Targeting of a tail-anchored protein to endoplasmic reticulum and mitochondrial outer membrane by independent but competing pathways

Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b(5), a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.     

Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc(3)Man(9)GlcNAc(2)-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12-14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Ribophorin I associates with a subset of membrane proteins after their integration at the sec61 translocon

The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.     

Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.     

Characterization and subcellular localization of murine and human magnesium-dependent neutral sphingomyelinase

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, an essential lipid constituent of the plasma membrane, lysosomal membranes, endoplasmic reticulum, and the Golgi membrane stacks of mammalian cells. In this study, we report the biochemical and functional characterization and subcellular localization of magnesium-dependent nSMase1 from overexpressing human embryonic kidney (HEK293) cells. Site-directed mutagenesis of conserved residues probably involved in the enzymatic sphingomyelin cleavage as well as the removal of one or both putative transmembrane domains lead to the complete loss of enzymatic activity of human nSMase1 expressed in HEK293 cells. Polyclonal antibodies raised against recombinant mammalian nSMase1 immunoprecipitated and inactivated the enzyme in membrane extracts of overexpressing HEK293 cells and different murine tissues. Cell fractionation combined with immunoprecipitation studies localized the nSMase1 protein predominantly in the microsomal fraction. The enzyme colocalized with marker proteins of the endoplasmic reticulum and the Golgi apparatus in immunocytochemistry. Anti-nSMase1 antibodies did not affect the nSMase activity in the plasma membrane fraction and membrane extracts from murine brain. Our study leads to the conclusion that nSMase1 is one of at least two mammalian neutral sphingomyelinases with different subcellular localization, tissue specificity, and enzymatic properties.     

A window of opportunity: timing protein degradation by trimming of sugars and ubiquitins

Of the many post-translational modifications of proteins, ubiquitination and N-glycosylation stand out because they are polymeric additions. In contrast to single-unit modifications, the fate of the modified protein is determined by the dynamic equilibrium of polymerization versus depolymerization, rather than by the initial addition itself. Notably, it is the trimming of sugar chains and elongation of polyubiquitin that target the protein to degradation. Recent research suggests that, for each process, special receptors recognize chains that reach an appropriate length and commit the conjugated substrate for proteasomal disposal. We propose that the 'magic numbers' are loss of at least three mannose residues from the initial chain, or extension to at least four ubiquitins. Although these processes are compartmentalized to either side of the endoplasmic reticulum (ER) membrane, some proteins are sequentially subjected to both because they transverse this membrane for ER-associated degradation.     

Protein N-Myristoylation Plays a Critical Role in the Endoplasmic Reticulum Morphological Change Induced by Overexpression of Protein Lunapark,an Integral Membrane Protein of the Endoplasmic Reticulum

N-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human N-myristoylated proteins with predicted transmembrane domains. As a result, it was found that protein Lunapark, the human ortholog of yeast protein Lnp1p that has recently been found to be involved in network formation of the endoplasmic reticulum (ER), is an N-myristoylated polytopic integral membrane protein. Analysis of tumor necrosis factor-fusion proteins with each of the two putative transmembrane domains and their flanking regions of protein Lunapark revealed that transmembrane domain 1 and 2 functioned as type II signal anchor sequence and stop transfer sequence, respectively, and together generated a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. Immunofluorescence staining of HEK293T cells transfected with a cDNA encoding protein Lunapark tagged with FLAG-tag at its C-terminus revealed that overexpressed protein Lunapark localized mainly to the peripheral ER and induced the formation of large polygonal tubular structures. Morphological changes in the ER induced by overexpressed protein Lunapark were significantly inhibited by the inhibition of protein N-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein N-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark.     

Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST

The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER–Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.     

The ALS8-associated mutant VAPB(P56S) is resistant to proteolysis in neurons

VAMP/synaptobrevin associated proteins A and B (VAPA and VAPB), are type IV membrane proteins enriched on ER and Golgi membranes. Both VAPA and B interact with cytoplasmic lipid transport proteins and cytoskeletal elements to maintain the structure and composition of ER and Golgi membranes. Truncated forms of both proteins are present in some tissues but the functional significance of this is not clear. In rodents processing of VAPA occurs in most tissues, however, truncated forms of VAPB have only been reported in brain tissue. It is demonstrated here that the extent of VAPB processing in rat increases during postnatal development and that it is restricted to neurons. The C-terminal polypeptide generated by this cleavage reaction remains associated with cell membranes, but its subcellular distribution is distinct from the full-length protein. A mutant form of VAPB is associated with a familial form of neurodegenerative disease, amyotrophic lateral sclerosis type 8. The mutant protein, VAPB(P56S) , is resistant to truncation in primary neuronal cultures, although remains sensitive to some form of proteolysis when over-expressed in HEK293 cells. These data suggest that neuronal cells have a particular requirement for VAPB proteolysis and that reduced levels of processed polypeptides may contribute to the neurodegeneration associated with amyotrophic lateral sclerosis type 8.     

Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4.

Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.     

Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.     

Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system.

Assembly of glycosylphosphatidylinositol (GPtdIns)-anchored proteins requires translocation of the nascent polypeptide chain across the endoplasmic reticulum (ER) membrane and replacement of the C-terminal signal sequence with a GPtdIns moiety. The anchoring reaction is carried out by an ER enzyme, GPtdIns transamidase. Genetic studies with yeast indicate that the transamidase consists of a dynamic complex of at least two subunits, Gaa1p and Gpi8p. To study the GPtdIns-anchoring reaction, we used a small reporter protein that becomes GPtdIns-anchored when the corresponding mRNA is translated in the presence of microsomes, in conjunction with site-specific photocrosslinking to identify ER membrane components that are proximal to the reporter during its conversion to a GPtdIns-anchored protein. We generated variants of the reporter protein such that upon in vitro translation in the presence of Nepsilon-(5-azido-2-nitrobenzoyl)-lysyl-tRNA, photoreactive lysine residues would be incorporated in the protein specifically near the GPtdIns-attachment site. We analyzed photoadducts resulting from UV irradiation of the samples. We show that proproteins can be crosslinked to the transamidase subunit Gpi8p, as well as to ER proteins of molecular mass approximately 60 kDa, approximately 70 kDa, and approximately 120 kDa. The identification of a photoadduct between a proprotein and Gpi8p provides the first direct evidence of an interaction between a proprotein substrate and one of the genetically identified transamidase subunits. The approximately 70-kDa protein that we identified may correspond to the other subunit Gaa1p, while the other proteins possibly represent additional, hitherto unidentified subunits of the mammalian GPtdIns transamidase complex.     

Molecular and cellular characterization of the Down syndrome critical region protein 2

Down syndrome (DS) is caused by trisomy for human chromosome 21 and is the most common genetic cause of mental retardation. The distal 10 Mb region of the long arm of the chromosome has been proposed to be associated with many of the abnormalities seen in DS. This region is often referred to as the Down syndrome critical region (DSCR). We report here the results of our analyses of the DSCR protein 2 (DSCR2). Results from transiently transfected COS-1 and HEK293 cells suggest that DSCR2 is synthesized as a 43 kDa precursor protein, from which the N-terminus is cleaved resulting in a polypeptide of 41 kDa. The polypeptide is modified by still uncharacterized co- or post-translational modifications increasing the predicted molecular weight of 32.8 kDa by about 10 kDa. Analyses of the only putative N-glycosylation site by in vitro mutagenesis excluded the possibility of the contribution of N-glycosylation to this increase in molecular weight. Further, the results of intracellular localization studies and membrane fractionation assays indicate that DSCR2 is targeted to a cytoplasmic compartment as a soluble form.     

A mutant Kex2 enzyme with a C-terminal HDEL sequence releases correctly folded human insulin-like growth factor-1 from a precursor accumulated in the yeast endoplasmic reticulum.

Mutations in the pro region of the yeast DNA hybrid of prepro-alpha-factor and human insulin-like growth factor-1 (IGF-1) cause the accumulation, in the yeast Saccharomyces cerevisiae, of an unglycosylated precursor protein where the pre sequence is missing. The prepro sequence of the prepro-alpha-factor consists of a pre or signal sequence and a proregion which possesses three sites for N-glycosylation. Isolation of a precursor, where the pro region is still linked to IGF-1 through a pair of dibasic amino acid residues, implies that the polypeptide may have translocated into the endoplasmic reticulum (ER) but has not been processed by the Golgi membrane-bound Kex2 endoprotease. However, the lack of any N-glycosylation in the translocated polypeptide is surprising. The mutated pro region, can be processed, in vitro, by treatment with a soluble form of the Kex2 enzyme. It is also possible to release the pro region, in vivo, by coexpressing a mutant Kex2 protease which is partially retained in the ER with the help of the C-terminal tetrapeptide sequence, HDEL. The mature IGF-1, which is secreted from the intracellular pool of precursor proteins, is predominantly an active, monomeric molecule, corroborating observations that early removal of the pro region before folding in the ER helps to prevent aberrant intermolecular disulfide-bond formation in IGF-1. These results have revealed the utility of the ER-retained Kex2 enzyme as a novel in vivo biochemical tool.     

Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP-Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP-Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP-Dad1 in the ER membranes, but to a level that is still lower than that of free GFP-Dad1. This suggests that GFP-Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.     

Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane- spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

Characterization of V-ATPase inhibitor-induced secretion of cysteine-rich with EGF-like domains 2

We previously demonstrated that cysteine-rich with EGF-like domains 2 (CRELD2), a novel ER stress-inducible factor, is a secretory glycoprotein; however, the stimuli that induce CRELD2 secretion have not yet been characterized. In this study, we found that the perturbation of intravesicular acidification of cytoplasmic organelles in HEK293 cells stably expressing wild-type (wt) CRELD2 induced its secretion. In particular, Concanamycin A (CMA) and Bafilomycin A1 (Baf), inhibitors of vacuolar ATPase (V-ATPase), increased the secretion of CRELD2 without relying on its C-terminal structure. The levels of secretion of EGFP-fused CRELD2 (SP-EGFP-CRELD2), which consists of EGFP following the putative signal peptide (SP) sequence of CRELD2, from COS7 cells transiently transfected with this construct were also increased after each of the treatments, but their intracellular localization was barely affected by CMA treatment. Transient overexpression of 78-kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) also increased the secretion of CRELD2 from HEK293 cells expressing wt CRELD2, whereas the perturbation of intravesicular acidification did not alter the expression of GRP78 and PDI in the HEK293 cells. We further studied the roles of intracellular calcium ions and the Golgi apparatus in the secretion of CRELD2 from HEK293 cells in which intravesicular acidification was perturbed. The treatment with calcium ionophore increased the secretion of wt CRELD2, while that with BAPTA-AM, an intracellular calcium chelator, did not reduce the CMA-induced CRELD2 secretion. By contrast, treatment with brefeldin A (BFA), which inhibits the transportation of proteins from the ER to the Golgi apparatus, almost completely abolished the secretion of wt CRELD2 from the HEK293 cells. In conclusion, we demonstrated that the intravesicular acidification by V-ATPase regulates the secretion of CRELD2 without relying on the balance of intracellular calcium ions and the expression of ER chaperones such as GRP78 and PDI. These findings concerning the role of V-ATPases in modulating the secretion of CRELD2, a novel ER stress-inducible secretory factor, may provide new insights into the prevention and treatment of certain ER stress-related diseases.     

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.