Defining components required for transport from the ER to the Golgi complex in yeast

Several complementary approaches have been fruitful in the study of transport from the ER to the Golgi complex in yeast. Mutational analysis has led to the identification of genes required for this process, many of which are now being studied at the molecular and biochemical level. In the case of SEC18, DNA sequence analysis has demonstrated homology to a factor needed for transport in mammalian in vitro systems. In addition, the events that take place at this stage of the secretory pathway have been reconstituted in vitro.     

Dynamics of Golgi matrix proteins after the blockage of ER to Golgi transport

When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.     

Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes

Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP-binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed.     

Identification of a novel ADAMTS9/GON-1 function for protein transport from the ER to the Golgi

A disintegrin-like and metalloprotease with thrombospondin type I motif (ADAMTS9) is a member of the secreted metalloprotease family that is believed to digest extracellular matrix (ECM) proteins outside of cells. Its Caenorhabditis elegans orthologue, GON-1, is involved in ECM degradation and is required for gonad morphogenesis. ADAMTS9 and GON-1 have similar domain structures, and both have a unique C-terminal domain called the "GON domain," whose function remains unknown. Here we show that down-regulation of human ADAMTS9 and C. elegans GON-1 results in the inhibition of protein transport from the endoplasmic reticulum (ER) to the Golgi. This phenotype was rescued by the expression of the GON domain localizing in the ER in human cells and C. elegans. We propose a novel function of ADAMTS9 and GON-1 in the ER that promotes protein transport from the ER to the Golgi. This function is GON-domain dependent but protease activity independent.     

Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.     

Evidence for an ER to Golgi to chloroplast protein transport pathway

Chloroplast protein import is generally believed to occur posttranslationally through the interaction of a precursor protein with the Toc and Tic transport apparatus in the plastid envelope membranes. The cleavable N-terminal transit peptide present on translocated proteins has been considered to be essential and sufficient for targeting. This idea was recently challenged when an analysis of the chloroplast proteome revealed many proteins without a predicted transit peptide. A recent study demonstrates the existence of a novel chloroplast targeting pathway, starting with protein entry into the endoplasmic reticulum and involving the Golgi apparatus.     

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH₂-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.     

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.     

Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast.

Transport and sorting of lipids must occur with specific mechanisms because the membranes of intracellular organelles differ in lipid composition even though most lipid biosynthesis begins in the ER. In yeast, ceramide is synthesized in the ER and transferred to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed. These two facts imply that ceramide can be transported to the Golgi independent of vesicular traffic because IPC synthesis still continues when vesicular transport is blocked in sec mutants. Nonvesicular IPC synthesis in intact cells is not affected by ATP depletion. Using an in vitro assay that reconstitutes the nonvesicular pathway for transport of ceramide, we found that transport is temperature and cytosol dependent but energy independent. Preincubation of ER and Golgi fractions together at 4 degrees C, where ceramide transport does not occur, rendered the transport reaction membrane concentration independent, providing biochemical evidence that ER-Golgi membrane contacts stimulate ceramide transport. A cytosolic protease-sensitive factor is required after establishment of ER-Golgi contacts.     

Biogenesis of endoplasmic reticulum transport vesicles transferring gastric apomucin from ER to Golgi.

Rough endoplasmic reticulum (RER) transport vesicles were generated from gastric mucous cell RER microsomes in the presence of labeled precursors of phospholipids. The vesicles contained 7-10% of their proteins in the form of apomucin (cargo), and 80% of de novo synthesized phosphatidylcholine (PC) was incorporated into the vesicular membrane. In the absence of choline and ethanolamine precursors or in the presence of 3 mM N-ethylmaleimide (NEM), an inhibitor of CTP:phosphocholine cytidylyltransferase, formation of the transport vesicles, their enrichment in the newly synthesized PC, and the total synthesis of PC decreased by 86%, whereas in the presence of 3 mM Zn2+, complete blockage of vesicle formation and PC synthesis was observed. Analysis of the mucin-transporting vesicles indicated that the CTP:phosphocholine cytidylyltransferase and 1,2-diacyl-sn-glycerol:CDP-choline phosphotransferase remained associated with transport vesicles released from ER. The enzymes and other proteins separated from the vesicle surface prior to vesicle fusion with Golgi and the process was induced by phosphorylation. Based on the results of this study, it is proposed that the formation of the ER transport vesicles of gastric mucosal cells is in concert with synthesis of phospholipids and thus in part is regulated by phospholipid-synthesizing enzymes that reside on the membrane during its biogenesis and dissociate from its surface once the task is completed.     

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.     

Ceramide traffic in C6 glioma cells: evidence for CERT-dependent and independent transport from ER to the Golgi apparatus

Intracellular movements of ceramide are strongly limited by its hydrophobic nature, and the mechanisms involved in ceramide transport can represent a crucial aspect of sphingolipid metabolism and signaling. The recent identification of the ceramide specific carrier protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin biosynthesis. In this study we investigated the metabolic and functional role of CERT in C6 glioma cells. These cells were found to constitutively express CERT, the protein being mainly associated with the cytosolic fraction. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids demonstrated that the down regulation of CERT by RNAi technology resulted in a significant but not complete reduction of ceramide metabolism to sphingomyelin, without affecting its utilization for glycosphingolipid biosynthesis. Since nitric oxide is an inhibitor of ceramide ER-to-Golgi traffic and metabolism in C6 glioma cells, we evaluated the possibility that the CERT-mediated transport of ceramide might represent a target for nitric oxide. The data obtained demonstrate that CERT down regulation does not affect the inhibitory activity of nitric oxide on Cer metabolism, and the effects of nitric oxide and CERT silencing on ceramide utilization were additive. These results strongly suggest that a CERT-mediated and a CERT-independent, nitric oxide-sensitive Cer transport coexist in C6 glioma cells and can separately contribute to the control of sphingolipid metabolism and Cer levels in these cells.     

Function of intracellular phospholipase A2 in vectorial transport of apoproteins from ER to Golgi.

1. The cytosolic fraction required in in vitro reconstituted intracellular transport of mucus glycoprotein apopeptide (apomucin) was isolated and its potential as transport supporting factor assessed by the quantitation of the gastric apomucin transferred to Golgi. 2. The experiments with the fraction promoting transport and delivery of apomucin to Golgi revealed that the active protein has the property of phospholipase A2 (PLA2) which assists ER vesicles fusion with Golgi. 3. The ability of the 76 kDa PLA2 to hydrolyze phospholipids and to support transport and fusion of ER vesicles with Golgi was abolished by phosphorylation and regained following dephosphorylation. 4. The data provide evidence that 76 kDa intracellular PLA2 is responsible for the fusion of ER-transport vesicles with Golgi. The process of fusion is accomplished by generation of lysophospholipids in fusing membranes.     

Site-1 and site-2 proteases: A team of two in regulated proteolysis

The site-1 and site-2 proteases (S1P and S2P) were identified over 20 years ago, and the functions of both have been addressed in numerous studies ever since. Whereas S1P processes a set of substrates independently of S2P, the latter acts in concert with S1P in a mechanism, called regulated intramembrane proteolysis, that controls lipid metabolism and response to unfolded proteins. This review summarizes the molecular roles that S1P and S2P jointly play in these processes. As S1P and S2P deficiencies mainly affect connective tissues, yet with varying phenotypes, we discuss the segregated functions of S1P and S2P in terms of cell homeostasis and maintenance of the connective tissues. In addition, we provide experimental data that point at S2P, but not S1P, as a critical regulator of cell adaptation to proteotoxicity or lipid imbalance. Therefore, we hypothesize that S2P can also function independently of S1P activity.     

Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.