The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Emp47p and its close homolog Emp46p have a tyrosine-containing endoplasmic reticulum exit signal and function in glycoprotein secretion in Saccharomyces cerevisiae

The yeast open reading frame YLR080w/EMP46 encodes a homolog of the Golgi protein Emp47p. These two proteins are 45% identical and have a single transmembrane domain in their C-terminal regions and a carbohydrate recognition domain signature in the N-terminal region. The C-terminal tail of Emp46p includes a dilysine signal. This protein is localized to Golgi membranes at steady state by subcellular fractionation and green fluorescent protein labeling. On block of forward transport in sec12-4 cells, redistribution of Emp46p from the Golgi to the endoplasmic reticulum is observed. These localization features are similar to those previously reported for Emp47p. In addition, mutagenesis of the C-terminal region identified a tyrosine-containing motif as a critical determinant of the Golgi-localization and interaction with both COPI and COPII components. Similar motifs are also observed in the C-terminal tail of Emp47p and other mammalian homologs. Disruption of Emp47p displays a growth defect at a high temperature or on Ca(2+)-containing medium, which is rescued by overexpression of Emp46p, suggesting a partially overlapping function between Emp46p and Emp47p. In addition, we found that the disruption of both Emp46p and Emp47p show a marked defect in the secretion of a subset of glycoproteins. Analysis of the C-terminal mutants for Ca(2+) sensitivity revealed that the forward transport of Emp46/47p is essential for their function, whereas the retrograde transport is not. We propose that Emp46p and Emp47p are required for the export of specific glycoprotein cargo from the endoplasmic reticulum.     

Physiological Functions of the COPI Complex in Higher Plants

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β′-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β′-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β′-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β′-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.     

Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP.     

In situ localization and in vitro induction of plant COPI-coated vesicles

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.     

RGS4 and RGS2 bind coatomer and inhibit COPI association with Golgi membranes and intracellular transport

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit beta'-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant beta'-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of beta'-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on G(ialpha). In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi-plasma membrane or intra-Golgi transport.     

COPI-independent Anterograde Transport: Cargo-selective ER to Golgi Protein Transport in Yeast COPI Mutants

The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (γ-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and α-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY–invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi–ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.     

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5′-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.     

Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer

In mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins. Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat. The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells. Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells. We show that a dilysine motif in the -3,-4 position (relative to the cytosolic C-terminus) recruits in a very specific manner all the subunits of the plant coatomer complex. Lysines cannot be replaced by arginines or histidines to bind plant coatomer. A diphenylalanine motif in the -7,-8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif. Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells. The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs. ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgammaS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form.     

Golgi inheritance: rab rides the coat-tails

A recent study describes a role for a Rab GTPase previously implicated in endoplasmic reticulum and mitochondrial inheritance and for a COPI coatomer subunit in the targeting of a type V myosin to the late Golgi in yeast.     

Multiple features within the syntaxin Sed5p mediate its Golgi localization

Protein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi—the Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells, the syntaxin SNARE Sed5p oversees membrane fusion events. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the steady‐state localization of Sed5p to the Golgi appears to be primarily conformation‐based relying on intra‐molecular associations between the Habc domain and SNARE‐motif while its tribasic COPI‐coatomer binding motif plays a role in intra‐Golgi retention.     

Molecular structure and flexibility of the yeast coatomer as revealed by electron microscopy

Coat protein complex I (COPI)-coated vesicles, one of three major types of vesicular carriers in the cell, mediate the early secretory pathway and retrograde transport from the Golgi to the endoplasmic reticulum. COPI vesicles are generated through activation of the regulatory GTPase Arf1 at the donor membrane and the subsequent recruitment of coatomer, a coat protein complex consisting of seven stably associated components. Coatomer functions in binding and sequestering cargo molecules and assembles into a polymeric protein shell that encompasses the surface of COPI vesicles. Little is known about the structural properties of this heptameric complex. We have isolated native yeast coatomer and examined its structure and subunit organization by single-particle electron microscopy. Our analyses provide the first three-dimensional picture of the complete coatomer and reveal substantial conformational flexibility likely to be critical for its scaffolding function.     

The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmic reticulum retrieval in yeast

Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a C-terminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.     

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.     

The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex

The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif.     

ArfGAP1 interacts with coat proteins through tryptophan-based motifs

The Arf1 GTPase-activating protein ArfGAP1 regulates vesicular traffic through the COPI system. This protein consists of N-terminal catalytic domain, lipid packing sensors (the ALPS motifs) in the central region, and a carboxy part of unknown function. The carboxy part contains several diaromatic sequences that are reminiscent of motifs known to interact with clathrin adaptors. In pull-down experiments using GST-fused peptides from rat ArfGAP1, a peptide containing a ³²⁹WETF sequence interacted strongly with clathrin adaptors AP1 and AP2, whereas a major coatomer-binding determinant was identified within the extreme carboxy terminal peptide (⁴⁰⁵AADEGWDNQNW). Mutagenesis and peptide competition experiments revealed that this determinant is required for coatomer binding to full-length ArfGAP1, and that interaction is mediated through the δ-subunit of the coatomer adaptor-like subcomplex. Evidence for a role of the carboxy motif in ArfGAP1-coatomer interaction in vivo is provided by means of a reporter fusion assay. Our findings point to mechanistic differences between ArfGAP1 and the other ArfGAPs known to function in the COPI system.     

Membrane recruitment of coatomer and binding to dilysine signals are separate events

It has previously been shown that transport of newly synthesized proteins and the structure of the Golgi complex are affected in the Chinese hamster ovary cell line ldlF, which bears a temperature-sensitive mutation in the Coat protein I (COPI) subunit epsilon-COP (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224; Hobbie, L., Fisher, A. S., Lee, S., Flint, A., and Krieger, M. (1994) J. Biol. Chem. 269, 20958-20970). Here, we pinpoint the site of the secretory block to an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi complex and show that the distributions of ER-Golgi recycling proteins, such as KDEL receptor and p23, as well as resident Golgi proteins, such as mannosidase II, are accordingly affected. At the nonpermissive temperature, neither the stability of the COPI complex nor its recruitment to donor Golgi membranes is affected. However, the binding of coatomer to the dilysine-based ER-retrieval motif is impaired in the absence of epsilon-COP, suggesting that dilysine signal binding is not the major means of COPI recruitment. Because expression of the exogenous chimera of epsilon-COP and green fluorescent protein in ldlF cells at nonpermissive temperature rapidly restores the wild type properties, epsilon-COP is likely to play an important role in the cargo selection events mediated by COPI.     

A kinetic proof-reading mechanism for protein sorting

Resident proteins of the exocytic pathway are maintained at various levels through coatomer protein I (COPI)-mediated recycling. Sorting of cargo by COPI requires GTP hydrolysis by ADP-ribosylation factor 1 (ARF-1). This small GTPase recruits coatomer onto Golgi membranes and upon hydrolysis, is thought to release coatomer back into the cytosol. This step requires the activating protein, ARFGAP1. By coupling sorting to a cargo-induced sequestering of ARFGAP1, we have formulated a kinetic proof-reading model that explains how a GTP hydrolysis-driven coat release can yield an active sorting event. The sorting scheme predicts a dependency on the amount of ARFGAP1 and explains the recent experimental findings that ARF-1 and COPI detach with different time constants from the Golgi membrane in vivo .     

Association of tapasin and COPI provides a mechanism for the retrograde transport of major histocompatibility complex (MHC) class I molecules from the Golgi complex to the endoplasmic reticulum

Tapasin is a subunit of the transporter associated with antigen processing (TAP). It associates with the major histocompatibility complex (MHC) class I. We show that tapasin interacts with beta- and gamma-subunits of COPI coatomer. COPI retrieves membrane proteins from the Golgi network back to the endoplasmic reticulum (ER). The COPI subunit-associated tapasin also interacts with MHC class I molecules suggesting that tapasin acts as the cargo receptor for packing MHC class I molecules as cargo proteins into COPI-coated vesicles. In tapasin mutant cells, neither TAP nor MHC class I are detected in association with the COPI coatomer. Interestingly, tapasin-associated MHC class I molecules are antigenic peptide-receptive and detected in both the ER and the Golgi. Our data suggest that tapasin is required for the COPI vesicle-mediated retrograde transport of immature MHC class I molecules from the Golgi network to the ER.