ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.     

Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells

To analyze the role of glucose trimming and reglucosylation in the binding of substrate proteins to calnexin in the endoplasmic reticulum (ER) of living cells, we made use of the thermosensitive vesicular stomatitis virus tsO45 glycoprotein (G protein). At nonpermissive temperature the G protein failed to fold completely and remained bound to calnexin. When the cells were shifted to permissive temperature, complete folding occurred accompanied by glucosidase-mediated elimination of calnexin-G protein complexes. If release from calnexin was blocked during the temperature shift by inhibiting the glucosidases, folding occurred, albeit at a reduced rate. In contrast, when unfolded by a shift from permissive to nonpermissive temperature, the G protein was reglucosylated rapidly and became capable of rebinding to calnexin. The rate at which calnexin binding occurred showed a 20-min delay that was explained by accumulation of the G protein in calnexin-free exit sites of the ER. These contained the glucosyltransferase responsible for reglucosylation of misfolded glycoproteins but had little or no calnexin. After unfolding and reglucosylation, the G proteins moved slowly from these structures back to the ER where they reassociated with the chaperone. Taken together, these results in live cells fully supported the lectin-only model of calnexin function. The ER exit sites emerged as a potentially important location for components of the quality control system.     

Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus

Proteins synthesized in the ER are generally transported to the Golgi complex and beyond only when they have reached a fully folded and assembled conformation. To analyze how the selective retention of misfolded proteins works, we monitored the long-term fate of a membrane glycoprotein with a temperature-dependent folding defect, the G protein of tsO45 vesicular stomatitis virus. We used indirect immunofluorescence, immunoelectron microscopy, and a novel Nycodenz gradient centrifugation procedure for separating the ER, the intermediate compartment, and the Golgi complex. We also employed the folding and recycling inhibitors dithiothreitol and AIF4-, and coimmunoprecipitation with calnexin antibodies. The results showed that the misfolded G protein is not retained in the ER alone; it can move to the intermediate compartment and to the cis-Golgi network but is then recycled back to the ER. In the ER it is associated with calnexin and BiP/GRP78. Of these two chaperones, only BiP/GRP78 seems to accompany it through the recycling circuit. Thus, the retention of this misfolded glycoprotein is the result of multiple mechanisms including calnexin binding in the ER and selective retrieval from the intermediate compartment and the cis-Golgi network.     

Endoplasmic reticulum retention,degradation, and aggregation of olfactory G-protein coupled receptors

The mammalian olfactory G-protein coupled receptor family is comprised of hundreds of proteins that mediate odorant binding and initiate signal transduction cascades leading to the sensation of smell. However, efforts to functionally express olfactory receptors and identify specific odorant ligand–olfactory receptor interactions have been severely impeded by poor olfactory receptor surface expression in heterologous systems. Therefore, experiments were performed to elucidate the cellular mechanism(s) responsible for inefficient olfactory receptor cell surface expression. We determined that the mouse odorant receptors mI7 and mOREG are not selected for export from the ER and therefore are not detectable at the Golgi apparatus or plasma membrane. Specifically, olfactory receptors interact with the ER chaperone calnexin, are excluded from ER export sites, do not accumulate in ER–Golgi transport intermediates at 15 °C, and contain endoglycosidase H-sensitive oligosaccharides, consistent with olfactory receptor exclusion from post-ER compartments. A labile pool of ER-retained olfactory receptors are post-translationally modified by polyubiquitination and targeted for degradation by the proteasome. In addition, olfactory receptors are sequestered into ER aggregates that are degraded by autophagy. Collectively, these data demonstrate that poor surface expression of olfactory receptors in heterologous cells is attributable to a combination of ER retention due to inefficient folding and poor coupling to ER export machinery, aggregation, and degradation via both proteasomal and autophagic pathways Plasmids .     

Sec7p directs the transitions required for yeast Golgi biogenesis

Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56–68). The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function. The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins. Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature. This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites. These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype. Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells. These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition. Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins. These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation.     

Structural integrity of the Golgi is temperature sensitive in conditional-lethal mutants with no detectable GM130

At 39.5 °C in the temperature-sensitive, conditional-lethal mutant ldlG, glycoprotein processing is disrupted and secretion is blocked. The ultrastructure of the Golgi apparatus in ldlG cells was examined using immunofluorescence and immunoelectron microscopy. At 34 °C the structure of the Golgi apparatus was normal, whereas after incubation at 39.5 °C for 12 h it disassembled into dispersed vesicles. These reassembled into stacks when cells were returned to 34 °C for 6 h. At both 34 and 39.5 °C, all Golgi markers examined were present at wild-type levels except GM130, which was undetectable (<5% of control). Transfection with GM130 corrected the mutant phenotypes. Although the endogenous gene encoding NSF is apparently normal in ldlG cells, all mutant phenotypes were corrected by transfection with NSF, suggesting that NSF functioned as an extragenic suppressor. These findings provide additional support for a role of GM130 in determining the properties of the Golgi apparatus and for NSF in influencing GM130 stability and function. They also suggest that, at 34 °C, detectable levels of GM130 are not required for normal Golgi morphology and function, but that GM130 – or a GM130-dependent protein(s) – does play a role in protecting the Golgi, and thus the cells, from stress at higher temperatures .     

ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.     

Roles for alpha(2)p24 and COPI in endoplasmic reticulum cargo exit site formation.

A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg(2+)ATP and Mg(2+)GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins alpha(2)p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of alpha(2)p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of alpha(2)p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPgammaS, inhibition by Brefeldin A (BFA), or depletion of beta-COP from cytosol. Therefore, the p24 family member, alpha(2)p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the generation of ER cargo exit sites.     

Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment

What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum–derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.     

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

Release from Endoplasmic Reticulum Matrix Proteins Controls Cell Surface Transport of MHC Class I Molecules

The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi‐step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES) and retrieval from the ER‐Golgi intermediate compartment or the cis‐Golgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine major histocompatibility complex (MHC) class I peptide receptor, H‐2D^b and H‐2K^b. We find that a novel pre‐ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix.      

A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound-activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of beta-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated beta-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with beta-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, beta-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15 degrees C. These data suggest that the Rab2 protein plays a role in the low-temperature-sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.     

Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.     

Dynamic equilibrium between vesicular stomatitis virus glycoprotein monomers and trimers in the Golgi and at the cell surface.

Previous studies have shown that trimers of the vesicular stomatitis virus glycoprotein (VSV G protein) are in rapid equilibrium with monomeric subunits after folding and assembly in the endoplasmic reticulum (ER). To determine whether G protein trimers were in equilibrium with monomers in other cellular compartments, we studied heterotrimer formation between VSV G protein and a mutant G protein (G mu protein) containing a 3-amino-acid cytoplasmic domain replacing the normal 29-amino-acid domain. The G mu protein is transported from the ER much more slowly than G protein, although both G and G mu proteins form trimers rapidly in the ER. In coexpression experiments, we observed that VSV G protein molecules exited the ER about sixfold faster than G mu protein molecules, and we observed no heterotrimer formation in the ER, probably because of rapid reassortment of the mutant and wild-type trimers. However, heterotrimer formation between the two proteins was observed after long chase periods that allowed time for trimers of the mutant protein to reach the plasma membrane and reassort with the G protein subunits. Additional studies showed that heterotrimers of the two proteins could form in the Golgi or in the ER if exit of the G protein from either compartment was blocked.     

Identification of a Site in Sar1 Involved in the Interaction with the Cytoplasmic Tail of Glycolipid Glycosyltransferases

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1^D198A) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1^D189A also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.     

Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum

Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.     

An Ordered Inheritance Strategy for the Golgi Apparatus: Visualization of Mitotic Disassembly Reveals a Role for the Mitotic Spindle

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.     

Diacylglycerol Kinase δ Suppresses ER-to-Golgi Traffic via Its SAM and PH Domains

We report here that the anterograde transport from the endoplasmic reticulum (ER) to the Golgi was markedly suppressed by diacylglycerol kinase delta (DGKdelta) that uniquely possesses a pleckstrin homology (PH) and a sterile alpha motif (SAM) domain. A low-level expression of DGKdelta in NIH3T3 cells caused redistribution into the ER of the marker proteins of the Golgi membranes and the vesicular-tubular clusters (VTCs). In this case DGKdelta delayed the ER-to-Golgi traffic of vesicular stomatitis virus glycoprotein (VSV G) and also the reassembly of the Golgi apparatus after brefeldin A (BFA) treatment and washout. DGKdelta was demonstrated to associate with the ER through its C-terminal SAM domain acting as an ER-targeting motif. Both of the SAM domain and the N-terminal PH domain of DGKdelta were needed to exert its effects on ER-to-Golgi traffic. Kinase-dead mutants of DGKdelta were also effective as the wild-type enzyme, suggesting that the catalytic activity of DGK was not involved in the present observation. Remarkably, the expression of DGKdelta abrogated formation of COPII-coated structures labeled with Sec13p without affecting COPI structures. These findings indicate that DGKdelta negatively regulates ER-to-Golgi traffic by selectively inhibiting the formation of ER export sites without significantly affecting retrograde transport.