Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.     

The luminal domain of p23 (Tmp21) plays a critical role in p23 cell surface trafficking

p23 (Tmp21 or p24δ), a member of the p24 family, is important for maintaining the integrity of the secretory pathway in mammals. It is a type I protein with a receptor-like luminal domain and a short cytoplasmic tail. This cytoplasmic tail carries an atypical endoplasmic reticulum (ER) retention KKXX motif that binds to coat protein I. The trafficking of p23 has been thought to be restricted to the early secretory pathway. However, recent findings as well as this study demonstrate that p23 is also found in the plasma membrane. By tagging different domains of p23 with green fluorescent protein, it is shown that it is the luminal domain that is primarily responsible for the appearance of p23 in the plasma membrane, despite the presence of a functional KKXX-ER retention and retrieval motif. When the KKXX motif is abolished, p23 shows an extremely increased trafficking to the plasma membrane. These experiments reveal the presence of two fractions of p23 with distinct trafficking destinations. One fraction cycles through the ER–Golgi pathway using its functional KKXX retrieval motif. The transient appearance of p23 in the plasma membrane is supported by the luminal domain. These results help to explain the functional presence of p23 in plasma membrane protein complexes and post-Golgi compartments.     

New COP1-binding motifs involved in ER retrieval.

Coatomer-mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins. For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER). Making use of the two-hybrid system, we characterized a new sequence (deltaL) which interacts specifically with the delta-COP subunit of the COP1 complex. Transfer of deltaL to the cytoplasmic domain of a reporter membrane protein resulted in its localization in the ER, in yeast and mammalian cells. This was due to continuous retrieval of tagged proteins from the Golgi back to the ER, in a manner similar to the ER retrieval of KKXX-tagged proteins. Extensive mutagenesis of deltaL identified an aromatic residue as a critical determinant of the interaction with COP1. Similar COP1-binding motifs containing an essential aromatic residue were identified in the cytoplasmic domain of an ER-resident protein, Sec71p, and in an ER retention motif previously characterized in the CD3epsilon chain of the T-cell receptor. These results emphasize the role of the COP1 complex in retrograde Golgi-to-ER transport and highlight its functional similarity with clathrin-adaptor complexes.     

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.     

ARMER,apoptotic regulator in the membrane of the endoplasmic reticulum,a novel inhibitor of apoptosis

We have identified a novel protein, apoptotic regulator in the membrane of the endoplasmic reticulum (ARMER), which protects HT1080 fibrosarcoma cells from apoptosis induced by various stimuli. We demonstrate that ARMER is an endoplasmic reticulum (ER) integral membrane protein with four predicted transmembrane domains and a COOH-terminal KKXX ER retrieval motif. We used an inducible expression system (pIND) to study the effects of regulated ARMER overexpression. Cells in which ARMER was overexpressed exhibited protection from multiple apoptotic inducers including serum starvation, doxorubicin, UV irradiation, tumor necrosis factor alpha, and the ER stressors brefeldin A, tunicamycin, and thapsigargin. Analysis of the caspase proteolytic cascade reveals that ARMER inhibits proteolysis of the caspase-9-specific fluorogenic substrate LEHD-AFC as well as endogenous substrates downstream of caspase-9; however, it does not inhibit cytochrome c release or cleavage of caspase-9 itself. Apoptotic stimuli cause endogenous levels of ARMER protein and RNA to decrease, leading to cell death; however, sustaining ARMER protein levels through exogenous expression inhibits apoptosis. These data suggest that ARMER is a novel ER integral membrane protein which protects cells by inhibiting caspase-9 activity and reveal a possible role for ARMER in cell survival.     

Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.     

Structural basis of FFAT motif-mediated ER targeting

The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain. We have solved crystal structures of the rat VAP-A MSP homology domain alone and in complex with an FFAT motif. The co-crystal structure was used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant also blocked function of the VAP homolog Scs2p in yeast. Finally, overexpression of the FFAT binding-defective VAP in COS7 cells dramatically altered ER morphology. Our data establish the structural basis of FFAT-mediated ER targeting and suggest that FFAT-targeted proteins play an important role in determining ER morphology.     

Cytoplasmic tail motifs mediate endoplasmic reticulum localization and export of transmembrane reporters in the protozoan parasite Toxoplasma gondii

In mammalian cells and yeasts, amino acid motifs in the cytoplasmic tails of transmembrane proteins play a prominent role in protein targeting in the early secretory pathway by mediating localization to or rapid export from the endoplasmic reticulum (ER). However, early sorting events are poorly characterized in protozoan parasites. Here, we show that a C-terminal QKTT sequence mediates the ER localization of chimeric reporter constructs consisting of bacterial alkaline phosphatase (BAP) fused to the transmembrane domain (TMD) and truncated cytoplasmic tail of the human low-density lipoprotein receptor (LDL) receptor or of murine lysosome-associated membrane protein (lamp-1) in Toxoplasma gondii . The cytoplasmic tail of human TGN46 also determines ER localization of BAP chimeras in the parasite, but this can be overcome by the addition at the C-terminus of the tail of an acidic patch, which functions as an ER export signal in conjunction with an upstream tyrosine motif. These results suggest that COPI-dependent ER retrieval and COPII-dependent export mechanisms mediated by KKXX and DXE motifs of mammalian cells are generally conserved in T. gondii . In contrast, the failure of the QKTT motif and TGN46 cytoplasmic tail to induce steady-state ER localization of vesicular stomatitis virus glycoprotein (VSVG) chimeras in HeLa and NRK cells indicates that significant differences in early secretory trafficking also exist.     

An Endoplasmic Reticulum Retrieval Signal Partitions Human Foamy Virus Maturation to Intracytoplasmic Membranes

Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes. The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER. This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif. Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses. Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses. Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers. We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.     

Rer1p,a retrieval receptor for ER membrane proteins,recognizes transmembrane domains in multiple modes

The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Deltarer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.     

Intracellular Interaction of Simian Immunodeficiency Virus Gag and Env Proteins

In polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.     

Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N-terminal nucleotide-binding fold

The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.     

Rer1p, a retrieval receptor for endoplasmic reticulum membrane proteins, is dynamically localized to the Golgi apparatus by coatomer

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.     

Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated

The Cf-9 resistance gene from tomato confers resistance to races of the fungal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf-9 function, we tagged the Cf-9 protein with a triple myc epitope at either the amino- or carboxy-terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf-9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c-myc antibodies, c-myc : Cf-9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf-9 protein is highly glycosylated. Plasma membranes were isolated using two-phase partitioning, and c-myc : Cf-9 was enriched in these fractions, indicating that Cf-9 is a plasma membrane protein. This was confirmed by silver-enhanced immunogold labelling of tobacco protoplasts carrying the amino-terminal c-myc tag; a higher labelling density was observed on the surface of protoplasts derived from c-myc : Cf-9 tobacco compared to untransformed control. The presence of Cf-9 in the plasma membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.     

Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins

Membrane proteins destined for the vacuolar or lysosomal lumen are typically ubiquitinated, the ubiquitin serving as a targeting signal for the multivesicular body pathway. The RING-domain ubiquitin ligase Tul1 is an integral membrane protein that modifies the yeast vacuolar enzyme carboxypeptidase S (Cps1), the polyphosphatase Ppn1/Phm5 and other proteins containing exposed hydrophilic residues within their transmembrane domains (TMDs). Here we show that Bsd2 provides an alternative ubiquitination mechanism for Cps1, Phm5 and other proteins. Bsd2 is a three-TMD protein with a PPXY motif that binds the HECT domain ubiquitin ligase Rsp5. It can thus act as a specific adaptor linking Rsp5 to its substrates. Like Tul1, the Bsd2 system recognises polar TMDs. Bsd2 also controls the vacuolar targeting of a manganese transporter and a mutant plasma membrane ATPase, and together with the ER retrieval receptor Rer1, it protects cells from stress. We suggest that Bsd2 has a wide role in the quality control of membrane proteins. Bsd2 is the yeast homologue of human NEDD4 binding protein N4WBP5, which may therefore have similar functions.     

Diacidic motifs influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.     

The Cf-9 disease resistance protein is present in an approximately 420-kilodalton heteromultimeric membrane-associated complex at one molecule per complex

The tomato Cf-9 gene confers race-specific resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In tobacco, Cf-9 confers a hypersensitive response to the Avr9 peptide. To investigate Cf-9 protein function in initiating defense signaling, we engineered a functional C-terminal fusion of the Cf-9 gene with the TAP (Tandem Affinity Purification) tag. In addition, we established a transient expression assay in Nicotiana benthamiana leaves for the production of functional Cf-9:myc and Cf-9:TAP. Transiently expressed Cf-9:myc and Cf-9:TAP proteins induced an Avr9-dependent hypersensitive response, consistent with previous results with stably transformed tobacco plants and derived cell suspension cultures expressing c-myc-tagged Cf-9. Gel filtration of microsomal fractions solubilized with octylglucoside revealed that the Cf-9 protein, either as c-myc or TAP fusions, migrated at a molecular mass of 350 to 475 kD. By using blue native gel electrophoresis, the molecular size was confirmed to be approximately 420 kD. Our results suggest that only one Cf-9 protein molecule is present in the Cf-9 complex and that Cf-9 is part of a membrane complex consisting of an additional glycoprotein partner(s). The high structural similarity between Cf proteins and Clavata2 (CLV2) of Arabidopsis, together with the similarity of molecular mass between Cf-9 and CLV complexes (420 and 450 kD, respectively), led us to investigate whether Cf-9 is integrated into membrane-associated protein complexes like those formed by CLV1 and CLV2. Unlike CLV2, the Cf-9 protein did not form disulfide-linked heterodimers, no ligand (Avr9)-dependent shift in the molecular mass of the Cf-9 complex was detected, and no Rho-GTPase-related proteins were found associated with Cf-9 under the conditions tested. Thus, Cf-9-dependent defense signaling and CLV2-dependent regulation of meristem development seem to be accomplished via distinct mechanisms, despite the structural similarity of their key components Cf-9 and CLV2.     

The C-terminal dilysine motif for targeting to the endoplasmic reticulum is not required for Cf-9 function

The tomato resistance gene Cf-9 encodes a membrane-anchored, receptor-like protein that mediates specific recognition of the extracellular elicitor protein AVR9 of Cladosporium fulvum. The C-terminal dilysine motif (KKRY) of Cf-9 suggests that the protein resides in the endoplasmic reticulum. Previously, two conflicting reports on the subcellular location of Cf-9 were published. Here we show that the AARY mutant version of Cf-9 is still functional in mediating AVR9 recognition, suggesting that functional Cf-9 resides in the plasma membrane. The data presented here and in reports by others can be explained by masking the dilysine signal of Cf-9 with other proteins.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.