Membrane topology and nicastrin-enhanced endoproteolysis of APH-1, a component of the gamma-secretase complex

APH-1, presenilin, nicastrin, and Pen-2 are proteins with varying membrane topologies that compose the gamma-secretase complex, which is responsible for the intramembrane proteolysis of several substrates including the amyloid precursor protein. APH-1 is known to be necessary for gamma-secretase activity, but its precise function in the complex is not fully understood, and its membrane topology has not been described, although it is predicted to traverse the membrane seven times. To investigate this, we used selective permeabilization of the plasma membrane and immunofluorescence microscopy to show that the C terminus of the APH-1 resides in the cytosolic space. Insertion of N-linked glycosylation sites into each of the hydrophilic loop domains and the N terminus of APH-1 showed that the N-terminal domain as well as loops 2, 4, and 6 could be glycosylated, whereas loops 1, 3, and 5 were not. Thus, APH-1 topologically resembles a seven-transmembrane domain receptor with the N terminus and even-numbered loops facing the endoplasmic reticulum lumen, and the C terminus and odd-numbered loops reside in the cytosolic space. By using these glycosylation mutants, we provide evidence that the association between nicastrin and APH-1 may occur very soon after APH-1 synthesis and that the interaction between these two proteins may rely more heavily on the transmembrane domains of APH-1 than on the loop domains. Furthermore, we found that APH-1 can be processed by several endoproteolytic events. One of these cleavages is strongly up-regulated by co-expression of nicastrin and generates a stable C-terminal fragment that associates with nicastrin.     

Transmembrane topology of pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases

The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.     

Membrane topology of gamma-secretase component PEN-2

PEN-2 is an integral membrane protein that is a necessary component of the gamma-secretase complex, which is central in the pathogenesis of Alzheimer's disease and is also required for Notch signaling. In the absence of PEN-2, Notch signaling fails to guide normal development in Caenorhabditis elegans, and amyloid beta peptide is not generated from the amyloid precursor protein. Human PEN-2 is a 101-amino acid protein containing two putative transmembrane domains. To understand its interaction with other gamma-secretase components, it is important to know the membrane topology of each member of the complex. To characterize the membrane topology of PEN-2, we introduced single amino acid changes in each of the three hydrophilic regions of PEN-2 to generate N-linked glycosylation sites. We found that the N-linked glycosylation sites present in the N- and C-terminal domains of PEN-2 were utilized, whereas a site in the hydrophilic "loop" region connecting the two transmembrane domains was not. The addition of a carbohydrate structure in the N-terminal domain of PEN-2 prevented association with presenilin 1, whereas glycosylation in the C-terminal region of PEN-2 did not, suggesting that the N-terminal domain is important for interactions with presenilin 1. Immunofluorescence microscopy with selective permeabilization of the plasma membrane of cells expressing epitope-tagged forms of PEN-2 confirmed the lumenal location of both the N and C termini. A protease protection assay also demonstrated that the loop domain of PEN-2 is cytosolic. Thus, PEN-2 spans the membrane twice, with the N and C termini facing the lumen of the endoplasmic reticulum.     

Presenilin-1 maintains a nine-transmembrane topology throughout the secretory pathway

Presenilin-1 is a polytopic membrane protein that assembles with nicastrin, PEN-2, and APH-1 into an active gamma-secretase complex required for intramembrane proteolysis of type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology remains an issue of strong controversy. We revisited presenilin-1 topology by inserting glycosylation consensus sequences in human PS1 and expressing the obtained mutants in a presenilin-1 and 2 knock-out background. Based on the glycosylation status of these variants we provide evidence that presenilin-1 traffics through the Golgi after a conformational change induced by complex assembly. Based on our glycosylation variants of presenilin-1 we hypothesize that complex assembly occurs during transport between the endoplasmic reticulum and the Golgi apparatus. Furthermore, our data indicate that presenilin-1 has a nine-transmembrane domain topology with the COOH terminus exposed to the lumen/extracellular surface. This topology is independently underscored by lysine mutagenesis, cell surface biotinylation, and cysteine derivation strategies and is compatible with the different physiological functions assigned to presenilin-1.     

Membrane protein topology of oleosin is constrained by its long hydrophobic domain.

Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membrane-integrated hydrophobic (H) domain of 72 residues, flanked by two cytosolic hydrophilic domains. We have investigated the targeting and topological determinants present within the oleosin polypeptide sequence using ER-derived canine pancreatic microsomes. Our data indicate that oleosins are integrated into membranes by a cotranslational, translocon-mediated pathway. This is supported by the identification of two independent functional signal sequences in the H domain, and by demonstrating the involvement of the SRP receptor in membrane targeting. Oleosin topology was manipulated by the addition of an N-terminal cleavable signal sequence, resulting in translocation of the N terminus to the microsomal lumen. Surprisingly, the C terminus failed to translocate. Inhibition of C-terminal translocation was not dependent on either the sequence of hydrophobic segments in the H domain, the central proline knot motif or charges flanking the H domain. Therefore, the topological constraint results from the length and/or the hydrophobicity of the H domain, implying a general case that long hydrophobic spans are unable to translocate their C terminus to the ER lumen.     

Membrane topology of Bves/Pop1A,a cell adhesion molecule that displays dynamic changes in cellular distribution during development

We investigated the membrane topology of Bves/Pop1A as a foundation to dissect the molecular basis and function of Bves/Pop1A trafficking during development. Bves contains two asparagine-linked glycosylation sites within the amino terminus and three putative membrane domains. Therefore, glycosylation assays were performed to determine if the amino terminus of Bves is delivered into the endoplasmic reticulum lumen and glycosylated. We establish that Bves from chick heart and transfected cells is glycosylated, implying that the amino terminus of cell surface molecules is extracellular. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves. First, glycosylation of Bves at exogenous sites within the carboxyl terminus was only observed in a construct that lacked the third membrane domain, which presumably reversed the orientation of the carboxyl terminus. Second, co-expression of full-length Bves with soluble, carboxyl-terminal Bves constructs that reside in different subcellular compartments revealed that Bves-Bves interactions occur in the cytoplasm. Third, the immunoreactivity of endogenous Bves at the cell surface of epicardial cells was dramatically enhanced with detergent. These results suggest that the membrane topology of cell surface Bves/Pop1A is composed of an extracellular amino terminus, three transmembrane domains, and a cytoplasmic carboxyl terminus. We therefore hypothesize that the carboxyl terminus regulates the cellular distribution of Bves/Pop1A during coronary vessel development.     

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357–418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357–418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357–418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.     

Membrane topology and dimerization of the two subunits of the transporter associated with antigen processing reveal a three-domain structure

Presentation of peptides derived from cytosolic and nuclear proteins by MHC class I molecules requires their translocation across the membrane of the endoplasmic reticulum (ER) by a specialized ABC (ATP-binding cassette) transporter, TAP. To investigate the topology of the heterodimeric TAP complex, we constructed a set of C-terminal deletions for the TAP1 and TAP2 subunits. We identified eight and seven transmembrane (TM) segments for TAP1 and TAP2, respectively. TAP1 has both its N and C terminus in the cytoplasm, whereas TAP2 has its N terminus in the lumen of the ER. A putative TM pore consists of TM1-6 of TAP1 and, by analogy, TM1-5 of TAP2. Multiple ER-retention signals are present within this region, of which we positively identified TM1 of both TAP subunits. The N-terminal domain containing TM1-6 of TAP1 is sufficient for dimerization with TAP2. A second, independent dimerization domain, located between the putative pore and the nucleotide-binding cassette, lies within the cytoplasmic peptide-binding domains, which are anchored to the membrane via TM doublets 7/8 and 6/7 of TAP1 and TAP2, respectively. We present a model in which TAP is composed of three subdomains: a TM pore, a cytoplasmic peptide-binding pocket, and a nucleotide-binding domain.     

Human Vitamin K Epoxide Reductase and Its Bacterial Homologue Have Different Membrane Topologies and Reaction Mechanisms

Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.     

Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking

The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N- and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (delta384-444) variants and the new TRPV4-C (delta237-284), TRPV4-D (delta27-61), and TRPV4-E (delta237-284 and delta384-444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo- and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum.     

Membrane topography and topogenesis of prenylated Rab acceptor (PRA1)

The mouse prenylated Rab acceptor (mPRA1) is associated with the Golgi membrane at steady state and interacts with Rab proteins. It contains two internal hydrophobic domains (34 residues each) that have enough residues to form four transmembrane (TM) segments. In this study, we have determined the membrane topography of mPRA1 in both intact cells and isolated microsomes. The putative TM segments of mPRA1 were used to substitute for a known TM segment of a model membrane protein to determine whether the mPRA1 segments integrate into the membrane. Furthermore, N-linked glycosylation scanning methods were used to distinguish luminal domains from cytoplasmic domains of mPRA1. The data demonstrate that mPRA1 is a polytopic membrane protein containing four TM segments. These TM segments act cooperatively during the translocation and integration at the endoplasmic reticulum membrane. All hydrophilic domains are in the cytoplasm, including the N-terminal domain, the linker domain between the two hydrophobic domains, and the C-terminal domain. As a result, the bulk of mPRA1 is located in the cytoplasm, supporting its postulated role in regulating Rab membrane targeting and intracellular trafficking.     

Effect of PKD1 gene missense mutations on polycystin-1 membrane topogenesis

Polycystin-1 (PC1), the product of the polycystic kidney disease-1 (PKD1) gene, has a number of reported missense mutations whose pathogenicity is indeterminate. Previously, we utilized N-linked glycosylation reporter tags along with membrane insertion and topology assays to define the 11 membrane-spanning domains (I-XI) of PC1. In this report, we utilize glycosylation assays to determine whether two reported human polymorphisms/missense mutations within transmembrane (TM) domains VI and X affect the membrane topology of PC1. M3677T within TM VI had no effect on the topology of this TM domain as shown by the ability of two native N-linked glycosylation sites within the extracellular loop following TM VI to be glycosylated. In contrast, G4031D, within TM X, decreased the glycosylation of TM X reporter constructs, demonstrating that the substitution affected the C-terminal translocating activity of TM X. Furthermore, G4031D reduced the membrane association of TM X and XI together. These results suggest that G4031D affects the membrane insertion and topology of the C-terminal portion of polycystin-1 and represents a bona fide pathogenic mutation.     

Mutagenesis mapping of the presenilin 1 calcium leak conductance pore

Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the γ-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The water-filled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to γ-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca(2+) leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca(2+) conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca(2+) leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca(2+) conductance pore.     

Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix

The NTB-VPg protein of Tomato ringspot nepovirus is an integral membrane protein found in association with endoplasmic reticulum (ER)-derived membranes active in virus replication. A transmembrane helix present in a hydrophobic region at the C terminus of the NTB domain was previously shown to traverse the membranes, resulting in the translocation of the VPg domain in the lumen. We have now conducted an in planta analysis of membrane-targeting domains within NTB-VPg using in-frame fusions to the green fluorescent protein (GFP). As expected, the entire NTB-VPg protein directed the GFP fluorescence to ER membranes. GFP fusion proteins containing the C-terminal 86 amino acids of NTB-VPg also associated with ER membranes, resulting in ER-specific glycosylation at a naturally occurring glycosylation site in the VPg domain. Deletion of the hydrophobic region prevented the membrane association. The N-terminal 80 amino acids of NTB were also sufficient to direct the GFP fluorescence to intracellular membranes. A putative amphipathic helix in this region was necessary and sufficient to promote membrane association of the fusion proteins. Using in vitro membrane association assays and glycosylation site mapping, we show that the N terminus of NTB can be translocated in the lumen at least in vitro. This translocation was dependent on the presence of the putative amphipathic helix, suggesting that oligomeric forms of this helix traverse the membrane. Taken together, our results suggest that at least two distinct elements play a key role in the insertion of NTB-VPg in the membranes: a C-terminal transmembrane helix and an N-terminal amphipathic helix. An updated model of the topology of the protein in the membrane is presented.     

Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.     

Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11beta-hydroxysteroid dehydrogenase and the 50-kDa esterase.

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11beta-hydroxysteroid dehydrogenase, isozyme 1 (11beta-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11beta-HSD and E3 may constitute a lumenal targeting signal (LTS). To investigate this hypothesis we created chimeric fusions using the putative targeting sequences and the reporter gene, Aequorea victoria green fluorescent protein. Transfected COS cells expressing LTS-green fluorescent protein chimeras were examined by fluorescent microscopy and electron microscopic immunogold labeling. The orientation of expressed chimeras was established by immunocytofluorescent staining of selectively permeabilized COS cells. In addition, protease protection assays of membranes in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To investigate the general applicability of the proposed LTS, we fused the N terminus of E3 to the N terminus of the NADH-cytochrome b5 reductase lacking the myristoyl group and N-terminal 30-residue membrane anchor. The orientation of the cytochrome b5 reductase was reversed, from cytosolic to lumenal projection of the active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed by a specific array of hydrophobic residues terminating with acidic residues, is sufficient for lumenal targeting of single-pass proteins that are structurally and functionally unrelated.     

Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.     

Identification of sequence determinants that direct different intracellular folding pathways for aquaporin-1 and aquaporin-4

Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function.     

Membrane topology of S2P, a protein required for intramembranous cleavage of sterol regulatory element-binding proteins.

In sterol-depleted mammalian cells, a two-step proteolytic process releases the NH(2)-terminal domains of sterol regulatory element-binding proteins (SREBPs) from membranes of the endoplasmic reticulum (ER). These domains translocate into the nucleus, where they activate genes of cholesterol and fatty acid biosynthesis. The SREBPs are oriented in the membrane in a hairpin fashion, with the NH(2)- and COOH-terminal domains facing the cytosol and a single hydrophilic loop projecting into the lumen. The first cleavage occurs at Site-1 within the ER lumen to generate an intermediate that is subsequently released from the membrane by cleavage at Site-2, which lies within the first transmembrane domain. A membrane protein, designated S2P, a putative zinc metalloprotease, is required for this cleavage. Here, we use protease protection and glycosylation site mapping to define the topology of S2P in ER membranes. Both the NH(2) and COOH termini of S2P face the cytosol. Most of S2P is hydrophobic and appears to be buried in the membrane. All three of the long hydrophilic sequences of S2P can be glycosylated, indicating that they all project into the lumen. The HEIGH sequence of S2P, which contains two potential zinc-coordinating residues, is contained within a long hydrophobic segment. Aspartic acid 467, located approximately 300 residues away from the HEIGH sequence, appears to provide the third coordinating residue for the active site zinc. This residue, too, is located in a hydrophobic sequence. The hydrophobicity of these sequences suggests that the active site of S2P is located within the membrane in an ideal position to cleave its target, a Leu-Cys bond in the first transmembrane helix of SREBPs.     

Characterization of membrane association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein

Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.