident plant N -glycan processing enzymes occurs through a selective mechanism based on recognition of single basic amino acids present in their cytoplasmic tails.     

Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites

Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP‐A and VAP‐B, interact with proteins from other organelles that possess a small VAP‐interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain‐containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro. MOSPD2 is an ER‐anchored protein, and it interacts with several FFAT‐containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle‐bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.     

Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.     

gp25L/emp24/p24 Protein Family Members of the cis-Golgi Network Bind Both COP I and II Coatomer

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.     

Endothelial receptor tyrosine kinases involved in angiogenesis

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge- independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p- Emp47p hybrid protein was mislocalized to the cell surface in the alpha- COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di- lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.     

ER Export of KAT1 is Correlated to the Number of Acidic Residues within a Triacidic Motif

For a number of ion channels, including the potassium (K⁺) inward rectifying channel from Arabidopsis thaliana (KAT1), diacidic endoplasmic reticulum (ER) export motifs have been identified. These motifs consist of two acidic amino acids (aspartate (D) and/or glutamate (E)) separated by any amino acid. To specify the role of single acidic amino acids for efficiency of ER export, we analysed a sequence of KAT1 that included the originally identified diacidic ER export motif (DxE) plus an additional D just upstream of the diacidic motif. Analysis of single, double and triple mutations of the acidic amino acids of the DxDxE motif revealed a gradual reduction of ER export depending on the number of mutated acidic residues. The amount of reduction in ER export was not related to the position, but only to the number of mutated acidic amino acids. These results show that a triacidic motif is essential for efficient ER export of KAT1. Function of the triacidic motif probably involves cooperative binding to Sec24.     

Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site-directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein

Like other coronaviruses, severe acute respiratory syndrome coronavirus (SARS CoV) assembles at and buds into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). Accumulation of the viral envelope proteins at this compartment is a prerequisite for virus assembly. Previously, we reported the identification of a dibasic motif (KxHxx) in the cytoplasmic tail of the SARS CoV spike (S) protein that was similar to a canonical dilysine ER retrieval signal. Here we demonstrate that this motif is a novel and functional ER retrieval signal which reduced the rate of traffic of the full-length S protein through the Golgi complex. The KxHxx motif also partially retained two different reporter proteins in the ERGIC region and reduced their rates of trafficking, although the motif was less potent than the canonical dilysine signal. The dibasic motif bound the coatomer complex I (COPI) in an in vitro binding assay, suggesting that ER retrieval may contribute to the accumulation of SARS CoV S protein near the virus assembly site for interaction with other viral structural proteins. In support of this, we found that the dibasic motif on the SARS S protein was required for its localization to the ERGIC/Golgi region when coexpressed with SARS membrane (M) protein. Thus, the cycling of SARS S through the ER-Golgi system may be required for its incorporation into assembling virions in the ERGIC.     

Sorting competition with membrane-permeable peptides in intact epithelial cells revealed discrimination of transmembrane proteins not only at the trans-Golgi network but also at pre-Golgi stages

Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif.     

A molecular specificity code for the three mammalian KDEL receptors

AC-terminal KDEL-like motif prevents secretion of soluble endoplasmic reticulum (ER)–resident proteins. This motif interacts with KDEL receptors localized in the intermediate compartment and Golgi apparatus. Such binding triggers retrieval back to the ER via a coat protein I–dependent pathway. To date, two human KDEL receptors have been reported. Here, we report the Golgi localization of a third human KDEL receptor. Using a reporter construct system from a screen of 152 variants, we identified 35 KDEL-like variants that result in efficient ER localization but do not match the current Prosite motif for ER localization ([KRHQSA]-[DENQ]-E-L). We cloned 16 human proteins with one of these motifs and all were found in the ER. A subsequent screen by bimolecular fluorescence complementation determined the specificities of the three human KDEL receptors. Each KDEL receptor has a unique pattern of motifs with which it interacts. This suggests a specificity in the retrieval of human proteins that contain different KDEL variants.     

Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+ transporter Gdt1p are required for calcium transport

The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E‐φ‐G‐D‐[KR]‐[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca²⁺ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site‐directed mutagenesis, we generated 17 mutations in the yeast Golgi‐localized Ca²⁺ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca²⁺‐ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation.     

Plant ERD2s self-interact and interact with GTPase-activating proteins and ADP-ribosylation factor 1

ERD2s (ER luminal protein receptors)-mediated retrograde transport is one of the most substantial processes to maintain the endoplasmic reticulum (ER) homeostasis. It is completed by the recognition of the escaped ER luminal proteins, the gathering into COP I vesicle, and the fusion and releasing into the ER. ERD2s can recognize HDEL/KDEL motifs at the C-terminal of the escaped ER luminal proteins at the Golgi to initiate the retrograde transport. However, these mechanisms remain largely unknown in plants. We recently found that two Nicotiana benthamiana homologs, ERD2a and ERD2b, functioned as ER luminal protein receptors, were required for both HDEL/KDEL motifs-mediated ER retrieval and participated in cell death triggered by ER stress and nonhost pathogens. Here, we provide a set of new data that ERD2a/2b can form homo- or hetero-oligomerization and interact with both the ADP-ribosylation factor 1 (ARF1) and its potential GTPase-activating proteins (GAP) indicated by the firefly luciferase complementation imaging assay (LCI). These evidences further support the ER luminal protein receptor function of ERD2a/2b in plants and suggest their evolutionarily conserved mechanism during the retrograde trafficking. We also analyze the characteristics of ERD2s within a species and among different species.     

GGA Autoinhibition Revisited

The cytosolic adaptors GGA1‐3 mediate sorting of transmembrane proteins displaying a C‐terminal acidic dileucine motif (DXXLL) in their cytosolic domain. GGA1 and GGA3 contain similar but intrinsic motifs that are believed to serve as autoinhibitory sites activated by the phosphorylation of a serine positioned three residues upstream of the DXXLL motif. In the present study, we have subjected the widely acknowledged concept of GGA1 autoinhibition to a thorough structural and functional examination. We find that (i) the intrinsic motif of GGA1 is inactive, (ii) only C‐terminal DXXLL motifs constitute active GGA binding sites, (iii) while aspartates and phosphorylated serines one or two positions upstream of the DXXLL motif increase GGA1 binding, phosphoserines further upstream have little or no influence and (iv) phosphorylation of GGA1 does not affect its conformation or binding to Sortilin and SorLA. Taken together, our findings seem to refute the functional significance of GGA autoinhibition in particular and of intrinsic GGA binding motifs in general.