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.     

An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum.

Use of alternative initiator methionines in human invariant (Ii) chain mRNA results in the synthesis of two polypeptides, Iip33 and Iip31. After synthesis both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. Subsequently, Iip31 is transported out of the ER, guiding MHC class II to the endocytic pathway, whereas Iip33, which differs by only a 16 residue extension at the N-terminus, becomes an ER resident. Mutagenesis of this extension showed that multiple arginines close to the N-terminus were responsible for ER targeting. The minimal requirements of this targeting motif were found to be two arginines (RR) located at positions 2 and 3, 3 and 4 or 4 and 5 or split by a residue at positions 2 and 4 or 3 and 5. Transplanting an RR motif onto transferrin receptor demonstrated that this motif can target other type II membrane proteins to the ER. The characteristics of this RR motif are similar to the KK ER targeting motif for type I membrane proteins. Indeed, RR-tagged transferrin receptor partially localized to the intermediate compartment, suggesting that like the KK motif, the RR motif directs the retrieval of membrane proteins to the ER via a retrograde transport pathway.     

Cellular response to unfolded proteins in the endoplasmic reticulum of plants

Secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER) in eukaryotic cells. Nascent polypeptide chains, which are translated on the rough ER, are translocated to the ER lumen and folded into their native conformation. When protein folding is inhibited because of mutations or unbalanced ratios of subunits of hetero-oligomeric proteins, unfolded or misfolded proteins accumulate in the ER in an event called ER stress. As ER stress often disturbs normal cellular functions, signal-transduction pathways are activated in an attempt to maintain the homeostasis of the ER. These pathways are collectively referred to as the unfolded protein response (UPR). There have been great advances in our understanding of the molecular mechanisms underlying the UPR in yeast and mammals over the past two decades. In plants, a UPR analogous to those in yeast and mammals has been recognized and has recently attracted considerable attention. This review will summarize recent advances in the plant UPR and highlight the remaining questions that have yet to be addressed.     

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.     

Retention of membrane proteins by the endoplasmic reticulum

We have used a monoclonal antibody specific for a hydrocarbon-induced cytochrome P450 to localize, by electron microscopy, the epitope- specific cytochrome P450. The cytochrome was found in the rough and smooth endoplasmic reticulum (ER) and the nuclear envelope of hepatocytes. Significant quantities of cytochrome P450 were not found in Golgi stacks. We also could not find any evidence of Golgi- associated processing of the Asn-linked oligosaccharide chains of two well-characterized ER membrane glycoprotein enzymes (glucosidase II and hexose-6-phosphate dehydrogenase), or of the oligosaccharides attached to the bulk of the glycoproteins of the ER membrane. We conclude that these ER membrane proteins are efficiently retained during a process of highly selective export from this organelle.     

Topogenesis of membrane proteins at the endoplasmic reticulum

Most eukaryotic membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane by the Sec61 translocation complex. They are targeted to the translocon by hydrophobic signal sequences, which induce the translocation of either their N- or their C-terminal sequence. Signal sequence orientation is largely determined by charged residues flanking the apolar sequence (the positive-inside rule), folding properties of the N-terminal segment, and the hydrophobicity of the signal. Recent in vivo experiments suggest that N-terminal signals initially insert into the translocon head-on to yield a translocated N-terminus. Driven by a local electrical potential, the signal may invert its orientation and translocate the C-terminal sequence. Increased hydrophobicity slows down inversion by stabilizing the initial bound state. In vitro cross-linking studies indicate that signals rapidly contact lipids upon entering the translocon. Together with the recent crystal structure of the homologous SecYEbeta translocation complex of Methanococcus jannaschii, which did not reveal an obvious hydrophobic binding site for signals within the pore, a model emerges in which the translocon allows the lateral partitioning of hydrophobic segments between the aqueous pore and the lipid membrane. Signals may return into the pore for reorientation until translation is terminated. Subsequent transmembrane segments in multispanning proteins behave similarly and contribute to the overall topology of the protein.     

A single C-terminal peptide segment mediates both membrane association and localization of lysyl hydroxylase in the endoplasmic reticulum

Hydroxylation of lysyl residues is crucial for the unique glycosylation pattern found in collagens and for the mechanical strength of fully assembled extracellular collagen fibers. Hydroxylation is catalyzed in the lumen of the endoplasmic reticulum (ER) by a specific enzyme, lysyl hydroxylase (LH). The absence of the known ER-specific retrieval motifs in its primary structure and its association with the ER membranes in vivo have suggested that the enzyme is localized in the ER via a novel retention/retrieval mechanism. We have identified here a 40-amino acid C-terminal peptide segment of LH that is able to convert cathepsin D, normally a soluble lysosomal protease, into a membrane-associated protein. The same segment also markedly slows down the transport of the reporter protein from the ER into post-ER compartments, as assessed by our pulse-chase experiments. The retardation efficiency mediated by this C-terminal peptide segment is comparable with that of the intact LH but lower than that of the KDEL receptor-based retrieval mechanism. Within this 40-amino acid segment, the first 25 amino acids appear to be the most crucial ones in terms of membrane association and ER localization, because the last 15 C-terminal amino acids did not possess substantial retardation activity alone. Our findings thus define a short peptide segment very close to the extreme C terminus of LH as the only necessary determinant both for its membrane association and localization in the ER.     

C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae.

We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end. These fusion proteins contain at least one of the internal signal sequences of arginine permease. When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint. The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane. These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane.     

Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval.

Dilysine signals confer localization of type I membrane proteins to the endoplasmic reticulum (ER). According to the prevailing model these signals target proteins to the ER by COP I-mediated retrieval from post-ER compartments, whereas the actual retention mechanism in the ER is unknown. We expressed chimeric membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF constructs, which had access to post-ER compartments, the KKAA chimeras were localized to the ER by confocal microscopy and were neither processed by cis-Golgi-specific enzymes in vivo nor included into ER-derived transport vesicles in an in vitro budding assay, suggesting that KKAA-bearing proteins are permanently retained in the ER. The ER localization was nonsaturable and exclusively mediated by the dilysine signal because mutating the lysines to alanines led to cell surface expression of the chimeras. Although the KKAA signal avidly binds COP I in vitro, the ER retention by this signal does not depend on intact COP I in vivo because it was not affected in an epsilon-COP-deficient cell line. We propose that dilysine ER targeting signals can mediate ER retention in addition to retrieval.     

Steric masking of a dilysine endoplasmic reticulum retention motif during assembly of the human high affinity receptor for immunoglobulin E

Signals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the alpha chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the gamma chain, allowing transport out of the ER of the fully assembled receptor. Juxtaposition of the cytoplasmic domains of the alpha and gamma subunits during assembly is responsible for this loss of ER retention. Substitution of the gamma chain cytoplasmic domain with cytoplasmic domains of irrelevant proteins resulted in efficient transport out of the ER of the alpha chain, demonstrating that nonspecific steric hindrance by the cytoplasmic domain of the gamma chain accounts for the masking of the ER retention signal present in the cytoplasmic domain of the alpha chain. Such a mechanism allows the ER retention machinery to discriminate between assembled and nonassembled receptors, and thus participates in quality control at the level of the ER.     

14-3-3 proteins: regulation of endoplasmic reticulum localization and surface expression of membrane proteins

The density and composition of cell surface proteins are major determinants for cellular functions. Regulation of cell surface molecules occurs at several levels, including the efficiency of surface transport, and is therefore of great interest. As the major phosphoprotein-binding modules, 14-3-3 proteins are known for their crucial roles in a wide range of cellular activities, including the subcellular localization of target proteins. Accumulating evidence suggests a role for 14-3-3 in surface transport of membrane proteins, in which 14-3-3 binding reduces endoplasmic reticulum (ER) localization, thereby promoting surface expression of membrane proteins. Here, we focus on recent evidence of 14-3-3-mediated surface transport and discuss the possible molecular mechanisms.     

The Arabidopsis thaliana RER1 gene family: its potential role in the endoplasmic reticulum localization of membrane proteins

Many endoplasmic reticulum (ER) proteins are known to be localized to the ER by a mechanism called retrieval, which returns the molecules that are exported from the ER to the Golgi apparatus back to the ER. Signals are required to be recognized by this retrieval system. In the work on yeast Saccharomyces cerevisiae, we have demonstrated that transmembrane domains of a subset of ER membrane proteins including Sec12p, Sec71p and Sec63p contain novel ER retrieval signals. For the retrieval of these proteins, a Golgi membrane protein, Rer1p, is essential (Sato et al., Mol. Biol. Cell 6 (1995) 1459–1477; Proc. Natl. Acad. Sci. USA 94 (1997) 9693–9698). To address the role of Rer1p in higher eukaryotes, we searched for homologues of yeast RER1 from Arabidopsis thaliana. We identified three cDNAs encoding Arabidopsis counterparts of Rer1p with an amino acid sequence identity of 39–46% to yeast Rer1p and named AtRER1A, AtRER1B, and AtRER1C1. AtRer1Ap and AtRer1Bp are homologous to each other (85% identity), whereas AtRer1C1p is less similar to AtRer1Ap and AtRer1Bp (about 50%). Genomic DNA gel blot analysis indicates that there are several other AtRER1-related genes, implying that Arabidopsis RER1 constitutes a large gene family. The expression of these three AtRER1 genes is ubiquitous in various tissues but is significantly higher in roots, floral buds and a suspension culture in which secretory activity is probably high. All the three AtRER1 cDNAs complement the yeast rer1 mutant and remedy the defect of Sec12p mislocalization. However, the degree of complementation differs among the three with that of AtRER1C1 being the lowest, again suggesting a divergent role of AtRer1C1p.     

Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants

Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.     

The endoplasmic reticulum membrane is permeable to small molecules

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of approximately 5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.     

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Background  

Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.     

Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein-protein and protein-lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail-anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER-Golgi boundary by simple receptor-independent mechanisms based on partitioning.     

Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins

The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane.     

A class of membrane proteins shaping the tubular endoplasmic reticulum

How is the characteristic shape of a membrane bound organelle achieved? We have used an in vitro system to address the mechanism by which the tubular network of the endoplasmic reticulum (ER) is generated and maintained. Based on the inhibitory effect of sulfhydryl reagents and antibodies, network formation in vitro requires the integral membrane protein Rtn4a/NogoA, a member of the ubiquitous reticulon family. Both in yeast and mammalian cells, the reticulons are largely restricted to the tubular ER and are excluded from the continuous sheets of the nuclear envelope and peripheral ER. Upon overexpression, the reticulons form tubular membrane structures. The reticulons interact with DP1/Yop1p, a conserved integral membrane protein that also localizes to the tubular ER. These proteins share an unusual hairpin topology in the membrane. The simultaneous absence of the reticulons and Yop1p in S. cerevisiae results in disrupted tubular ER. We propose that these "morphogenic" proteins partition into and stabilize highly curved ER membrane tubules.