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PubMed1996

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Based on sequence homologies with leguminous lectins, the intermediate compartment marker ERGIC-53 was proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independent, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of ERGIC-53 and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannose-column binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glyco-proteins in the early secretory pathway.

Amino Acid Sequence↗
PubMed1995

Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway.

ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.

Amino Acid Sequence↗
PubMed1994

A dual role for COOH-terminal lysine residues in pre-Golgi retention and endocytosis of ERGIC-53.

ERGIC-53 (former designation, p53) is a 53-kDa nonglycosylated, dimeric, and hexameric type I membrane protein that has been established as a marker protein for a tubulovesicular intermediate compartment in which protein transport from the endoplasmic reticulum to the Golgi apparatus is blocked at 15 degrees C. Although ERGIC-53 is not a resident protein of the rough endoplasmic reticulum its cDNA sequence carries a double lysine endoplasmic reticulum retention motif at the cytoplasmically exposed COOH terminus. Here we report that overexpression of ERGIC-53 in COS cells saturates its intracellular retention system leading to the appearance of ERGIC-53 at the cell surface. Cell surface ERGIC-53 is efficiently endocytosed by a mechanism that is disturbed when the two critical lysines of the endoplasmic reticulum retention motif are replaced by serines. The results suggest a mechanistic similarity of pre-Golgi retention by the double lysine motif and lysine-based endocytosis.

Amino Acid Sequence↗
PubMed1993

ERGIC-53, a membrane protein of the ER-Golgi intermediate compartment, carries an ER retention motif.

Overlapping cDNAs encoding the entire human ERGIC-53, a 53 kDa membrane protein of the ER-Golgi intermediate compartment, have been isolated and their nucleotide sequence determined. The isolated cDNA is about 2.7 kb in length. The deduced polypeptide chain for ERGIC-53 consists of 510 amino acids (M(r) 54217) including an N-terminal signal sequence of 30 amino acids, a single putative transmembrane segment of 18 amino acids, and a short cytoplasmic domain of 12 amino acids. Surprisingly, the cytoplasmic segment contains two lysines positioned three and four residues from the C-terminus. Such a double lysine motif is known to function as a retention signal for a group of membrane proteins associated with the ER. Expression of a full-length cDNA of ERGIC-53 in Vero cells revealed intracellular localization similar but not always identical to the endogenously expressed ERGIC-53. The presence of an ER retention motif in a protein of the ER-Golgi intermediate compartment has important implications for the retention mechanism mediated by this signal.

Amino Acid Sequence↗
PubMed1995

ERGIC-53, a membrane protein of the endoplasmic reticulum-Golgi intermediate compartment, is identical to MR60, an intracellular mannose-specific lectin of myelomonocytic cells.

A mannose-specific membrane lectin (MR60) isolated from human myelomonocytic HL60 cells by affinity chromatography is expressed in intracellular organelles of immature monocytes (Pimpaneau, V., Midoux, P., Monsigny, M., and Roche, A. C. (1991) Carbohydr. Res. 213, 95-108). It is not present at the cell surface and is immunochemically and structurally distinct from the M(r) 175,000 mannose receptor of mature macrophages. MR60 cDNA was isolated and characterized; on the basis of its sequence, MR60 is not related to any known mammalian lectins. Surprisingly, MR60 was found to be identical to ERGIC-53 (Schindler, R., Itin, C., Zerial, M., Lottspeich, F., and Hauri, H.P. (1993) Eur. J. Cell Biol. 61, 1-9), a type I integral membrane protein, defined as a marker of the intermediate compartment that recycles between the Golgi apparatus and endoplasmic reticulum; MR60/ERGIC-53 shares with VIP-36 significant homologies with leguminous plant lectins (Fiedler, K., and Simmons, K. (1994) Cell 77, 625-626). We extend these findings in evidencing a structural homology between MR60/ERGIC-53 and mammalian galectins (soluble beta galactose binding proteins). MR60/ERGIC-53 is the first lectin characterized as an endoplasmic reticulum-Golgi protein. Accordingly, this intracellular mannose binding protein could be involved in the traffic of glycoproteins between endoplasmic reticulum and the Golgi apparatus.

Amino Acid Sequence↗
PubMed1994

Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylceramide synthesis in Vero cells with respect to the endoplasmic reticulum-Golgi intermediate compartment.

The identification of an endoplasmic reticulum-Golgi intermediate compartment (ERGIC), defined by the 53-kDa transmembrane marker protein ERGIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the isolation of the ERGIC from Vero cells (Schweizer, A., Matter, K., Ketcham, C. M., and Hauri, H.-P. (1991) J. Cell Biol. 113, 45-54) which provides a means to study more precisely the compartmentalization of the various enzymic functions along the early secretory pathway. Here, we have investigated if O-glycan initiation and sphingomyelin synthesis are associated with the ERGIC by analyzing both the responsible enzyme activities and their corresponding products. Moreover, the synthesis of glucosylceramide, the precursor of most glycosphingolipids, was also analyzed. In the purified ERGIC fraction UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) was only minimally enriched, sphingomyelin synthase was not enriched, and UDP-glucose:ceramide-glucosyl transferase specific activity was lower than in the homogenate. On Percoll gradients all three enzymes cofractionated with Golgi markers rather than ERGIC-53. Accordingly, sphingomyelin concentrations were extremely low in the ERGIC fraction. Double immunofluorescence localization of core N-acetylgalactosamine, the product of GalNAc transferase, by monoclonal antibodies against GalNAc-Ser/Thr (Tn antigen) revealed only little apparent overlap with ERGIC-53. This was particularly evident in brefeldin A-treated cells which showed entirely different patterns of Tn antigens and ERGIC-53. The results suggest that in the secretory pathway of Vero cells O-glycan initiation and sphingomyelin as well as glucosylceramide synthesis mainly occur beyond the ERGIC in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1995

A novel endocytosis signal related to the KKXX ER-retrieval signal.

Membrane proteins often contain a sorting signal in their cytoplasmic tail that promotes their clustering into coated vesicles at a specific cellular site. ERGIC-53 contains a cytoplasmic ER-retrieval signal, KKFF. However, overexpressed ERGIC-53 is transported to the cell surface and rapidly endocytosed. Here we report that ERGIC-53 carries a previously undescribed endocytosis signal. Surprisingly, the signal was KKFF and like the ER-retrieval signal required a C-terminal position. In fact, the minimal consensus sequence determined by substitutional mutagenesis (K-K/R-F/Y-F/Y) was related to the ER-retrieval consensus (K-K-X-X). Furthermore, we provide evidence that internalization of VIP36, a protein that cycles between plasma membrane and Golgi, is mediated by a signal at its C-terminus that matches the internalization consensus sequence. The relatedness of the two signals suggests that coatomer-mediated retrieval of proteins may be mechanistically more related to clathrin-dependent sorting than previously anticipated.

Amino Acid Sequence↗
PubMed1996

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.

Animals↗
PubMed1993

Characterization of a novel 63 kDa membrane protein. Implications for the organization of the ER-to-Golgi pathway.

Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.

Animals↗
PubMed1997

Characterization of brefeldin A induced vesicular structures containing cycling proteins of the intermediate compartment/cis-Golgi network.

Residence of luminal ER proteins is mediated by a cyclic process which involves binding of escaped proteins to a KDEL receptor in a post-ER compartment and redistribution of the ligand-receptor complex back to the ER. We examined the relocation of the KDEL receptor after treatment with the fungal metabolite brefeldin A and compared this with the retrograde transport of the KDEL receptor observed after ligand or receptor overexpression. Incubation with brefeldin A led to the formation of vesicular structures containing the KDEL receptor and ERGIC-53, a marker for the ER-Golgi intermediate compartment. Immunoelectron microscopy revealed that these structures are composed of tubulo-vesicular clusters. The brefeldin A induced vesicular structures were morphologically and biochemically distinct from the ER-Golgi hybrid compartment as demonstrated by double immunofluorescence microscopy and subcellular fractionation. Overexpression of the receptor itself or a lysozyme-KDEL construct led to a shift of the KDEL receptor together with ERGIC-53, an intermediate compartment marker to the ER but not to structures resembling BFA induced vesicular structures. Moreover, overexpression of the receptor resulted in the partial redistribution of marker proteins of the medial Golgi and the trans-Golgi network to ER-like structures. We conclude that the effects of brefeldin A on the redistribution of the KDEL receptor do not reflect physiological events occurring during increased occupancy of the receptor with ligands.

Animals↗
PubMed1997

Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI.

Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP[ct]). Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI. These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex. Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.

Animals↗
PubMed1996

Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein.

An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo-vesicular elements at the endoplasmic reticulum-Golgi interface and the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllöf, B., and Saraste, J. (1992) J. Cell Sci. 103, 321-333). Here we report the purification of p58 by two-dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex, as well as its leakage to the plasma membrane. Immunolocalization using antibodies raised against a lumenal peptide stained the total cellular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex.

Amino Acid Sequence↗
PubMed1995

The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP.

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.

Amino Acid Sequence↗
PubMed1996

Sorting by COP I-coated vesicles under interphase and mitotic conditions.

COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.

Amino Acid Sequence↗
PubMed1997

Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617-634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

Antigens, CD↗
PubMed1994

CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment.

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Söling, H.D. and Hauri, H.P. (1993) Eur. J. Cell Biol. 60, 366-370). Our conclusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and alpha-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In addition experiments, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.

Amino Acid Sequence↗
PubMed1995

Reassessment of the subcellular localization of p63.

p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.

Amino Acid Sequence↗