Recycling of proteins from the Golgi compartment to the ER in yeast

In the yeast Saccharomyces cerevisiae, the carboxyl terminal sequence His-Asp-Glu-Leu (HDEL) has been shown to function as an ER retention sequence (Pelham, H. R. B., K. G. Hardwick, and M. J. Lewis. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1757-1762). To examine the mechanism of retention of soluble ER proteins in yeast, we have analyzed the expression of a preproalpha factor fusion protein, tagged at the carboxyl terminus with the HDEL sequence. We demonstrate that this fusion protein, expressed in vivo, accumulates intracellularly as a precursor containing both ER and Golgi-specific oligosaccharide modifications. The Golgi-specific carbohydrate modification, which occurs in a SEC18-dependent manner, consists of alpha 1-6 mannose linkages, with no detectable alpha 1-3 mannose additions, indicating that the transit of the HDEL-tagged fusion protein is confined to an early Golgi compartment. Results obtained from the fractionation of subcellular organelles from yeast expressing HDEL-tagged fusion proteins suggest that the Golgi-modified species are present in the ER. Overexpression of HDEL-tagged preproalpha factor results in the secretion of an endogenous HDEL-containing protein, demonstrating that the HDEL recognition system can be saturated. These results support the model in which the retention of these proteins in the ER is dependent on their receptor-mediated recycling from the Golgi complex back to the ER.     

An acidic sequence of a putative yeast Golgi membrane protein binds COPII and facilitates ER export.

We previously identified Sys1p as a high copy number suppressor of Ypt6 GTPase-deficient yeast mutants that are defective in endosome-to-Golgi transport. Here, we show that Sys1p is an integral membrane protein that resides on a post-endoplasmic reticulum (ER) organelle(s). Affinity studies with detergent- solubilized yeast proteins showed that the C-terminal 53 amino acid tail of Sys1p binds effectively to the cytoplasmic Sec23p-Sec24p COPII subcomplex. This binding required a di-acidic Asp-Leu-Glu (DXE) motif, previously shown to mediate efficient ER export of the vesicular stomatitis virus glycoprotein in mammalian cells. In Sys1p, a Glu-Leu-Glu (EXE) sequence could not substitute for the (DXE) motif. Mutations of the (DXE) sequence resulted in ER retention of approximately 30% of the protein at steady state, whereas addition of the Sys1p tail to an ER-resident membrane protein led to an intracellular redistribution of the chimeric protein. Our study demonstrates for the first time that, in yeast, a di-acidic sequence motif can act as a sorting signal for cargo selection during the formation of transport vesicles at the ER by direct binding to COPII component(s).     

Bos1p, a membrane protein required for ER to Golgi transport in yeast, co-purifies with the carrier vesicles and with Bet1p and the ER membrane.

BOS1 and BET1 are required for transport from the ER to the Golgi complex in yeast and genetically interact with each other and a subset of the other genes, whose products function at this stage of the secretory pathway. In a previous study, we reported that BOS1 encodes a putative 27 kDa membrane protein. Here we show that BET1 is structurally similar to the synaptobrevins and identical to the SLY12 gene product. Overexpression of SLY12 compensates for the loss of function of the ras-like GTP-binding protein Ypt1. Both Bos1p and Bet1p are cytoplasmically oriented membrane proteins. Bos1p co-purifies with the ER to Golgi transport vesicles and co-fractionates with Bet1p and the ER membrane.     

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.     

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.     

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.     

The BOS1 gene encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast

We recently described the identification of BOS1 (Newman, A., J. Shim, and S. Ferro-Novick. 1990. Mol. Cell. Biol. 10:3405-3414.). BOS1 is a gene that in multiple copy suppresses the growth and secretion defect of bet1 and sec22, two mutants that disrupt transport from the ER to the Golgi complex in yeast. The ability of BOS1 to specifically suppress mutants blocked at a particular stage of the secretory pathway suggested that this gene encodes a protein that functions in this process. The experiments presented in this study support this hypothesis. Specifically, the BOS1 gene was found to be essential for cellular growth. Furthermore, cells depleted of the Bos1 protein fail to transport pro-alpha-factor and carboxypeptidase Y (CPY) to the Golgi apparatus. This defect in export leads to the accumulation of an extensive network of ER and small vesicles. DNA sequence analysis predicts that Bos1 is a 27-kD protein containing a putative membrane-spanning domain. This prediction is supported by differential centrifugation experiments. Thus, Bos1 appears to be a membrane protein that functions in conjunction with Bet1 and Sec22 to facilitate the transport of proteins at a step subsequent to translocation into the ER but before entry into the Golgi apparatus.     

The yeast orthologue of GRASP65 forms a complex with a coiled-coil protein that contributes to ER to Golgi traffic

The mammalian Golgi protein GRASP65 is required in assays that reconstitute cisternal stacking and vesicle tethering. Attached to membranes by an N-terminal myristoyl group, it recruits the coiled-coil protein GM130. The relevance of this system to budding yeasts has been unclear, as they lack an obvious orthologue of GM130, and their only GRASP65 relative (Grh1) lacks a myristoylation site and has even been suggested to act in a mitotic checkpoint. In this study, we show that Grh1 has an N-terminal amphipathic helix that is N-terminally acetylated and mediates association with the cis-Golgi. We find that Grh1 forms a complex with a previously uncharacterized coiled-coil protein, Ydl099w (Bug1). In addition, Grh1 interacts with the Sec23/24 component of the COPII coat. Neither Grh1 nor Bug1 are essential for growth, but biochemical assays and genetic interactions with known mediators of vesicle tethering (Uso1 and Ypt1) suggest that the Grh1-Bug1 complex contributes to a redundant network of interactions that mediates consumption of COPII vesicles and formation of the cis-Golgi.     

Isolation of a functional vesicular intermediate that mediates ER to Golgi transport in yeast

We have used an in vitro assay that reconstitutes transport from the ER to the Golgi complex in yeast to identify a functional vesicular intermediate in transit to the Golgi apparatus. Permeabilized yeast cells, which serve as the donor in this assay, release a homogeneous population of vesicles that are biochemically distinct from the donor ER fraction. The isolated vesicles, containing a post-ER/pre-Golgi form of the marker protein pro-alpha-factor, were able to bind to and fuse with exogenously added Golgi membranes. The ability to isolate fusion competent vesicles provides direct evidence that ER to Golgi membrane transport is mediated by a discrete population of vesicular carriers.     

Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyses have suggested that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early region of the Golgi apparatus. The rer1 mutant we isolated in a previous study mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene and determined its reading frame. RER1 encodes a hydrophobic protein of 188 amino acid residues containing four putative membrane spanning domains. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER, implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an epitope derived from the influenza hemagglutinin was added to the C-terminus of Rer1p and the cells expressing this tagged but functional protein were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern that is typical for Golgi proteins and clearly distinct from the ER staining. This punctate staining was in fact exaggerated in the sec7 mutant that accumulates the Golgi membranes at the restrictive temperature. Furthermore, double staining of Rer1p and Ypt1p, a GTPase that is known to reside in the Golgi apparatus, showed good colocalization. Subcellular fractionation experiments indicated that the fractionation pattern of Rer1p was similar to that of an early Golgi protein, Och1p. From these results, we suggest that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.     

Biochemical and genetic evidence for the involvement of yeast Ypt6-GTPase in protein retrieval to different Golgi compartments

Yeast Ypt6p, the homologue of the mammalian Rab6 GTPase, is not essential for cell viability. Based on previous studies with ypt6 deletion mutants, a regulatory role of the GTPase either in protein retrieval to the trans-Golgi network or in forward transport between the endoplasmic reticulum (ER) and early Golgi compartments was proposed. To assess better the primary role(s) of Ypt6p, temperature-sensitive ypt6 mutants were generated and analyzed biochemically and genetically. Defects in N-glycosylation of proteins passing the Golgi and of Golgi-resident glycosyltransferases as well as protein sorting defects in the trans-Golgi were recorded shortly after functional loss of Ypt6p. ER-to-Golgi transport and protein secretion were delayed but not interrupted. Mis-sorting of the vesicular SNARE Sec22p to the late Golgi was also observed. Combination of the ypt6-2 mutant allele with a number of mutants in forward and retrograde transport between ER, Golgi, and endosomes led to synthetic negative growth defects. The results obtained indicate that Ypt6p acts in endosome-to-Golgi, in intra-Golgi retrograde transport, and possibly also in Golgi-to-ER trafficking.     

The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the Golgi in yeast

Gcs1 is an Arf GTPase-activating protein (Arf-GAP) that mediates Golgi-ER and post-Golgi vesicle transport in yeast. Here we show that the Snc1,2 v-SNAREs, which mediate endocytosis and exocytosis, interact physically and genetically with Gcs1. Moreover, Gcs1 and the Snc v-SNAREs colocalize to subcellular structures that correspond to the trans-Golgi and endosomal compartments. Studies performed in vitro demonstrate that the Snc-Gcs1 interaction results in the efficient binding of recombinant Arf1Delta17N-Q71L to the v-SNARE and the recruitment of purified coatomer. In contrast, the presence of Snc had no effect on Gcs1 Arf-GAP activity in vitro, suggesting that v-SNARE binding does not attenuate Arf1 function. Disruption of both the SNC and GCS1 genes results in synthetic lethality, whereas overexpression of either SNC gene inhibits the growth of a distinct subset of COPI mutants. We show that GFP-Snc1 recycling to the trans-Golgi is impaired in gcs1Delta cells and these COPI mutants. Together, these results suggest that Gcs1 facilitates the incorporation of the Snc v-SNAREs into COPI recycling vesicles and subsequent endosome-Golgi sorting in yeast.     

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.     

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.     

A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.     

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward‐bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi‐to‐plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal‐deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail‐anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy‐ and Rab6‐dependent, and Rab6 inhibition accelerated signal‐deleted VSVG's transport to the cell surface. Our results extend the dynamic bi‐directional relationship between the Golgi and ER to include surface‐directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.     

SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex

The ERD2 gene, which encodes the yeast HDEL (His-Asp-Glu-Leu) receptor, is essential for growth (Semenza, J. C., K. G. Hardwick, N. Dean, and H. R. B. Pelham. 1990. Cell. 61:1349-1357; Lewis, M. J., D. J. Sweet, and H. R. B. Pelham. 1990. Cell. 61:1359-1363). SED5, when present in multiple copies, enables cells to grow in the absence of Erd2p. Sequence analysis of SED5 reveals no significant homology with ERD2 or other known genes. We have raised antibodies to Sed5p which specifically recognize a 39-kD integral membrane protein. A stretch of hydrophobic residues at the COOH terminus is predicted to hold Sed5p on the cytoplasmic face of intracellular membranes. Cells that are depleted of Sed5p are unable to transport carboxypeptidase Y to the Golgi complex, and stop growing after a dramatic accumulation of ER membranes and vesicles. We conclude that the SED5 gene is essential for growth and that Sed5p is required for ER to Golgi transport. When Sed5p is overexpressed the efficiency of ER to Golgi transport is reduced, vesicles accumulate, and cellular morphology is perturbed. Immunofluorescence studies reveal that the bulk of Sed5p is not found on ER membranes but on punctate structures throughout the cytoplasm, the number of which increases upon SED5 overexpression. We suggest that Sed5p has an essential role in vesicular transport between ER and Golgi compartments and that it may itself cycle between these organelles.     

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.