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1、A Specific Transmembrane Domain of a Coronavirus E1 Glycoprotein Is Required for ItsRetention in the Golgi RegionAuthor(s): Carolyn E. Machamer and John K. RoseSource: The Journal of Cell Biology, Vol. 105, No. 3 (Sep., 1987), pp. 1205-1214Published by: The Rockefeller University PressStable URL: ht
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4、rating with JSTOR to digitize, preserve and extend access to TheJournal of Cell Biology.http:/www.jstor.org This content downloaded from 185.2.32.21 on Sun, 22 Jun 2014 20:27:03 PMAll use subject to JSTOR Terms and ConditionsAbstract. The E1 glycoprotein of the avian coronavi- rus infectious bronchi
5、tis virus contains a short, glycosylated amino-terminal domain, three membrane- spanning domains, and a long carboxy-terminal cyto- plasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in in- fected cells. E1 proteins with precise deletions of the first and s
6、econd or the second and third membrane- spanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain rT iHE intracellular transport and targeting of proteins from their site of synthesis to their correct destination is a process instrumental to maintainence
7、 of cellular integrity. Membrane-bound and secreted proteins are sorted from cytoplasmic proteins and those destined for the nucleus or mitochrondria by virtue of a signal or leader sequence which directs insertion into the lumen of the endoplasmic reticulum (Walter et al., 1984). Plasma membrane an
8、d se- creted proteins follow a similar pathway through the cell, from the rough endoplasmic reticulum through the Golgi complex en route to the plasma membrane (Sabatini et al., 1982). Other proteins in this exocytic pathway are retained at points along the way, such as cytochrome P-450 in the en- d
9、oplasmic reticulum (Brands et al., 1985) and galactosyl transferase in the Golgi complex (Roth and Berger, 1982). Lysosomal enzymes are sorted to lysosomes after passage through the Golgi complex. Some lysosomal enzymes are sorted via a specific marker, a mannose-6-phosphate modi- fication of aspara
10、gine-linked (N-linked)1 oligosaccharides of these enzymes, which is recognized by a receptor (Sly and Fischer, 1982). The process of intracellular transport to the plasma mem- brane could be governed by positive sorting signals; i.e. pro- C. E. Machamers present address is Department of Pathology, Y
11、ale Univer- sity School of Medicine, New Haven, Connecticut 06510. J. K. Roses pres- ent address is Departments of Pathology and Cell Biology, at the same in- stitute. 1. Abbreviations used in this paper: IBV, infectious bronchitis virus; MHV, mouse hepatitis virus; N-linked, asparagine-linked; VSV,
12、 vesicular stomati- tis virus. ? The Rockefeller University Press, 0021-9525/87/09/1205/10 $2.00 The Journal of Cell Biology, Volume 105, September 1987 1205-1214 can function as an internal signal sequence. The mu- tant protein with only the first transmembrane domain accumulated intracellularly li
13、ke the wild-type protein, but the mutant protein with only the third transmem- brane domain was transported to the cell surface. This result suggests that information specifying accumula- tion in the Golgi region resides in the first transmem- brane domain, and provides the first example of an in- t
14、racellular membrane protein that is transported to the plasma membrane after deletion of a specific domain. teins with these signals would be transported to the cell sur- face via a type of receptor-mediated process, like that involved in targeting of lysosomal enzymes. Alternatively, as recently su
15、ggested by Kelly (1985) and Rothman (1986), pro- teins that are destined for the plasma membrane or for con- stitutive secretion could move passively with the bulk flow of lipids, while proteins secreted in a regulated manner or those retained in intracellular membranes would possess sig- nals which
16、 selectively retain them from moving with the bulk flow of membranes. A third model could invoke both positive signals that enhance the rate of incorporation of proteins into transport vesicles, and negative retention signals that ensure intracellular proteins to not be transported beyond a certain
17、point. Recombinant DNA technology that uses gene expression and site-directed mutagenesis offers a powerful approach to the study of intracellular transport and targeting. For exam- ple, using this approach, an involvement of cytoplasmic do- mains of proteins has been suggested in facilitating both
18、exocytosis (Rose and Bergmann, 1983) and endocytosis (Lehrman et al., 1985; Roth et al., 1986), and in targeting of proteins to basolateral membranes of polarized epithelial cells (Mostov et al., 1986; Puddington et al., 1987). Al- though specific molecules interacting with these domains have not ye
19、t been identified, these results suggest that impor- tant interactions with cytoplasmic domains do occur. Viruses provide simple and useful model systems for studying the signals involved in targeting of membrane pro- teins in cells. The coronaviruses provide an especially inter- esting model becaus
20、e they bud from intracellular membranes rather than from the plasma membrane. The best-studied 1205 A Specific Transmembrane Domain of a Coronavirus E1 Glycoprotein Is Required for Its Retention in the Golgi Region Carolyn E. Machamer and John K. Rose Molecular Biology and Virology Laboratory, The S
21、alk Institute for Biological Studies, San Diego, California 92138 This content downloaded from 185.2.32.21 on Sun, 22 Jun 2014 20:27:03 PMAll use subject to JSTOR Terms and Conditionsmembers of the coronavirus group are mouse hepatitis virus (MHV) and avian infectious bronchitis virus (IBV). Both vi
22、ruses specify two glycoproteins called E1 and E2 (reviewed by Sturman and Holmes, 1985). E2 forms the virion spike and can be detected in the plasma membrane as well as intra- cellular membranes of infected cells. In contrast, E1 ac- cumulates intracellularly, and thus appears to play a critical rol
23、e in intracellular budding of the virus. The viral nucleo- capsid interacts with the MHV E1 protein, presumably the cytoplasmic tail, and virion budding occurs at the site of E1 accumulation in infected cells (Sturman et al., 1980; Dubois- Dalque et al., 1982; Tooze et al., 1984). The sequences of c
24、DNAs encoding E1 proteins from both MHV and IBV have been recently reported (Armstrong et al., 1984; Boursnell et al., 1984). These sequences predict polypeptides with a similar structure: a short, glycosylated amino-terminal domain, three hydrophobic domains be- lieved to span the membrane three ti
25、mes, and a long cytoplas- mic domain at the carboxy terminus. Studies that use pro- tease treatment of intact virus (Sturman and Holmes, 1977; Cavanagh et al., 1986) or of E1 inserted into microsomal membranes (Rottier et al., 1984) support the model for the structure of El. Since there is no cleave
26、d amino-terminal sig- nal sequence (Rottier et al., 1984), and the protein is inserted into microsomal membranes in a signal recognition parti- cle-dependent manner (Rottier et al., 1985), it has been sug- gested that one or more of the putative membrane-spanning domains functions as an internal, un
27、cleaved signal sequence. We report here that the IBV E1 glycoprotein is retained in the Golgi region of the cell in the absence of the other viral proteins when expressed from cDNA. In an earlier study using gene expression and mutagenesis techniques, we deter- mined that the cytoplasmic domain of t
28、he IBV E1 protein did not contain a signal that was capable of retaining the G pro- tein of vesicular stomatitis virus (VSV) in intracellular mem- branes (Puddington et al., 1986). Thus, in the present study we chose to search for a retention signal in the amino- terminal half of the IBV E1 protein.
29、 We report that the first of the three membrane-spanning domains may constitute such a signal, and our results are discussed in terms of cur- rent models for protein transport to the plasma membrane. Materials and Methods Construction of an Expression Vector Encoding IBV E1 A cDNA clone encoding the
30、 E1 protein of the Beaudette strain (M42) of IBV was derived from viral genomic RNA and kindly provided by D. Stern (pIBV-5; Stern, 1983). A subclone containing the entire coding region was prepared (p57-6), and the nucleotide sequence was determined by the proce- dure of Maxam and Gilbert (1977). T
31、his sequence predicts a polypeptide with 225 amino acids, and differs at only two nucleotides from the sequence published by Boursnell et al. (1984) for the E1 protein of the Beaudette strain of IBV. These differences were a T instead of a C at nucleotide 167 and a C instead of a T at nucleotide 375
32、, changing the codon for Pro 2 and for Thr 71 to that for Ser and Ile, respectively. A fragment that contains the en- tire coding sequence of IBV E1 (773 bp) was excised from p57-6 with Hpa I and Hha I, and incubated with the Klenow fragment of DNA polymerase I and deoxynucleoside triphosphates to r
33、emove the 3 overhang. After liga- tion with Xho I linkers, the fragment was cloned into the unique Xho I site of the SV40-based expression vector, pJC119 (Sprague et al., 1983), and a clone with the insert in the proper orientation for expression from the late SV40 promoter was selected (pSV/IBVE1).
34、 This construct includes a 5 un- translated region of 52 nucleotides, and a 3 untranslated region of 45 nucleotides. The Journal of Cell Biology, Volume 105, 1987 1206 Oligonucleotide-directed Mutagenesis Synthetic oligonucleotides were used to precisely delete coding sequences for putative membrane
35、-spanning domains of El. The negative strand of the IBV E1 gene was cloned into the Bam HI site of M13 mp8 as described (Rose et al., 1984), and single-stranded phage DNA was purified for use as the template for mutagenesis. Synthetic oligonucleotides (33 mers) were de- signed to loop out specific r
36、egions of the coding sequence by hybridizing with 16 nucleotides on the 5 side of the loop and 17 nucleotides on the 3 side. The domain junctions were assigned on the basis of secondary struc- ture and hydrophobicity predictions for IBV E1 (Boursnell et al., 1984; Rot- tier et al., 1986). The oligon
37、ucleotide used to generate the coding sequence for the mutant protein that lacked the second and third hydrophobic domains (Am2,3) was 5GTATGGCTATGCAACAAGAClU 1 1 AAGCGGTG-3 and that for the mutant protein that lacked the first and second hydrophobic domains (Aml,2) was 5-TCAGCIlIITIAAAGAGGGAGGLIUlC
38、GCAGC-3. They were synthesized and purified as previously described (Rose et al., 1984; Machamer et al., 1985). After hybridization of the oligonucleotide with the template DNA, primer extension was carried out with the Klenow fragment of DNA polymerase I (Boehringer Mannheim Biochemicals, Indi- ana
39、polis, IN) by the procedure previously described (Adams and Rose, 1985; Machamer et al., 1985). After transfection of competent Escherichia coli JM103 with the primer extension mixture, plaques containing the muta- tions were identified by differential hybridization with the 5-32P-labeled oligonucle
40、otide. DNA containing the mutation was excised and subcloned into the expression vector pJC119, and the mutation confirmed by DNA se- quence analysis (Maxam and Gilbert, 1977). The mutant protein Am2,3 was created by fusion of the codon for Thr 42 to that for Arg 102 (deleting 156 nucleotides), and
41、Aml,2 by fusion of the codon for Glu 20 to that for Gly 77 (deleting 168 nucleotides). Cells and Virus Both COS-1 and HeLa cells were maintained in Dulbecco-Vogts modified Eagles medium with 5% FCS. The IBV used in these experiments was adapted to growth in monkey cells by nine consecutive passages
42、of egg- grown virus at low multiplicity of infection in Vero cells, and was obtained from B. Sefton (The Salk Institute). Tissue culture supernatant from infected Vero cells was used as the inoculum to infect COS-1 cells for radiolabeling and immunofluorescence. The multiplicity of infection was app
43、roximately 0.1, and infected cells were assayed for IBV-specific polypeptides at 29 and 48 h postinfection. Preparation of Recombinant Vaccinia Viruses Encoding El and A ml,2 DNA encoding wild-type E1 or the mutant protein Aml,2 was excised from the SV40 expression vector with Xho I, and the ends we
44、re filled in with the Klenow fragment of DNA polymerase I. The inserts were then subcloned into the vector pSCll (Chakrabarti et al., 1985) at the unique Sma I site. After selection of clones with inserts in the correct orientation for expres- sion from the vaccinia promoter P7.5K, supercoiled DNA w
45、as transfected onto HeLa cells by calcium phosphate coprecipitation. The HeLa cells had previously been infected at a multiplicity of 0.05 with wild-type vaccinia vi- rus 1 h before addition of the DNA. The medium was replaced 18 h later, and incubation continued another 2 d. Recombinant virus was i
46、solated by including X-gal (5-bromo-4-chloro-3-indolyl-PB-D-galactopyranoside; Boeh- ringer Mannheim Biochemicals) in the plaquing overlay as described (Chakrabarti et al., 1985). The pSCll vector contains the E. coli lacz gene under control of a late vaccinia promoter; thus ,B-galactosidase activit
47、y results in formation of blue plaques by recombinant virus. Recombinant viruses encoding E1 or Aml,2 proteins (VVE1 and VVAml,2) were plaque- purified three times in HeLa cells, and large stocks were prepared. HeLa cells (5 x 105) in 35-mm dishes were infected with the recombinant vac- cinia viruse
48、s at a multiplicity of approximately 4, and analyzed for expres- sion 5 h later. Antibody Preparation A polyclonal rabbit antiserum was raised to a synthetic peptide correspond- ing to the carboxy-terminal 22 amino acids of IBV El. The peptide was con- jugated to BSA via the penultimate tyrosine res
49、idue with bis-diazobenzidine This content downloaded from 185.2.32.21 on Sun, 22 Jun 2014 20:27:03 PMAll use subject to JSTOR Terms and Conditions(DeCarvalho et al.,1964). Two New Zealand white rabbits were immunized with 1 mg conjugate each, emulsified with 0.75 ml complete Freunds adju- vant in a
50、total volume of 1.5 ml. About 30 sites were injected intradermally with 50 tl per site. Rabbits were boosted with 40.5 mg conjugate (0.75 ml total) in the same way every 4 wk. ELISA titers to the peptide were mea- sured in a solid-phase assay that uses a second antibody conjugated with horseradish p
51、eroxidase, and were approximately 1:10,000 after the second boost. This titer did not change significantly after repeated boosts. After the second boost, the synthetic peptide was discovered to be incorrect in that it contained an extra glycine (after the glycine at position 8 of the peptide), and s
52、ubsequent boosts were performed with the correct synthetic peptide. Ability of the antiserum to immunoprecipitate the hybrid protein G23 (Pud- dington et al., 1986) was detected after the third boost. This titer increased until the fifth boost, when one of the two rabbits was producing antibody with
53、 a slightly higher titer than the other with less background in im- munoprecipitates. This antiserum was used for all subsequent experiments. Anti-peptide antibodies were affinity purified on Affigel-histamine columns coupled with the peptide. Histamine was coupled to Affi-gel 10 (Bio-Rad Laboratori
54、es, Richmond, CA); the peptide was then coupled to the conjugated resin via the tyrosine residue with bis-diazobenzidine as de- scribed above. 10 mg of peptide was coupled to 1 ml of resin, and remaining active groups were blocked with 1 mg/ml ovalbumin. Serum (4 ml) contain- ing antibodies to the p
55、eptide was incubated with 1 ml of conjugated resin for 4 h at 4?C with end-over-end inversion. After transfer to a column, resin was first washed with 20 ml of 10 mM Tris, pH 8.0, with 0.2% deoxycholate, and then with 15 ml of 10 mM Tris, pH 8.0, containing 0.5 M NaC1. Specific antibodies were elute
56、d with 4 M MgCl2 and dialyzed immediately against 10 mM Tris, pH 7.4, containing 0.15 M NaC1 and 0.02% NaN3. Pooled fractions with the highest OD280 reading were stored at 4?C with 100 U/ml kallikrein inhibitor (Calbiochem-Behring Corp., La Jolla, CA) and 0.5 mg/ml ovalbumin. Approximately 1.5 mg of
57、 purified immunoglobulin was recovered from 4 ml of serum. Transfection of COS-I Cells, Radiolabeling and Immunoprecipitation COS-1 cells (4 x 105) plated in 35-mm dishes the previous day were trans- fected with 10 ug supercoiled DNA using DEAE-dextran followed by chlo- roquine treatment as previous
58、ly described (Adams and Rose, 1985). Ap- proximately 44 h after transfection, cells were labeled at 37?C with 50 uCi 35Scysteine (1,300 Ci/mmol; Amersham Corp., Arlington Heights, IL) in 0.5 ml cysteine-free Dulbecco-Vogts modified Eagles medium containing 4% dialyzed FCS for 1 h or for the time ind
59、icated. For treatment with tunicamycin, transfected cells were pretreated with 3 tg/ml tunicamycin (Sigma Chemical Co., St. Louis, MO) for 2 h, and then labeled in the pres- ence of the same concentration of the drug. COS-1 or HeLa cells infected with recombinant vaccinia viruses were labeled 5 h af
60、ter infection with 35Scysteine as described above. After labeling, cells were lysed at 0?C in 0.5 ml of a solution containing 50 mM Tris, pH 8.0, 1% NP-40, 0.4% deoxy- cholate, 62.5 mM EDTA, and 100 U/ml kallikrein inhibitor per ml. Nuclei were removed by centrifugation at 15,000 g for 1 min, and ly
61、sates were ad- justed to a final concentration of 0.3 % SDS. For immunoprecipitation of E1 proteins, 5 gl of anti-peptide serum (not affinity purified) was incubated for 2 h with 0.5 ml lysate at 4?C. Antigen-antibody complexes were isolated with protein A-bearing Staphylococcus aureus (Pansorbin; C
62、albiochem- Behring Corp.), and washed four times with RIPA buffer (10 mM Tris, pH 7.4, 0.15 M NaC1, 1% NP-40, 1% deoxycholate, and 0.1% SDS). Pellets were eluted by incubation in Laemmli sample buffer containing 2 % 2-mer- captoethanol at room temperature for 20 min (unless otherwise noted), and the
63、 S. aureus was removed by centrifugation. In the experiment shown in Fig. 2 B, pellets were eluted in 1 M Tris, pH 8.8, 2 % SDS, and 2 % 2-mer- captoethanol at 100?C for 1 min. After removal of the S. aureus cells, super- natants were incubated with a final concentration of 0.33 M iodoacetamide for
64、60 min at room temperature, and proteins were precipitated with 9 vol of acetone at -20?C for 2 h. After washing in acetone, precipitates were dried and resuspended in sample buffer without 2-mercaptoethanol. Im- munoprecipitates were subjected to electrophoresis in 10 or 15% poly- acrylamide gels c
65、ontaining SDS (Laemmli, 1970). Marker proteins were lnCmethylated standard molecular weight markers (Amersham Corp.). Labeled proteins were detected by fluorography (Bonner and Laskey, 1974). Indirect Immunofiuorescence Microscopy COS-1 cells grown on coverslips were fixed with paraformaldehyde and
66、per- meabilized with NP-40 as described (Rose and Bergmann, 1982). E1 pro- teins were detected by incubation with the affinity-purified anti-El peptide antibody described above (1:30) followed by affinity-purified fluorescein- conjugated goat anti-rabbit IgG (1:50; Southern Biotechnology Associates,
67、 Inc., Birmingham, AL). For localization of the Golgi complex, coverslips were incubated with rhodamine-conjugated wheat germ agglutinin (1:100, E-Y Laboratories, Inc., San Mateo, CA). Cells were visualized with a Nikon Optiphot microscope equipped with fluorescence epiillumination and a Nikon 40x o
68、il immersion plan apochromat objective. Treatment of Intact Cells with Proteases Transfected COS-1 cells were radiolabeled as described above with 35SJcysteine for 90 min, and incubated in the presence of excess unlabeled cysteine for 90 min. Cells were then incubated for 15 min at 37?C in 0.5 ml of
69、 PBS containing 1 mg/ml bromelain (Calbiochem-Behring Corp.) and 0.1 mM 2-mercaptoethanol. Parallel dishes of transfected cells were in- cubated for the same period of time in the absence of bromelain, then lysed as usual. Bromelain-treated cells were collected by centrifugation and washed three tim
70、es in Tris-buffered saline, lysed, and E1 proteins were im- munoprecipitated as described above. HeLa cells infected with VVE1 or VVAml,2 were labeled 5 h postinfection with 35Scysteine for 60 min. As described above, cells were either (a) mock digested for 15 min at 37?C, (b) treated with 1 mg/ml b
71、romelain, (c) treated with 1 mg/ml trypsin-TPCK (Worthington Biochemical Corp., Freehold, NJ) in PBS, or (d) treated in medium with 1% FCS containing 1 mg/ml pronase (from Streptomyces griseus; Boehringer Mannheim Biochemicals), which had been previously self digested for 15 min at 37?C. Results Con
72、struction of an Expression Vector Encoding IBVE1 A cDNA clone prepared from viral genomic RNA encoding the IBV E1 protein was obtained from D. Stern (Stern, 1983), and subjected to DNA sequence analysis. This nucleotide se- quence predicts a polypeptide of 225 amino acids, and is identical to that f
73、or the IBV E1 protein as reported by Bours- nell et al. (1984), with the exception of two nucleotides (Fig. 1; see Materials and Methods). This sequence includes two potential sites for N-linked glycosylation at Asn 3 and Asn 6, both of which are glycosylated in the E1 protein isolated from IBV, sin
74、ce the protein contains two N-linked oligosac- charides (Stern and Sefton, 1982b). In contrast, the MHV E1 protein contains only O-linked carbohydrate (Neimann and Klenk, 1981). The three potential membrane-spanning do- mains of IBV E1 include amino acids Tyr 21 through Thr 42, 10 20 30 40 50 MSNETN
75、CTLDFEQSVqLFK. E !SKVIYTL 60 70 80 90 1 00 110 120 130 140 150 :LFKRCRSWWSFNPESNAVGSILLTNGQQCNFAIESVPMVLSPIIKNGV 160 170 180 190 200 LYCEGqWLAKCEPDHLPKDIFVCTPDRRNIYRMVQKYTGDQSGNKKRFAT 210 220 FVYAKQSVDTGELESVATGGSSLY Figure 1. Predicted amino acid sequence of IBV El. Membrane- spanning domains are s
76、haded, and glycosylated asparagine residues are underlined. The nucleotide sequence previously published for this protein (Boursnell et al., 1984) differs at two nucleotides, which results in changing the codon for serine 2 and for isoleucine 71 to that for proline and threonine, respectively. 1207
77、Machamer and Rose Golgi Retention Signal This content downloaded from 185.2.32.21 on Sun, 22 Jun 2014 20:27:03 PMAll use subject to JSTOR Terms and ConditionsA c -69 -46 -30 - 14.3 or toxicity to cells, aberrant splicing from cryptic splice sites, and/or a low titer of antibodies to E1 in the anti-I
78、BV serum. Results obtained with hybrid proteins between IBV E1 and VSV G protein suggested that at least one of the prob- lems was an insufficient titer of antibodies to E1 in the anti-IBV serum (not shown). uninf IBV 11 _ aCTEl Detection of IBV E1 in Transfected Cells using an Anti-Peptide Antiseru
79、m We prepared a rabbit antiserum specific for a synthetic pep- tide corresponding to the carboxy-terminal 22 amino acids of IBV El. A peptide corresponding to this region of the polypeptide was chosen because it is likely to be the most accessible in the native protein. COS-1 cells infected with IBV
80、 were labeled 48 h after infection with 35Scysteine, and the cell lysate was subjected to immunoprecipitation with the anti-El peptide serum. A single polypeptide of 32 kD was specifically immunoprecipitated by the anti-El pep- tide serum from IBV-infected cells (Fig. 2 A). In contrast, the anti-IBV
81、 serum precipitated the nucleocapsid protein (N) as well as E1 and trace amounts of the spike glycoprotein, E2. The anti-peptide serum was therefore highly specific for IBV El. COS-1 cells transfected with DNA encoding IBV E1 were labeled 44 h after transfection with 35Scysteine and sub- jected to i
82、mmunoprecipitation. A polypeptide co-migrating with E1 from infected cells was precipitated from COS-1 cells transfected with pSV/IBVE1 but not with the vector pJC119 lacking the insert encoding E1 (Fig. 2 B). A small amount of a 61-kD protein can also be observed in the im- munoprecipitates from tr
83、ansfected cells (Fig. 2 B; see also Fig. 7 A). This band was specific for El-expressing COS-1 cells, but was not consistently observed. It is probable that it represents a dimer of El, as the MHV E1 protein has been shown to be susceptible to aggregation, especially when heated in the presence of SD
84、S and reducing agent (Sturman, 1977). In addition to the transient expression of IBV E1 in COS-1 cells using the SV40-based vector, expression of E1 was also achieved in cells infected with a recombinant vaccinia virus that encodes the protein. This expression system is advanta- geous since every ce
85、ll is infected and is thus expressing the protein of interest, and since replication in the cytoplasm eliminates the potential problem of aberrant splicing at cryp- tic splice sites (see Discussion). Recombinant vaccinia vi- rus encoding IBV E1 under control of an early vaccinia pro- moter was const
86、ructed as described (Mackett et al., 1984; Chakrabarti et al., 1985). COS-1 cells infected with this recombinant virus (VVE1), or one encoding the VSV G pro- tein (VVG), were labeled 5 h after infection with 35Scys- teine, and lysates were subjected to immunoprecipitation with the anti-E1 peptide se
87、rum. A polypeptide which co- migrates with IBV E1 was readily detected in immunoprecip- itates from cells infected with the recombinant virus VVE1, but not from control cells infected with VVG (Fig. 2 C). Localization of E1 in Transfected Cells by Indirect Immunofluorescence Localization of the IBV
88、E1 protein in transfected COS-1 cells was compared to that in IBV-infected cells using indirect immunofluorescence microscopy. Cells grown on coverslips were either infected with IBV or tansfected with pSV/IBVEl. Cells were fixed and permeabilized 29 h after infection or N N El- Figure 2. Detection
89、of E1 in infected and transfected COS-1 cells. (A) COS-1 cells were either mock-infected or infected with IBV and radiolabeled 48 h later with 35Scysteine for 2 h. IBV proteins were immunoprecipitated with a rabbit anti-IBV serum (alBV) or with a rabbit anti-peptide serum specific for the carboxy te
90、rminus of IBV E1 (aCTE/). Positions of the IBV proteins N and E1 are noted, and the arrow marks the position of a polypeptide that is most likely the spike glycoprotein, E2. (B) COS-1 cells were trans- fected with vector alone (pJCl9) or with pSV/IBVEl, and labeled 44 h later with 35Scysteine for 2
91、h. E1 was immunoprecipitated with aCTEl. (C) COS-1 cells were infected with a recombinant vaccinia virus encoding IBV E1 (VVE1) or as a control, with a recombinant vaccinia virus encoding VSV G protein (VVG). Cells were labeled 5 h after infection with 35Scysteine for 1 h, and E1 was immunoprecipita
92、ted with aCTEl. Samples in A and C were eluted in sample buffer at room temperature for 20 min, and those in B were eluted at 100?C, followed by alkylation with iodoaceta- mide (see Materials and Methods). Samples in A and B were sub- jected to electrophoresis in the same 15% polyacrylamide gel, and
93、 samples in C were subjected to electrophoresis in a 10% polyacryl- amide gel. Met 52 through Tyr 72, and Gly 78 through Ile 101 (Fig. 1). A fragment containing the complete coding sequence for IBV E1 was subcloned into the SV40-based expression vec- tor, pJCl19 (Sprague et al., 1983). When transfec
94、ted onto COS cells, which provide T antigen and thus support exten- sive replication of the vector (Gluzman, 1981), high levels of protein can be expressed transiently from the late SV40 pro- moter. Initial attempts to detect the IBV E1 polypeptide in COS-1 cells transfected with this construct (pSV
95、/IBVEl) were unsuccessful. These experiments were performed with a rabbit hyperimmune serum raised to purified virus, which immunoprecipitates IBV E1 from infected chick embryo kid- ney cells (Stem and Sefton, 1982a). Although E1 could not be detected in transfected COS-1 cells, El-specific RNA was
96、readily detected in dot blots (data not shown). Inability to de- tect the protein could have been the result of its instability The Journal of Cell Biology, Volume 105, 1987 1208 B zE *CTEl ,i,- IU I& COflurj aaaa This content downloaded from 185.2.32.21 on Sun, 22 Jun 2014 20:27:03 PMAll use subjec
97、t to JSTOR Terms and ConditionsaCTE1 WGA IBV pSV/IBVE1 Figure 3. Detection of IBV E1 in infected and transfected COS-1 cells by indirect immunofluorescence microscopy. COS-1 cells grown on coverslips were infected with IBV or transfected with pSV/IBVEl, and fixed and permeabilized 29 h after infecti
98、on or 44 h after transfec- tion. E1 was detected by incubation of the coverslips with the affinity-purified anti-E1 peptide serum followed by a fluorescein-conjugated goat anti-rabbit IgG. The Golgi complex of these cells was localized by staining with rhodamine-conjugated wheat germ agglutinin (WGA
99、). Each set of micrographs shows the same field photographed with the fluorescein (aCTE/) and the rhodamine (WGA) filters. Arrows indicate the Golgi region. Bar, ,10 Rm. 44 h after transfection, and E1 was detected by incubation with the affinity-purified anti-El peptide antibody followed by a fluor
100、escein-conjugated second antibody. The cells were then stained with rhodamine-conjugated wheat germ aggluti- nin, a marker for the Golgi region (Virtanen et al., 1978). In both infected and transfected cells, IBV E1 is localized in a perinuclear region which co-localizes with the region stained by w
101、heat germ agglutinin (Fig. 3). No staining of the plasma membrane was observed; this suggests that E1 ac- cumulates intracellularly in the absence of the other viral proteins, as well as in IBV-infected cells. This intracellular accumulation has been well documented for the E1 glycopro- tein from MH
102、V in infected cells (Dubois-Dalcq et al., 1982; Tooze et al., 1984; Tooze and Tooze, 1985). Expression of Mutant El Proteins Which Lack Two of the Three Putative Membrane-spanning Domains Our goal was to identify a structural feature of E1 that might be responsible for intracellular accumulation of
103、the protein. Results obtained with hybrid proteins of VSV G protein and IBV E1 suggested that the amino-terminal half of El might contain this information. The hybrid protein G23, with the extracellular and transmembrane domains of G protein and the cytoplasmic domain of E1 was transported efficient
104、ly to the cell surface (Puddington et al., 1986), whereas the reciprocal hybrid protein 23G (with the amino-terminal and hydrophobic domains of E1 and the cytoplasmic domain of G protein) was retained intracellularly like the wild-type E1 protein (data not shown). Oligonucleotide-directed mutagen- e
105、sis was performed to delete sequences encoding either the second and third or the first and second transmembrane do- mains. This would be expected to produce proteins with only one membrane-spanning domain (the first or the third hydro- phobic region, respectively) with the same membrane orien- tati
106、on as wild-type El. Two questions regarding the mutant proteins could be asked. First, can the first or third mem- brane-spanning domain mediate insertion of the protein into the membrane in the absence of the other two hydrophobic domains? Second, what is the effect of removing these re- gions of t
107、he protein on the intracellular accumulation of El? Oligonucleotides were designed to loop out regions of the coding sequence to precisely delete membrane-spanning do- mains and the intervening sequences. The mutagenesis was performed in phage M13 by standard methods (Zoller and Smith, 1982) as desc
108、ribed previously (Machamer et al., 1985; Adams and Rose, 1985). A schematic representation of the mutant proteins is presented in Fig. 4. The codon for threonine 42 is fused to that for arginine 102 in the mutant protein that lacks the second and third hydrophobic domains (Am2,3), such that only the
109、 first hydrophobic domain re- mains. Likewise, the codon for glutamic acid 20 is fused to that for glycine 77 in the mutant protein that lacks the first and second hydrophobic domains (Aml,2), leaving only the third hydrophobic domain. After DNA encoding the mutant proteins was subcloned into the ex
110、pression vector, the desired mutations were con- firmed by DNA sequence analysis. COS-1 cells were trans- Machamer and Rose Golgi Retention Signal 1209 This content downloaded from 185.2.32.21 on Sun, 22 Jun 2014 20:27:03 PMAll use subject to JSTOR Terms and Conditionslurren jJG IGu 20wGly 77 | Am1,
111、2 Figure 4. Schematic representation of wild-type IBV E1 and mutant proteins lacking two of the three membrane-spanning domains. The model depicts the predicted membrane orientation for the wild-type protein as well as the two mutants generated by oligonucleotide- directed mutagenesis which lack eit
112、her the second and third hydro- phobic domains (Am2,3) or the first and second hydrophobic do- mains (Aml,2). The point at which the coding sequence is fused to create the mutant proteins is also shown. i lipid bilayer E C Am2,3 + ;a% of - + - + - 30 - 21.5 -14.3 ligure 5. Expression of the mutant E
113、1 proteins in transfected COS-1 cells. COS-1 cells were transfected with DNA encoding the wild- type E1 protein, or the mutant E1 proteins Am2,3 or Aml,2. Cells were labeled 44 h after transfection with 35Scysteine for 1 h. A parallel set of transfected cells was pretreated with 3 Iag/ml tunicamycin
114、 (Tm) for 2 h and then labeled in the presence of the same concentration of the drug. E1 proteins were immunoprecipi- tated with the anti-El peptide serum and subjected to electrophore- sis in a 15% polyacrylamide gel. fected with these DNAs, and radiolabeled with 35Scysteine 44 h later. E1 proteins
115、 were immunoprecipitated and ana- lyzed on SDS-polyacrylamide gels (Fig. 5). Parallel dishes of transfected cells were pretreated with and labeled in the presence of tunicamycin to determine if the mutant proteins were glycosylated with N-linked oligosaccharides. As shown in Fig. 5, the relative mob
116、ilities of both of the mutant proteins (27 kD) is consistent with deletion of 52 or 56 amino acids. In addition, as seen by comparing the mobilities of the pro- teins produced in tunicamycin-treated cells, both are glycosylated with N-linked oligosaccharides, indicating that both are inserted in the
117、 membrane of the endoplasmic reticu- lum. The mutant protein Aml,2 may be less efficiently glycosylated than wild-type E1 and Am2,3, since bands representing nonglycosylated Aml,2 and Aml,2 with one oligosaccharide are also observed. The oligosaccharides on wild-type E1 and both of the mutant protei
118、ns appear to re- main in the high-mannose form, since they remain suscepti- ble to cleavage with endoglycosidase H (data not shown). Thus, it appears that either the first or the third hydropho- bic domains of IBV E1 can mediate insertion of the polypep- tide into the lipid bilayer in vivo. Neither
119、of the mutant pro- teins could be detected in the medium harvested from labeled transfected cells, even after a labeling period of 8 h (data not shown), suggesting that either the first or third hydrophobic domains can also serve to anchor the protein in the mem- brane by spanning the lipid bilayer.
120、 Localization of the Mutant EI Proteins in Transfected Cells The intracellular distribution.of the mutant E1 proteins was analyzed by indirect immunofluorescence microscopy. COS- 1 cells grown on coverslips were fixed and permeabilized 44 h after transfection. E1 proteins were visualized by staining
121、 with the affinity-purified anti-E1 peptide antibody, followed by a fluorescein-conjugated second antibody. The results are presented in Fig. 6. The mutant E1 protein with only the first hydrophobic domain (Am2,3) was localized intracellularly, in a pattern similar to that observed for the wild-type
122、 E1 pro- tein. Prominent perinuclear staining as well as some reticu- lar staining was observed for both proteins. However, the other mutant protein with only the third hydrophobic domain (Aml,2) appeared in a pattern which resembles that found for the hybrid protein G23, which is transported to the
123、 plasma membrane (Puddington et al., 1986). Staining of microvilli is readily detected in cells expressing both Aml,2 and G23 (Fig. 6). This surprising finding suggested that dele- tion of the first and second membrane-spanning domains of E1 results in removal of a structural feature of the protein
124、which is essential for its accumulation in intracellular mem- branes. Since the mutant protein with only the first mem- brane-spanning domain (A2,3) accumulates intracellularly The Journal of Cell Biology. Volume 105. 1987 1210 V El Am2.3 Am1.2 n: This content downloaded from 185.2.32.21 on Sun, 22
125、Jun 2014 20:27:03 PMAll use subject to JSTOR Terms and Conditionslike the wild-type protein, the information for retention probably resides in the first hydrophobic domain. Although the results with indirect immunofluorescence suggested that the mutant protein Aml,2 was transported to the plasma mem
126、brane, we sought a more definitive experi- ment to prove that the amino terminus of the protein was exposed at the cell surface. This was not possible with im- munofluorescence experiments, since our antibody recog- nizes a determinant found only on the cytoplasmic side of the membrane. This proof w
127、as obtained by assessing the sensi- tivity of the protein to proteolysis when intact, transfected cells were treated with bromelain. The amino-terminal, glycosylated domain of IBV E1 in purified virus was recently shown to be susceptible to digestion with this enzyme (Cavanagh et al., 1986). Transfe
128、cted COS-1 cells were la- beled for 90 min with 35Scysteine, followed by a 90-min chase period. Cells were then treated with bromelain for 15 min. A parallel set of dishes were incubated in the absence of bromelain. Cells were washed and lysed, and E1 proteins were immunoprecipitated and subjected t
129、o electrophoresis in an SDS-polyacrylamide gel. Fig. 7 A shows that both wild- type E1 and the mutant Am2,3 are unaffected when intact cells are treated with bromelain. However, a proportion of the mutant protein Aml,2 is digested to a form which mi- grates slightly faster (arrowhead; 418 kD) than t
130、he non- glycosylated protein, suggesting that a portion of the amino terminus, including the oligosaccharides, has been removed from the protein. The level of expression of the E1 proteins in transfected COS-1 cells was low, and these cells were fragile and suscep- tible to lysis during proteolysis.
131、 Thus to confirm the result above, we used recombinant vaccinia virus vectors to achieve a higher level of expression, and to allow use of another cell type. A recombinant vaccinia virus encoding the mutant E1 protein Aml,2 was constructed, and HeLa cells were in- fected with this virus or the recom
132、binant vaccinia virus en- coding wild-type El. The localization of both of these pro- teins in infected HeLa cells as determined by indirect immunofluorescence microscopy was identical to that ob- served in transfected COS-1 cells (data not shown). Cells were labeled 5 h after infection with 35Scyst
133、eine for 60 min, and then mock-treated, or treated with bromelain, pronase, or trypsin. After washing and lysing the cells, E1 proteins were immunoprecipitated and subjected to elec- trophoresis in an SDS- polyacrylamide gel. As shown in Fig. 7 B, the amino terminus of Aml,2, but not wild-type El, i
134、s susceptible to digestion with bromelain. The decrease in to- tal amount of protein observed in bromelain-treated cells ex- pressing both wild-type and mutant proteins (in Fig. 7, A and B) was a result of loss of cells and/or cell lysis during the digestion. For this reason, pronase and trypsin wer
135、e also in- cluded in the experiment shown in Fig. 7 B. When intact cells expressing Aml,2 were digested with pronase, a fragment of similar size to that obtained with bromelain was observed. Trypsin does not cleave the amino terminus of Aml,2, but since the only cleavage site in the amino-terminal d
136、omain is quite close to the membrane (Lys 19), this is not unexpected. Thus, the mutant E1 protein with only the third hydrophobic domain is indeed expressed at the cell surface. In a similar experiment using a chase period, virtually all of Aml,2 was digested to the smaller form (data not shown), s
137、uggesting E1 Figure 6. Localization of mutant El proteins in transfected cells by indirect immunofiuorescence microscopy. COS-1 cells grown on coverslips were transfected with DNA encoding wild-type El, the mutant E1 proteins Am2,3 or Aml,2, or the hybrid protein G23. Cells were fixed and permeabili
138、zed 44 h after transfection, and pro- teins were detected by incubation with the affinity-purified anti-E1 peptide serum followed by a fluorescein-conjugated goat anti-rabbit IgG. G23-expressing cells are included to show the pattern ob- served for a protein known to be transported to the plasma mem
139、- brane. Bar, ,10 tm. that this protein is efficiently transported to the plasma mem- brane. Discussion We have shown here that the E1 glycoprotein of the coronavi- rus IBV accumulates in the Golgi region when expressed Machamer and Rose Golgi Retention Signal 1211 This content downloaded from 185.2
140、.32.21 on Sun, 22 Jun 2014 20:27:03 PMAll use subject to JSTOR Terms and ConditionsFigure 7. Susceptibility of wild-type and mutant E1 pro- teins to digestion by exogenous proteases. (A) COS-1 cells transfected with DNA encod- ing wild-type or the mutant E1 proteins Am2,3 or Aml,2 were radiolabeled
141、44 h after transfection with 35Scysteine for 90 min, followed by incu- bation in excess unlabeled cysteine for 90 min. Intact cells were mock-treated or treated with bromelain for 15 min, and E1 proteins were im- munoprecipitated and subject- ed to electrophoresis in a 15 % polyacrylamide gel. (B) H
142、eLa cells were infected with re- combinant vaccinia viruses encoding wild-type E1 (VVE1) or the mutant protein Aml,2 (VVAml,2). Cells were la- beled 5 h after infection with 35Scysteine for 60 min, and intact cells were mock-treated or treated with bromelain, pronase, or trypsin for 15 min. E1 prote
143、ins were immunopre- cipitated and subjected to elec- trophoresis in a 15% poly- acrylamide gel. Arrowheads show the position of digested Aml,2, which migrates slight- ly faster than the nonglycosyl- ated form of the protein. A B E1 Am2,3 Am1,2 brom.: - + - + - + 69 - 46 - 30 - 21.5 - from cDNA in tr
144、ansfected cells, as it does in virus-infected cells. Concurrent experiments in this laboratory have shown that the E1 protein from another coronavirus, MHV, also ac- cumulates in the Golgi region when expressed in transfected COS cells (Rottier and Rose, 1987). This indicates that the information fo
145、r intracellular accumulation must reside in the E1 protein itself, and allows its use as a model for membrane proteins that are retained at this point in the exocytic pathway. The preparation of an antiserum specific for a synthetic peptide corresponding to the carboxy terminus of IBV E1 was instrum
146、ental to our ability to detect E1 in transfected COS-1 cells, since a rabbit antiserum prepared to IBV did not have a sufficient titer of antibodies to El. The level of expres- sion of IBV E1 obtained with the SV40-based expression vec- tor and COS-1 cells was lower than expected by comparison to th
147、e other proteins we have expressed with the same vector (Rose and Bergmann, 1982; Guan and Rose, 1984). Since IBV is an RNA virus which replicates in the cytoplasm of the cell, it is possible that aberrant splicing at cryptic splice sites occurs when the cDNA is introduced into the nucleus. Pre- lim
148、inary experiments using Northern blots suggested that a significant proportion of the El-specific message in trans- fected COS-1 cells lacks the 5 half of the coding sequence, including the initiation codon (our unpublished observa- tions). We have obtained higher levels of expression of E1 using a
149、recombinant vaccinia virus vector which replicates in the cytoplasm (Mackett et al., 1984; Chakrabarti et al., 1985), and thus avoids the problem of aberrant splicing. This expression system may therefore be the one of choice for fu- ture studies of IBV El. In this study, we have gained information
150、on two aspects of intracellular targeting of the E1 protein: that of insertion into membranes and that of intracellular accumulation. Mu- tant E1 proteins that lacked either the first and second or sec- ond and third membrane-spanning domains were generated by oligonucleotide-directed mutagenesis of
151、 the coding se- quence. We found that the mutant protein with only the first hydrophobic domain (Am2,3) or that with only the third hydrophobic domain (Aml,2) were both inserted into the membrane of the endoplasmic reticulum, since both were glycosylated with N-linked oligosaccharides. Thus, it ap-
152、pears that either the first or the third hydrophobic domain can function as an internal, uncleaved signal sequence. Clearly, the third hydrophobic domain must also function as a mem- brane anchor because the mutant protein with only this hy- drophobic domain is expressed on the plasma membrane. The
153、first hydrophobic domain probably also functions as a membrane anchor, but further experiments using in vitro translation in the presence of microsomes followed by pro- teolysis would be required to prove this. There are several examples of proteins with a single membrane-spanning do- main which ser
154、ves to mediate membrane insertion as well as to anchor the protein in the lipid bilayer (Bos et al., 1984; Holland et al., 1984; Lipp and Dobberstein, 1986). Also, some The Journal of Cell Biology, Volume 105, 1987 1212 WE1 VV a ml,2 This content downloaded from 185.2.32.21 on Sun, 22 Jun 2014 20:27
155、:03 PMAll use subject to JSTOR Terms and Conditionsproteins that span the membrane several times have been demonstrated to have more than one internal, uncleaved sig- nal sequence, as shown by site-directed mutagenesis and as- sessment of membrane insertion in vitro (Friedlander and Blobel, 1985; Mu
156、eckler and Lodish, 1986). The most novel information gained from the mutant E1 proteins concerns the intracellular accumulation of the poly- peptide. Deletion of the first and second, but not the second and third, hydrophobic domains resulted in efficient expres- sion of the protein at the cell surf
157、ace. This is the first demon- stration that an intracellular membrane protein can be trans- ported to the plasma membrane after elimination of a particular structural domain. Interestingly, Poruchynsky et al. (1985) have shown that a rotavirus protein normally re- tained in the endoplasmic reticulum
158、 is secreted when a por- tion of the region presumed to be the membrane anchor is deleted. This hydrophobic region thus appears to contain a signal capable of retaining the rotavirus protein in the en- doplasmic reticulum. The mechanism by which the IBV E1 protein is normally retained intracellularl
159、y could be envisioned in two ways: ei- ther (a) the lack of positive signal for transport to the plasma membrane or (b) the presence of a signal responsible for in- tracellular retention. The first explanation, lack of a positive signal for transport to the plasma membrane, seems unlikely. If this w
160、ere the case, fusion of Glu 20 to Gly 77 to delete the first and second hydrophobic domains would have to cre- ate a positive signal for transport. We therefore favor the hy- pothesis that IBV E1 possesses a signal for retention in the Golgi region. This could be an active type of signal, where a sp
161、ecific sequence is recognized by another protein or lipids which are specifically retained in this region of the cell. Al- ternatively, the retention could be passive, resulting from structural properties of the protein which sterically constrain it from moving in the exocytic pathway, perhaps by in
162、ability to be incorporated into transport vesicles. The transport of E1 to the plasma membrane after removal of a retention sig- nal would result if all membrane proteins move passively by bulk flow unless specifically held back. Alternatively, the protein could possess a positive signal for transpo
163、rt to the plasma membrane as well as a signal for intracellular reten- tion, the latter being stronger. Removal of the retention signal would then result in transport of the protein to the plasma membrane. Further experiments designed to determine if signals for transport to the plasma membrane exis
164、t in other domains of E1 should allow us to distinguish these possibil- ities. The intracellular accumulation of E1 in coronavirus- infected cells results in budding of virions from intracellular membranes. This offers an important advantage to the virus in evasion of the host immune system. Persist
165、ent infections are readily established by coronaviruses both in vivo and in vitro (reviewed by Sturman and Holmes, 1983). Perhaps conversion of the E1 protein from a plasma membrane pro- tein to one that is retained intracellularly was an important step in the successful evolution of the coronavirus
166、es. Such a conversion could have logically occurred by incorporation of a retention signal into a protein which already possessed signals for transport to the plasma membrane. In comparing the sequences of the E1 proteins from MHV and IBV, Rottier et al. (1986) observed that one of the most conserve
167、d regions between these proteins is found in the first and second hydrophobic domains. This implies that this re- gion plays an important role in the function of the polypep- tide. Our results suggest that this function is the retention of the protein in the Golgi region of the cell. The elucidation
168、 of the mechanism by which this occurs should provide valu- able clues to the complex process of intracellular transport and targeting of proteins. We thank Drs. David Stern and Bart Sefton for the cDNA clone encoding IBV E1 and for the rabbit anti-IBV antiserum; Nicholas Ling for the syn- thetic pe
169、ptide; Bernard Moss for pSCll and wild-type vaccinia virus; Gail Wertz for advice on selecting recombinant vaccinia viruses; and Bart Sefton, Lynn Puddington, Willy Spaan, Bob Florkiewicz, and Peter Rottier for helpful discussions. This work was supported by Public Health Service grants AI-15481, GM
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