Intracellular processing of the gp160 HIV-1 envelope precursor. Endoproteolytic cleavage occurs in a cis or medial compartment of the Golgi complex

The intracellular processing of the gp160 HIV-1 envelope precursor was characterized in acutely infected CD4+ T cells. Our data show that gp160 undergoes endoproteolytic cleavage by a nonacid dependent protease(s) in the rough endoplasmic reticulum-Golgi complex, within cis or medial cisternae, and is not transported to the cell surface. Two-dimensional electrophoretic pulse-chase analysis indicates that it takes greater than 2 h for gp160 to be transported from the rough endoplasmic reticulum to the site of action of sialyltransferases in the trans Golgi. Evidence is presented that gp160 is subject to mannose trimming in the Golgi complex, which is inhibited by 1-deoxymannojirimycin (a specific Golgi alpha-mannosidase I inhibitor). Preliminary data also suggest that gp120 is post-translationally modified by sialylated O-linked oligosaccharides.     

Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences.     

ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments

The oligosaccharide processing intermediates of the vesicular stomatitis virus strain ts045 G protein were used to identify ATP- and temperature-sensitive steps in the constitutive pathway of protein transfer to the cell surface. In addition to the initial ATP-sensitive step required for export from the endoplasmic reticulum (Balch, W. E., Elliott, M. M., and Keller, D. S. (1986) J. Biol. Chem. 261, 14681-14689), two distinct ATP-sensitive steps functionally dissect the Golgi into at least 3 compartments: a cis compartment containing the trimming enzyme mannosidase I, a medial compartment conferring resistance to endoglycosidase H, and a trans compartment containing terminal glycosyl transferases. A fourth ATP-sensitive step is required for export of G protein from the trans Golgi to the cell surface. A high threshold of cellular ATP (70% of the control) was required for maximal rates of transport between Golgi compartments. Transport between compartments is inhibited at 40% of the normal cellular ATP pool. Only a single temperature-sensitive step localized to the endoplasmic reticulum inhibited transport of ts045 G protein to the cell surface. The data suggest that ATP-sensitive steps punctuate transport of protein between compartmental boundaries of the secretory pathway.     

Endoplasmic reticulum retention of the large splice variant of the UDP-galactose transporter is caused by a dilysine motif

Nucleotide-sugar transporters supply mainly the Golgi glycosyltransferases with substrates. Some glycosyltransferases in the endoplasmic reticulum (ER), however, also use activated sugars. Recent studies have demonstrated that UDP-galactose (UDP-Gal) is the substrate for the ER resident ceramide-galactosyltransferase (cer-GalT) and cells expressing cer-GalT are able to retain the UDP-Gal transporter (UGT) by physical contacts formed between the two proteins. Here, we describe a second active mechanism for ER localization of the UGT. The UGT is produced in two splice forms UGT1 and UGT2. The proteins vary only at their extreme C-termini but show strikingly different intracellular distribution. Although N-terminally epitope tagged forms of UGT1 localize exclusively to the Golgi, similar constructs of UGT2 show both ER and Golgi localization. The dilysine motif KVKGS contained in UGT2 can be demonstrated to be responsible for the dual localization because: (1) disturbance of the signal via site specific mutation or C-terminal extension completely shifts the transporter to the Golgi, (2) transfer of the dilysine motif is sufficient to redistribute the Golgi CMP-sialic acid transporter to the ER, and (3) replacement of KVKGS by the strong ER retention signal KKNT is sufficient to completely retain UGT2 in the ER.     

MG-160. A novel sialoglycoprotein of the medial cisternae of the Golgi apparatus [published eeratum appears in J Biol Chem 1989 Mar 5;264(7):4264

A monoclonal antibody (mAb 10A8), derived from mice immunized with fractions of the Golgi apparatus from rat brain neurons, was exploited to isolate and partially characterize a novel glycoprotein of 160 kDa apparent molecular mass which was localized by immunoelectron microscopy in medial cisternae of the Golgi apparatus of neurons, glia, pituitary cells, and rat pheochromocytoma (PC 12). The yield of immunoaffinity purified protein was 0.9 microgram/g of rat brain and represented 3% of the Golgi protein; the protein contained asparagine-linked carbohydrates and sialic acid and N-acetylglucosamine residues; unreduced protein had a greater electrophoretic mobility (130 kDa) consistent with the presence of intrachain disulfide bonds. The bulk of the glycoprotein resided within the membrane and/or luminal face of the Golgi cisternae. After extraction with Triton X-114, the glycoprotein was found in both aqueous and detergent phases. The monoclonal antibody did not inhibit the activities of Golgi enzymes or the uptake of nucleotide sugars by intact Golgi vesicles. The findings indicate that the 160-kDa glycoprotein is a specific constituent of medial Golgi cisternae. The results of this study lend support to the hypothesis that the distributions of glycosyltransferases in the Golgi apparatus are cell specific, since in neurons this sialic acid containing glycoprotein is found in medial rather than in trans and/or in the trans Golgi reticulum cisternae, where sialyltransferases have been localized in other cells. Alternatively, resident neuronal Golgi sialoglycoproteins may acquire sialic acid in trans elements of the apparatus and then shuttle back in medial cisternae.     

The trans Golgi face in rat small intestinal absorptive cells

In the small intestine cell differentiation from immature crypt cells to mature absorptive cells localized along the villi is accompanied by alterations in the organization of the trans Golgi side. In immature crypt cells the transmost Golgi cisterna is usually located closely adjacent to the other cisternae thus being a component of the stack. Concomitantly with cell differentiation the transmost cisterna of an increasing number of Golgi stacks sets off from the other cisternae being then located at various distances to the stacks. This transmost cisterna has, as in several other cell types, been interpreted as "GERL" (Golgi associated endoplasmic reticulum lysosomes [20, 28]) and thus, has been postulated to represent a specialized region of the endoplasmic reticulum. Our results, however, have shown that the cytochemical staining pattern which has been used as a basis for the differentiation of GERL from Golgi components is not present in crypt cells nor in mature absorptive cells of the proximal small intestine: identical cisternae react for thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. Thiamine pyrophosphatase and inosine diphosphatase--enzymes characteristic for Golgi cisternae--are apparent over transmost cisternae defined as GERL, too, and in addition, acid phosphatase--postulated as GERL-marker--is demonstrable over stacked Golgi cisternae. This overlapping cytochemical reaction, as well as the alterations during cell differentiation, indicate that those structures which have been described as GERL are to be interpreted as Golgi components rather than as endoplasmic reticulum. On the other hand, endoplasmic reticulum is a constant component of the trans Golgi face in undifferentiated crypt-base cells and in maturing cells of the crypt-top region. From its localization closely adjacent to trans Golgi cisternae it may be termed "Golgi-associated endoplasmic reticulum"; however, these cisternae of endoplasmic reticulum are constantly devoid of acid phosphatase. No indications exist for continuities with the thiamine pyrophosphatase-, inosine diphosphatase-, and acid phosphatase-positive transmost Golgi cisternae, and for an engagement in production of lysosomes.     

Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER

In cells treated with brefeldin A (BFA), movement of newly synthesized membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Surprisingly, the glycoproteins retained in the ER were rapidly processed by cis/medial Golgi enzymes but not by trans Golgi enzymes. An explanation for these observations was provided from morphological studies at both the light and electron microscopic levels using markers for the cis/medial and trans Golgi. They revealed a rapid and dramatic redistribution to the ER of components of the cis/medial but not the trans Golgi in response to treatment with BFA. Upon removal of BFA, the morphology of the Golgi apparatus was rapidly reestablished and proteins normally transported out of the ER were efficiently and rapidly sorted to their final destinations. These results suggest that BFA disrupts a dynamic membrane-recycling pathway between the ER and cis/medial Golgi, effectively blocking membrane transport out of but not back to the ER.     

In vitro transport on cis and trans sides of the Golgi involves two distinct types of coatomer and ADP-ribosylation factor-independent transport intermediates

The cisternal maturation model proposes that secretory proteins transit the Golgi in cisternae that mature by the continuous retrograde transport of Golgi enzymes in vesicles. We have tested the hypothesis that de novo generation of transport intermediates containing medial, trans, and trans Golgi network (TGN) enzymes is reconstituted in vitro. Our analysis shows that the majority of transport is mediated by a steady state of transport intermediate production and consumption by Golgi cisternae, with only a minor contribution of pre-existing transport intermediates. Transport in the medial and trans regions of the stack involved intermediates containing Golgi enzymes, apparently moving in a retrograde direction. In contrast, transport between the trans Golgi and TGN was exclusively mediated by intermediates containing secretory protein, as expected for anterograde transport. These intermediates may be physiologically relevant, because only these two specific types of intermediates can be detected in cell homogenates. By analogy to the coatomer (COPI)-independent transport of Golgi enzymes to the endoplasmic reticulum, the steady-state production of intra-Golgi transport intermediates was not impaired by inhibition of COPI vesicle formation. These data suggest a model for COPI-independent intra-Golgi transport by cisternal maturation with a shift in mechanism to anterograde transport at the trans Golgi and TGN boundary.     

Biosynthesis of chondroitin/dermatan sulfate

Chondroitin sulfate and dermatan sulfate are synthesized as galactosaminoglycan polymers containing N-acetylgalactosmine alternating with glucuronic acid. The sugar residues are sulfated to varying degrees and positions depending upon the tissue sources and varying conditions of formation. Epimerization of any of the glucuronic acid residues to iduronic acid at the polymer level constitutes the formation of dermatan sulfate. Chondroitin/dermatan glycosaminoglycans are covalently attached by a common tetrasaccharide sequence to the serine residues of core proteins while they are adherent to the inner surface of endoplasmic reticulum/Golgi vesicles. Addition of the first sugar residue, xylose, to core proteins begins in the endoplasmic reticulum, followed by the addition of two galactose residues by two distinct glycosyl transferases in the early cis/medial regions of the Golgi. The linkage tetrasaccharide is completed in the medial/trans Golgi by the addition of the first glucuronic acid residue, followed by transfer of N-acetylgalactosamine to initiate the formation of a galactosaminoglycan rather than a glucosaminoglycan. This specific N-acetylgalactosaminyl transferase is different from the chondroitin synthase involved in generation of the repeating disaccharide units to form the chondroitin polymer. Sulfation of the chondroitin polymer by specific sulfotransferases occurs as the polymer is being formed. All the enzymes in the pathway for synthesis have been cloned, with the exception of the glucuronyl to iduronyl epimerase involved in the formation of dermatan residues.     

Membrane topology of the mammalian CMP-sialic acid transporter

Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgi apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP-sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters.     

Variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and Golgi in human hepatoma cells

We have previously shown that newly synthesized liver secretory proteins are exported at three distinct characteristic rates, with intracellular retention half-times of 110-120 min (e.g. transferrin), 75-80 min (e.g. ceruloplasmin), and 30-40 min (e.g. alpha 1-protease inhibitor) (J. B. Parent, H. Bauer, and K. Olden (1985) Biochim. Biophys. Acta, in press). In the present study we have determined the average time required for specific glycoproteins to move through the various compartments of the intracellular transport pathway, consisting of endoplasmic reticulum and Golgi complex. Localization in particular compartments was monitored by the use of the following complementary approaches: (i) Percoll density gradient fractionation of the subcellular organelles, (ii) sensitivity of the glycan moiety of N-linked glycosylation to endo-beta-N-acetylglucosaminidase H, and (iii) by the lectin-binding characteristics. The cell fractionation studies revealed that alpha 1-protease inhibitor, ceruloplasmin, and transferrin were transported from the rough endoplasmic reticulum with a retention half-time of 10, 30, or 45 min, respectively. Measurements of the rate at which newly synthesized glycoprotein became endo H-resistant (an event localized near the medial region of Golgi) demonstrated that it took 60-70, 30, and 18 min for 50% of transferrin, ceruloplasmin, and alpha 1-protease inhibitor, respectively, to reach the medial Golgi. Consistent with this finding, maximal binding of transferrin to wheat germ agglutinin (also a medial Golgi event) and Ricinus communis agglutinin I (a trans Golgi event) required 75 and 90 min, respectively, and maximal binding of ceruloplasmin to both lectins occurred in approximately 30 min. Maximal binding of alpha 1-protease inhibitor to wheat germ agglutinin and Ricinus communis agglutinin I required 15 and 30 min, respectively. The results presented here clearly indicate that (i) the time required for protein secretion cannot be entirely accounted for by lag in transport from the rough endoplasmic reticulum to the Golgi since the glycoproteins examined are retained in the former organelle for no more than two-fifths of the total intracellular retention half-time, and (ii) the variability in rates of protein secretion is not due solely to differences in rates of transport from the rough endoplasmic reticulum to the Golgi as variability in retention within the Golgi is also demonstrated. The results are discussed in terms of their compatibility with receptor-mediated transport of glycoproteins in both the endoplasmic reticulum and Golgi.     

Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole.

Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes

Summary The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogenous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

Endoplasmic Reticulum to Golgi Trafficking in Multinucleated Skeletal Muscle Fibers

The organization of membrane trafficking between endoplasmic reticulum and Golgi within multinucleated muscle fibers was analyzed. We found that markers for the compartment involved in endoplasmic reticulum to Golgi trafficking exhibited perinuclear as well as interfibrillar localization. Furthermore, these markers showed prominent colocalization with microtubules. To analyze membrane trafficking, we followed the temperature-controlled transport of the G protein of the mutant vesicular stomatitis virus, tsO45, in isolated myofibers. Perinuclear and cross-striated staining were seen at 39°C, while at 15°C a diffuse staining component appeared along a subset of interfibrillar microtubules. At 20°C, bright Golgi spots were seen to be associated with microtubules that appeared as circumnuclear rings and longitudinal bundles. Beneath the motor end plate, however, the organization of the Golgi elements and microtubules was found to be distinctive. Retrograde trafficking induced by brefeldin A resulted in the disappearance of the Golgi spots throughout the myofibers and the appearance of staining along microtubules. Thus, interfibrillar membranes seem to be active in protein export, and trafficking between endoplasmic reticulum and Golgi elements occurred throughout the myofibers. The results suggest that microtubules served as tracks for the two-way trafficking between the endoplasmic reticulum and the Golgi compartment.     

A phosphorylation-dependent export structure in ROMK (Kir 1.1) channel overrides an endoplasmic reticulum localization signal

The cell surface density of functional Kir1.1 (ROMK, KCNJ1) channels in the renal collecting duct is precisely regulated to maintain potassium balance. Here, we explore the mechanism by which phosphorylation of Kir1.1a serine 44 controls plasmalemma expression. Studies in Xenopus oocytes, expressing wild-type, phosphorylation mimic (S44D), or phosphorylation null (S44A) Kir1.1a, revealed that phosphorylation of serine 44 is required to stimulate traffic of newly synthesized channels to the plasma membrane through a brefeldin A-sensitive pathway. ROMK channels were found to acquire mature glycosylation in a serine 44 phosphorylation-dependent manner, consistent with a phosphorylation-dependent trafficking step within the endoplasmic reticulum/Golgi. Serine 44 neighbors a string of three "RXR" motifs, reminiscent of basic trafficking signals involved in directing early transport steps within the secretory pathway. Replacement of the arginine residues with alanine (R35A, R37A, R39A, R41A, or all Arg to Ala) did not restore cell surface expression of the phospho-null S44A channel, making it unlikely that phosphorylation abrogates a nearby RXR-type endoplasmic reticulum (ER) localization signal. Instead, analysis of the compound S44D phospho-mimic mutants revealed that the neighboring arginine residues are also necessary for cell surface expression, identifying a structure that determines export in the biosynthetic pathway. Suppressor mutations in a putative dibasic ER retention signal, located within the cytoplasmic C terminus (K370A, R371A), restored cell surface expression of the phospho-null S44A channel to levels exhibited by the phospho-mimic S44D channel. Taken together, these studies indicate that phosphorylation of Ser44 drives an export step within the secretory pathway to override an independent endoplasmic reticulum localization signal.     

Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation

The O-linked oligosaccharides of mucin-type glycoproteins contain N- acetyl-D-galactosamine (GalNAc) that is not found in N-linked glycoproteins. Because Helix pomatia lectin interacts with terminal GalNAc, we used this lectin, bound to particles of colloidal gold, to localize such sugar residues in subcellular compartments of intestinal goblet cells. When thin sections of low temperature Lowicryl K4M embedded duodenum or colon were incubated with Helix pomatia lectin- gold complexes, no labeling could be detected over the cisternal space of the nuclear envelope and the rough endoplasmic reticulum. A uniform labeling was observed over the first and several subsequent cis Golgi cisternae and over the last (duodenal goblet cells) or the two last (colonic goblet cells) trans Golgi cisternae as well as forming and mature mucin droplets. However, essentially no labeling was detected over several cisternae in the central (medial) region of the Golgi apparatus. The results strongly suggest that core O-glycosylation takes place in cis Golgi cisternae but not in the rough endoplasmic reticulum. The heterogenous labeling for GalNAc residues in the Golgi apparatus is taken as evidence that termination of certain O- oligosaccharide chains by GalNAc occurs in trans Golgi cisternae.     

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.     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes. Comparison with the lead technique

The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogeneous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER

Erv41p and Erv46p form an integral membrane protein complex that cycles between the endoplasmic reticulum (ER) and Golgi. Both proteins contain a large lumenal domain and short N- and C-terminal tail sequences exposed to the cytosol. The coat protein complex II (COPII) packages the Erv41p-Erv46p complex into ER-derived vesicles for delivery to the Golgi. We determined signals in the Erv41p-Erv46p complex that are required for COPII-dependent export from the ER. Mutants lacking the Erv41p or Erv46p C-terminus accumulated in the ER and were not packaged efficiently into vesicles. We identified an isoleucine-leucine sequence in the Erv41p tail that was required for COPII binding and inclusion of the complex into vesicles. This signal was sufficient for COPII binding but not for ER export. The Erv46p tail contains a phenylalanine-tyrosine sequence required together with the isoleucine-leucine signal in Erv41p for export of the complex. Surprisingly, Erv41p- Erv46p tail-swapped chimeras were not exported from the ER, indicating that signals in both the Erv41p and the Erv46p tail sequences are required in a specific orientation for efficient packaging of the Erv41p-Erv46p complex.