Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form

A cDNA was cloned coding for human placental 5'-nucleotidase. The 3547-bp cDNA contains an open reading frame that encodes a 574-residue polypeptide with calculated size of 63 375 Da. The NH2-terminal 26 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5'-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 510-523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the mature 5'-nucleotidase lacks the predicted COOH-terminal peptide extension (524-548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5'-nucleotidase.     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.     

Shedding of the glycoprotein from vesicular stomatitis virus-infected cells.

In a culture of Chinese hamster ovary cells infected with vesicular stomatitis virus, there is specific shedding of viral antigens into the medium. This shedding appears to be unrelated to progeny formation or to cell lysis. Although all five of the virus-specific proteins are detected in the extracellular soluble fraction, the major antigen is the Gs protein. This protein has a molecular weight of 54,000. Indirect analysis of the content of sialic acid as well as peptide analysis of the Gs and G proteins of vesicular stomatitis virus suggest that the Gs protein is derived from the G protein by proteolysis. Both proteins are hydrophobic when analyzed by charge-shift electrophoresis. The presence of phenylmethylsulfonyl fluoride in the culture medium or the removal of serum from the culture medium partially reduces the shedding of Gs protein. Increased shedding of the Gs protein is seen when there is an unstable M or matrix protein synthesized by a temperature-sensitive mutant, tsG31. These results indicate that the G protein is cleaved at the cell surface, thus releasing Gs protein into the medium. Furthermore, the stability of G protein at the cell surface appears to be dependent on its association with the M protein.     

Evidence for carboxyl-terminal processing and glycolipid-anchoring of human carcinoembryonic antigen

We have investigated the post-translational modification of carcinoembryonic antigen (CEA) for membrane-anchoring in QGP-1 cells derived from a human pancreatic carcinoma. Pulse-chase experiments with [3H]leucine demonstrated that CEA was initially synthesized as a precursor form with Mr 150,000 having N-linked high-mannose-type oligosaccharides, which was then converted to a mature form with Mr 200,000 containing the complex type sugar chains. The mature protein thus labeled was found to be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that CEA is a phosphatidylinositol-linked membrane protein. This was confirmed by metabolic incorporation into CEA of 3H-labeled compounds such as ethanolamine, myo-inositol, palmitic acid, and stearic acid. The 3H-labeled fatty acids incorporated were specifically removed from the protein by nitrous acid deamination as well as by phosphatidylinositol-specific phospholipase C treatment. Since the available cDNA sequence predicts that CEA contains a single methionine residue only in its carboxyl-terminal hydrophobic domain, processing of the carboxyl terminus was examined by pulse-chase experiments with [35S]methionine. It was found that CEA with Mr 150,000 was initially labeled with [35S]methionine but its radioactivity was immediately lost with chase. Taken together, these results suggest that CEA is anchored to the membrane by simultaneously occurring proteolysis of the carboxyl terminus and replacement by the glycophospholipid immediately after the synthesis.     

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.     

Testing topological models for the membrane penetration of the fusion peptide of influenza virus hemagglutinin

Low pH-induced binding of the bromelain-solubilized form of influenza virus hemagglutinin (BHA) to membranes occurs through the fusion peptide. From asymmetric hydrophobic photolabeling of membranes, evidence was obtained that this peptide penetrates only one leaflet of the bilayer. The asymmetrical labeling was achieved by employing a photoreactive analogue of a fatty acid whose transbilayer distribution can be manipulated by a membrane proton gradient.     

Determinants of membrane association in the SH4 domain of Fyn: Roles of N-terminus myristoylation and side-chain thioacylation

The SH4 domain of Fyn, a member of the Src family of tyrosine kinases, though rich in polar amino acid residues, anchors to the cytosolic face of membranes upon fatty acylation. In order to probe the requirement of specific fatty acylation at the N-terminus and at the side-chain of this domain for membrane-association, we have studied the interaction of peptides corresponding to the polar segment of the SH4 domain of Fyn and its mono- and dually fatty acylated analogs with model membranes. While the polar segment without covalently linked fatty acids (KDKEATKLTEW-amide) does not interact with lipid vesicles, peptides with one covalently linked fatty acid at the N-terminus or in the side-chain, associate with zwitterionic and anionic lipids to varying degrees. The interaction of dually acylated peptides (Myr-GK(ε-myr)KDKEATKLTEW-amide and Myr-GC(S-pal)KDKEATKLTEW-amide) with lipids depends on the linkage between fatty acyl side-chain and peptide backbone. The peptide chain associates with membranes only when the side-chain acylation is via an amide bond and not via a thioester bond. Our investigations indicate that acylation is essential for membrane targeting and unacylated polar stretch of the SH4 domain does not have a role in membrane-anchoring. Side-chain acylation via a thioester bond not only provides membrane anchorage but also directs the peptide chain away from the bilayer which might be important to enable the full length protein to interact with other signaling partners.     

Intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide

A deletion mutation, malE delta 12-18, removes seven residues from the hydrophobic core of the maltose binding protein (MBP) signal peptide and thus prevents secretion of this protein to the periplasm of E. coli. Intragenic suppressor mutations of malE delta 12-18 have been obtained, some highly efficient in their ability to restore proper MBP export. Twelve independently isolated suppressors represent six unique mutational events. Five result in alterations within the MBP signal peptide; one changes the amino acid at residue 19 of the mature MBP. Analysis of these suppressors indicates that the length of the hydrophobic core is a major determinant of signal peptide function. The experiments further suggest that the hydrophobic core region serves primarily a structural role in mediating protein secretion, and that other sequences outside of this region may be responsible for providing the initial recognition of the MBP nascent chain as a secreted protein.     

Mutational Analysis of the Putative Fusion Domain of Ebola Virus Glycoprotein

Ebola viruses contain a single glycoprotein (GP) spike, which functions as a receptor binding and membrane fusion protein. It contains a highly conserved hydrophobic region (amino acids 524 to 539) located 24 amino acids downstream of the N terminus of the Ebola virus GP2 subunit. Comparison of this region with the structural features of the transmembrane subunit of avian retroviral GPs suggests that the conserved Ebola virus hydrophobic region may, in fact, serve as the fusion peptide. To test this hypothesis directly, we introduced conservative (alanine) and nonconservative (arginine) amino acid substitutions at eight positions in this region of the GP2 molecule. The effects of these mutations were deduced from the ability of the Ebola virus GP to complement the infectivity of a vesicular stomatitis virus (VSV) lacking the receptor-binding G protein. Some mutations, such as Ile-to-Arg substitutions at positions 532 (I532R), F535R, G536A, and P537R, almost completely abolished the ability of the GP to support VSV infectivity without affecting the transport of GP to the cell surface and its incorporation into virions or the production of virus particles. Other mutations, such as G528R, L529A, L529R, I532A, and F535A, reduced the infectivity of the VSV-Ebola virus pseudotypes by at least one-half. These findings, together with previous reports of liposome association with a peptide corresponding to positions 524 to 539 in the GP molecule, offer compelling support for a fusion peptide role for the conserved hydrophobic region in the Ebola virus GP.     

Molecular dynamics study of a gelsolin-derived peptide binding to a lipid bilayer containing phosphatidylinositol 4,5-bisphosphate

Gelsolin is an actin-severing protein whose action is initiated by Ca(2+) and inhibited by binding to phosphorylated inositol lipid or phosphoinositides. The regions of gelsolin responsible for phosphoinositide binding are comprised of residues 150-169 (G150-169) and 135-142 (G135-142). The corresponding peptides possess similar binding potency as native gelsolin. Their common feature is the presence of arginine and lysine residues that can bind to negatively charged phosphate groups of phosphoinositides. In this work the binding of the G150-169 peptide to a phosphatidylinositol 4,5-bisphosphate (PIP2) cluster in a lipid membrane model was investigated by molecular dynamics calculations (MD) with the AMBER 4.1 force field, taking into account explicit solvent molecules. Initially the structure of G150-169 was simulated by using the electrostatically driven Monte Carlo (EDMC) and MD methods, and the resulting structure agreed within 3.7 A backbone-atom root mean square deviation with the corresponding experimentally derived structure (PDB code: 1SOL). Using this model for the peptide, a subsequent MD simulation of G150-169 in a periodic box containing a model of dimyristoyl-phosphatidylcholine (DMPC) lipids with a cluster of four PIP2 molecules was carried out. During the simulation G150-169 interacted strongly with PIP2 molecules, initially by formation of salt bridges between its N-terminal basic groups and the phosphate groups of PIP2, followed by formation of hydrophobic bonds between the hydrophobic side chains of the peptide and the fatty acid tail of the lipid. As a result of the formation of hydrophobic bonds, the PIP2 molecules were pulled out from the lipid bilayer. This mode of binding differs from those of other PIP2-binding protein motifs such as PH domains that interact solely with the hydrophilic head group of PIP2. These results suggest that dissociation of gelsolin from actin by PIP2 lipids may involve entering of the PIP2 molecules to the gelsolin-actin interface, thereby weakening the interactions between these proteins.     

Fatty acids can substitute the HIV fusion peptide in lipid merging and fusion: an analogy between viral and palmitoylated eukaryotic fusion proteins

Various fusion proteins from eukaryotes and viruses share structural similarities such as a coiled coil motif. However, compared with eukaryotic proteins, a viral fusion protein contains a fusion peptide (FP), which is an N-terminal hydrophobic fragment that is primarily involved in directing fusion via anchoring the protein to the target cell membrane. In various eukaryotic fusion proteins the membrane targeting domain is cysteine-rich and must undergo palmitoylation prior to the fusion process. Here we examined whether fatty acids can replace the FP of human immunodeficiency virus type 1 (HIV-1), thereby discerning between the contributions of the sequence versus hydrophobicity of the FP in the lipid-merging process. For that purpose, we structurally and functionally characterized peptides derived from the N terminus of HIV fusion protein - gp41 in which the FP is lacking or replaced by fatty acids. We found that fatty acid conjugation dramatically enhanced the capability of the peptides to induce lipid mixing and aggregation of zwitterionic phospholipids composing the outer leaflet of eukaryotic cell membranes. The enhanced effect of the acylated peptides on membranes was further supported by real-time atomic force microscopy (AFM) showing nanoscale holes in zwitterionic membranes. Membrane-binding experiments revealed that fatty acid conjugation did not increase the affinity of the peptides to the membrane significantly. Furthermore, all free and acylated peptides exhibited similar α-helical structures in solution and in zwitterionic membranes. Interestingly, the fusogenic active conformation of N36 in negatively charged membranes composing the inner leaflet of eukaryotic cells is β-sheet. Apparently, N-terminal heptad repeat (NHR) can change its conformation as a response to a change in the charge of the membrane head group. Overall, the data suggest an analogy between the eukaryotic cysteine-rich domains and the viral fusion peptide, and mark the hydrophobic nature of FP as an important characteristic for its role in lipid merging.     

Fluorescence anisotropy of a fatty acid covalently linked in vivo to the glycoprotein of vesicular stomatitis virus

The covalently-attached fatty acid of the membrane glycoprotein (G) of vesicular stomatitis virus was fluorescently labeled biologically by isolating vesicular stomatitis virus from infected baby hamster kidney clone 21 cells that had been grown in the presence of 16(9-anthroyloxy)palmitate. The fluorescent labeling was specific for the G protein; the other viral membrane protein, the matrix (M) protein, was not labeled. Steady state fluorescence anisotropy of the 16(9-anthroyloxy)palmitate-labeled G protein reconstituted into dipalmitoylphosphatidylcholine vesicles indicated that the fatty acid attached to G protein is located in a dipalmitoylphosphatidylcholine domain that does not undergo the gel to liquid-crystalline phase transition.     

Palmitoylation of cysteine 69 from the COOH-terminal of band 3 protein in the human erythrocyte membrane. Acylation occurs in the middle of the consensus sequence of F--I-IICLAVL found in band 3 protein and G2 protein of Rift Valley fever virus

One of the major physiologic functions of erythrocytes is the mediation of chloride-bicarbonate exchange in the transport of carbon dioxide from the tissues to the lungs. The anion exchange is mediated by a typical polytopic transmembrane protein in the cell membrane, designated Band 3. A carboxyl-terminal peptide of Band 3 was affinity-labeled with pyridoxal phosphate, a substrate for the anion transport system, and then sequenced (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The 10th amino acid residue of the peptide could not be determined, suggesting post-translational modification of the residue. In the present communication, we have investigated the molecular structure of human Band 3 and the COOH-terminal 8500-dalton peptide using gas-liquid chromatography-mass spectrometry. Band 3 was modified covalently by fatty acids and these acids were released from Band 3 by hydroxylamine treatment at either pH 7 or 11, indicating that the linkage between Band 3 and the fatty acid is a thio ester bond. 1 mol of Band 3 interacted with 1 mol of fatty acid at a cysteine residue located 69 residues from the COOH terminus of Band 3. The fatty acids used in the modification were myristate, palmitate, oleate, and stearate, with palmitate being the major component. The esterified site is close to the site affinity-labeled with pyridoxal phosphate (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The amino acid sequence including the acylation site was Phe-Thr-Gly-Ile-Gln-Ile-Ile-Cys-Leu-Ala-Val-Leu, which is conserved in the G2 protein of Rift Valley fever virus as Phe-Ser-Ser-Ile-Ala-Ile-Ile-Cys-Leu-Ala-Val-Leu. The G2 protein, like Band 3, is a polytopic transmembrane protein. Although acylation of the cysteine residue of G2 protein has not been examined, the Phe-X-X-Ile-X-Ile-Ile-Cys-Leu-Ala-Val-Leu sequence could be a common motif for fatty acylation of certain membrane proteins.     

Strong inhibition of peptide amyloid formation by a fatty acid

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer’s and Parkinson’s disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68–78 of the protein α-synuclein involved in Parkinson’s disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.     

Features of transmembrane segments that promote the lateral release from the translocase into the lipid phase

Topogenic sequences direct the membrane topology of proteins by being recognized and decoded by integral membrane translocases. In this paper, we have compared the minimal sequence characteristics of helical-hairpin, reverse signal-anchor, and stop-transfer sequences in bacterial membrane proteins that use either the YidC or SecYEG translocases for membrane insertion. We find that a stretch composed of 3 leucines and 16 alanines is required for efficient membrane-anchoring of the M13 procoat protein that inserts by a helical hairpin mechanism, and that a stretch composed of only 19 alanines has a detectable membrane-anchoring ability. Similar results were obtained for the reverse signal-anchor sequence of the single-spanning Pf3 coat protein and for stop-transfer segments engineered into leader peptidase. We have also determined the contribution to the apparent free energy of membrane insertion of M13 procoat for all 20 amino acids. The relative order of the contributions is similar to that determined for a stop-transfer sequence in the mammalian endoplasmic reticulum, but the absolute difference between the contributions for the most hydrophobic and most hydrophilic residues is somewhat larger in the E. coli system. These results are significant because they define the features of a membrane protein transmembrane segment that induce lateral release from the YidC and Sec translocases into the lipid bilayer in bacteria.     

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.     

Cell-free fatty acylation of microsomal integrated and detergent-solubilized glycoprotein of vesicular stomatitis virus

An enzymatic activity associated with intracellular membrane fractions of Merwin plasma cell tumor II, baby hamster kidney, and chicken embryo fibroblast cells and bovine kidney has been characterized which covalently links fatty acids onto the G protein of vesicular stomatitis virus. Exogenous G protein extracted from native vesicular stomatitis virus particles can be acylated in vitro only after it has been previously deacylated. The fatty acids transferred in vitro are sensitive to treatment with hydroxylamine, indicating an ester linkage. Cell-free acyl transfer was also observed with endogenous G protein present in membrane fractions prepared from vesicular stomatitis virus-infected cells. In this case, the fatty acids become linked to a G protein species (G1) which is not terminally glycosylated and therefore has not entered the trans-Golgi compartment. The same G protein species also becomes acylated in infected cells during short pulses with radioactive palmitic acid. Acylation of the G protein in vitro with free palmitic or myristic acid is energy-dependent, and the addition of ATP is specifically required. Other nucleoside triphosphates cannot substitute for ATP in the activation of free acyl chains. Alternatively, activated fatty acids linked in a high energy thioester bond to coenzyme A, e.g. [14C] palmitoyl-CoA, are suitable lipid donors in the in vitro acylation reactions. Palmitic acid transfer onto G protein shows the typical characteristics of an enzyme-catalyzed reaction.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

Influence of membrane anchoring and cytoplasmic domains on the fusogenic activity of vesicular stomatitis virus glycoprotein G.

Chimeric proteins in which the transmembrane anchoring sequence (TM) or both the TM and the cytoplasmic tail (CT) of vesicular stomatitis virus glycoprotein G were replaced with corresponding domains of viral or cellular integral membrane proteins were used to examine the influence of these domains on acidic-pH-induced membrane fusion by G protein. The TM and CT of G were also replaced with the lipid anchor glycosylphosphatidylinositol. Hybrids containing foreign TM or TM and CT sequences were fusogenic at acidic pH but glycosylphosphatidylinositol-anchored G was nonfusogenic at acidic pH. The results suggest that the fusogenic activity of G protein requires membrane anchoring by a hydrophobic peptide sequence and the specific amino acid sequence of the TM has no influence on fusogenic activity.     

Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Leu(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.