A Single-Amino-Acid Substitution Eliminates the StringentCarbohydrate Requirement for Intracellular Transport
of a Viral GlycoproteinANNE M. PITTA,t JOHN K. ROSE,t AND CAROLYN E. MACHAMER§*
Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, San Diego, California 92138
Received 9 January 1989/Accepted 15 May 1989
In this report, we have investigated the contribution of primary sequence to the carbohydrate requirementfor intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strainsof vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the twoconsensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G proteinrequired at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at37°C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differencesbetween these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major rolein determining the stringency for the carbohydrate requirement. The rates at which the glycosylated andnonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smallerfraction of the nonglycosylated protein was transported. These results suggest that the carbohydrate does notpromote intracellular transport directly but influences a polypeptide folding or oligomerization step which iscritical for transport.
The glycoprotein (G protein) of vesicular stomatitis virus(VSV) has been extensively studied as a model transmem-brane glycoprotein. The polypeptide is synthesized on mem-brane-bound ribosomes, where the amino-terminal signalsequence is cleaved, and two high-mannose asparagine-linked oligosaccharides are added as the protein is extrudedinto the lumen of the endoplasmic reticulum (23). Translo-cation of the polypeptide stops at the hydrophobic mem-brane anchor sequence near the carboxy terminus of theprotein, and a hydrophilic domain of 29 amino acids remainsin the cytoplasm (3, 9, 22). G protein is transported throughthe Golgi complex and then to the plasma membrane, apathway common to secreted and cell surface proteins (2).As it passes from the endoplasmic reticulum through thecisternae of the Golgi complex, the outer mannose residuesof the asparagine-linked (N-linked) oligosaccharides on Gprotein are trimmed and other sugars (galactose, N-acetyl-glucosamine, N-acetylneuraminic acid, and fucose) areadded (10, 11). The protein can also be expressed fromcloned cDNA in transfected cells, where in the absence ofthe other viral proteins it is synthesized, processed, andtransported to the plasma membrane in an identical manner(21).The N-linked oligosaccharides present on G protein have
been shown to be important for intracellular transport. InVSV-infected cells treated with tunicamycin, which blocksall N-linked glycosylation in the cell by interfering with thesynthesis of the precursor oligosaccharide (25, 26), nongly-cosylated G protein does not reach the plasma membraneand appears to exist in an aggregated state in intracellularmembranes (7, 14, 19). Virus assembly is severely inhibited
* Corresponding author.t Present address: Stratagene, La Jolla, CA 92037.t Present address: Departments of Pathology and Cell Biology,
Yale University School of Medicine, New Haven, CT 06510.§ Present address: Department of Cell Biology & Anatomy, The
Johns Hopkins University Medical School, Baltimore, MD 21205.
as a result. The G protein encoded by the San Juan strain ofthe Indiana serotype of VSV was shown to have a particu-larly stringent requirement for N-linked carbohydrate, inthat the nonglycosylated protein produced in tunicamycin-treated cells could not be detected on the cell surface whenthe cells were grown at a normal (37 to 38°C) or reduced(30°C) temperature (8). However, the G protein encoded bya closely related strain of VSV, Orsay, had a less stringentrequirement for carbohydrate when assayed in this manner,since some virus was produced when tunicamycin-treatedcells were grown at the lower temperature (8). The interpre-tation of these studies was that the carbohydrate was re-quired to maintain a polypeptide conformation which wascritical for intracellular transport of G protein, the aminoacid differences between the two proteins being responsiblefor the different stringencies of this requirement.We have used oligonucleotide-directed mutagenesis of the
coding sequence of the G protein from the San Juan strain ofVSV to study the role of N-linked oligosaccharides inintracellular transport. Consensus sequences of N-linkedglycosylation (Asn-X-Ser/Thr) were eliminated by changingthe codon for the third amino acid in this sequence. Thisapproach allowed us to assess the role of the individualoligosaccharides as well as to examine the transport ofnonglycosylated G protein without using drugs such as
tunicamycin. As found for tunicamycin-treated cells infectedwith the San Juan strain of VSV, the nonglycosylated Gprotein lacking both sites for N-linked oligosaccharide addi-tion was not transported to the surface of transfected cells.However, one oligosaccharide at either of the two sites wassufficient for transport of G protein to the plasma membrane(15). In addition, we have introduced new sites for N-linkedglycosylation in G protein lacking the normal sites and foundthat the locations in the polypeptide backbone at whicholigosaccharides will promote transport were somewhatflexible. Thus, the addition of an N-linked glycan at two newsites in the protein lacking the normal sites promoted trans-
port of G protein to the cell surface, but addition at fourother new sites did not (16).To study the role of N-linked glycosylation in the intra-
cellular transport of G protein further, we used the glyco-protein encoded by the Orsay strain of VSV. Comparison ofthe nucleotide sequences for the San Juan and Orsay G-protein genes predicts that only 10 amino acid differencesexist between the proteins (6). If the Orsay G protein couldindeed be transported to the plasma membrane in the ab-sence of glycosylation, as predicted by the studies ontunicamycin-treated VSV-infected cells (8), we could beginto ask which of the amino acid differences between theseclosely related proteins was responsible for this phenome-non.
MATERIALS AND METHODS
Oligonucleotide-directed mutagenesis and DNA constructsencoding recombinant proteins with one or two amino acidsubstitutions. To eliminate the two consensus sites for N-linked glycosylation in the wild-type Orsay VSV G protein,the negative strand of the gene encoding this protein (frompSV-OWT-9 [6]) was first cloned into the unique BamHI siteof M13mp8 as described before (20). The procedures foroligonucleotide synthesis and primer extension on single-stranded M13 DNA were exactly as described previously(15, 20). The oligonucleotide used to eliminate the firstsite was a 26-mer (5'-GTCCATAACTCGGCCACCTGGCATTC-3'), which introduced three nucleotide changes (under-lined) resulting in a single-amino-acid change, threonine 181to alanine. This oligonucleotide was used after a 22-mer,designed to change two nucleotides, failed repeatedly toproduce the desired changes without introduction of anadditional mutation, the insertion of a large repeated seg-ment of the gene. The second glycosylation site was elimi-nated with the same oligonucleotide (a 24-mer) used toeliminate the second site in San Juan G protein (15). In thiscase, two nucleotides were changed to produce a single-amino-acid substitution, threonine 338 to alanine. With bothmutations, new HaeIII sites were created to facilitatescreening. The frequency of mutations obtained at the firstsite was 3%, and that at the second site was 8%.DNA encoding the two mutant proteins was subcloned
into the simian virus 40-based expression vector pJC119 (24)at the unique XhoI site, and the mutations were confirmed byDNA sequence analysis (18). DNA encoding a mutant pro-tein which lacked both glycosylation sites was produced byrecombining a fragment from each of the DNAs encoding thesingle-site mutants at a central PstI site as described previ-ously (15). The mutant proteins encoded by these DNAs aretermed 0-TA1, O-TA2, and O-TA1,2.The recombinant encoding the N-terminal half of the San
Juan G protein lacking both glycosylation sites (S-TA1,2)and the C-terminal half of O-TA1,2 (termed SO-TA1,2) andthe reciprocal recombinant (termed OS-TA1,2) were con-structed by recombining the appropriate fragments at thecentral PstI site and subcloning back into the expressionvector.The synthetic oligonucleotide used to change the codon
for tyrosine 172 to aspartic acid in the San Juan S-TA1,2protein was a 22-mer (5'-AATGCAGCAATGACATATGCCC-3'), which introduced the single-nucleotide changepresent in the Orsay G protein at this site. Primer extensionwas carried out on single-stranded M13 DNA carrying thenegative strand of the gene encoding S-TA1,2. The fre-quency of this mutation was 7%. The DNA encoding this
mutant protein (S-TA1,2-YD), was subcloned into theexpression vector and sequenced as described above.To produce the mutant encoding S-TA1,2, with the codon
for glycine 231 changed to that for aspartic acid, a recombi-nant was constructed with the DNA encoding a revertant(pSVO45-R) of the temperature-sensitive mutant (tsO45) ofOrsay G protein (6). This DNA was used instead of DNAencoding the wild-type protein (pOWT-9) because it containsthe NcoI site at the codon for methionine 200, found also inthe San Juan G sequence, whereas pOWT-9 has a nucleotidesubstitution at this position which eliminates the NcoI siteand results in a methionine-to-threonine substitution. TheNcoI to PstI fragment excised from pSVO45-R contains twonucleotide differences from that of the San Juan G protein,but only one of them results in an amino acid substitution,that of glycine 231 to aspartic acid (6). Thus, the XhoI toNcoI fragment (630 base pairs) containing the 5' codingregion of S-TA1,2 and the PstI to XhoI fragment (865 basepairs) containing the 3' coding region of S-TA1,2 wererecombined with the NcoI to PstI fragment (188 base pairs)from pSVO45-R to construct the DNA encoding S-TA1,2-GD. The single-amino-acid substitution in this mutant pro-tein was confirmed by DNA sequence analysis.
Transfection, radiolabeling, and immunoprecipitation. Themethods for transfection of COS-1 cells and detection ofexpressed G proteins by immunoprecipitation and indirectimmunofluorescence have been described in detail previ-ously (1, 15). COS-1 cells in 35-mm dishes or 16-mm wells ofa multiwell plate (Nunc multiwell 4) were transfected withplasmid DNA (10 p.g/ml) in Tris-buffered saline containing0.5 mg of DEAE-dextran per ml and treated with chloro-quine for 3 h. Expression was assayed 42 to 48 h aftertransfection. Metabolic labeling was performed in 0.5 ml for35-mm dishes or in 0.25 ml for 16-mm wells with L-[35S]methionine (100 pXCi/ml) or D-[2-3H]mannose (250 ,uCi/ml) exactly as described previously (15). Lactoperoxidase-catalyzed iodination of transfected cells was performed asdescribed before (15), except 150 to 200 pCi of Na125I wasused per 35-mm dish. Immunoprecipitation with rabbit anti-VSV serum was performed as described previously (15), andimmunoprecipitated proteins were analyzed on 10% poly-acrylamide gels (13). Densitometry of the fluorograms andautoradiograms was used to quantitate immunoprecipitationdata. Several exposures were scanned to ensure that valueswere in the linear range of the film.
Surface immunoprecipitation. For determination of therates at which the proteins reached the plasma membrane, asurface immunoprecipitation assay was used. Transfectedcells in 16-mm wells were pulse-labeled for 15 min withL-[35S]methionine and incubated for various times in excessunlabeled methionine. After being washed several times incold phosphate-buffered saline containing 1% bovine serumalbumin, cells were incubated on a platform rocker for 2 h at4°C in 0.2 ml of the same solution containing 10 Rl ofanti-VSV antibody. After washing out unbound antibody,cells were lysed and surface G protein-antibody complexeswere isolated with protein A-bearing Staphylococcus au-reus. The remaining lysate was subjected to another round ofimmunoprecipitation to recover intracellular G protein. The16-mm wells (5 x 104 cells) were used in this assay to ensureantibody excess at each step. No difference in the level ofsurface G protein was detected when cells were lysed in thepresence of excess unlabeled G protein (threefold the num-ber of COS-1 cells transfected with pSVGL), indicating thatantibody prebound to surface G protein did not exchangewith internal G protein after lysis. Thus, lysis was performed
GLYCOSYLATION REQUIREMENT FOR INTRACELLULAR TRANSPORT 3803
without addition of excess unlabeled G protein to allowisolation of internal G protein from the same samples.
Indirect immunofluorescence microscopy. Localization ofG proteins by double-label indirect immunofluorescencemicroscopy in transfected COS-1 cells grown on cover slipswas performed essentially as described before (15), exceptthat a mixture of rabbit anti-VSV antiserum (1:200) and arabbit antiserum raised to a synthetic peptide correspondingto the 15 carboxy-terminal amino acids of G protein (1:10,affinity purified [16]) was used to detect intracellular Gproteins. This antibody combination allowed improved de-tection of all G-protein mutants.
RESULTS
Elimination of the sites for N-linked glycosylation in theOrsay G protein. To ascertain whether the G protein en-coded by the Orsay strain of VSV could be transported totY v plasma membrane of transfected cells in the absence ofglycosylation as predicted by studies of VSV-infected cellsthat used tunicamycin (8), the two sites of N-linked oligosac-charide addition were eliminated from the coding sequenceof the protein. The two consensus sites for glycosylation areconserved between the two G proteins, the first being atasparagine 179, in the sequence Asn-Ser-Thr, and the secondbeing at asparagine 336, in the sequence Asn-Gly-Thr. Thesame amino acid changes introduced into the San Juan Gprotein to eliminate the glycosylation sites (15) were usedhere. The codon for the third amino acid in the consensussite, threonine in both cases, was changed to that for alanine,using oligonucleotide-directed mutagenesis to introduce sev-eral nucleotide changes. A recombinant plasmid encoding aprotein with both sites eliminated was then constructed. Thestrategy used to produce and isolate the mutant DNAs andsubclone them into the simian virus 40-based expressionvector pJC119 was described previously (15). The wild-typeOrsay G protein is termed O-G; that lacking the first glyco-sylation site, O-TA1; that lacking the second glycosylationsite, O-TA2; and that lacking both sites, O-TA1,2. The SanJuan G proteins previously called G and TA1,2 (15-17) willbe referred to here as S-G and S-TA1,2 to avoid confusion.Lack of glycosylation at the mutated sites in the Orsay G
protein. DNAs encoding the mutant G proteins were trans-fected onto COS-1 cells, and expression was analyzed andcompared with expression in cells transfected with DNAencoding the wild-type Orsay G protein. Cells were labeled44 h after transfection with L-[35S]methionine, and G pro-teins were immunoprecipitated and electrophoresed on poly-acrylamide gels. Figure 1A shows that the relative mobilitiesof the proteins are consistent with the absence of one(0-TAl and O-TA2) or both (O-TA1,2) N-linked oligosac-charides. The lower level of expression of the mutant Gproteins relative to wild-type G protein is unexplained buthas been observed for other mutant G proteins expressed inCOS cells and does not appear to be due to increaseddegradation (15, 21). When transfected cells were pretreatedand labeled in the presence of tunicamycin, all of theproteins comigrated (Fig. 1B), suggesting that the mobilitydifferences were indeed due to carbohydrate. In the experi-ment shown, the inhibition of glycosylation by tunicamycinwas incomplete, as the glycosylated proteins can also bedetected, especially O-TA2. To confirm that the mutatedsites were not glycosylated, cells were labeled with D-[2-3H]mannose. Labeling of O-TA1,2 could not be detected,and 0-TAl and O-TA2 incorporated about half as much labelas the wild-type protein relative to the level of labeling with
A.35S met
C\Ir--r-I C\l l
H Hi-
56 6 6
B.35S met +TmII
C\j
Cl <<c,5'I-Io666
_
C.3H man
C\M
5, i5666O
a _
FIG. 1. Mutant Orsay G proteins are not glycosylated at themutated oligosaccharide addition sites. COS-1 cells transfected withplasmid DNA specifying wild-type or mutant G proteins wereradiolabeled 44 h after transfection, and detergent lysates of the cellswere subjected to immunoprecipitation with anti-VSV antiserumfollowed by SDS-polyacrylamide gel electrophoresis. Cells werelabeled with 100 ,uCi of L-[35S]methionine per ml either for 1 h (A) orin the presence of 2 ,ug of tunicamycin (Tm) per ml after a 2-hpretreatment in the same concentration of the drug (B) or with 250,uCi of D-[2-3H]mannose per ml for 4 h (C). VSV protein markers (Gand N) are shown in the first lane of panel A.
L-[35S]methionine (Fig. 1C). Thus, as found previously forthe corresponding mutations in the San Juan G protein (15),changing the third amino acid in the consensus sequence forN-linked glycosylation eliminated glycosylation of the aspar-agine residue.
Expression of the glycosylation mutants of Orsay G proteinon the cell surface. The transport of the mutant proteins tothe plasma membrane of transfected cells was analyzed byindirect immunofluorescence microscopy, using a doublelabel (Fig. 2). As found for the San Juan G protein glycosyl-ation mutants, Orsay G proteins with only one oligosaccha-ride were readily detected on the surface of transfected cells.In contrast to the nonglycosylated San Juan G proteinlacking both sites, the nonglycosylated Orsay protein (0-TA1,2) could be detected on the plasma membrane in abouthalf of the cells which were expressing the protein. Twoexamples of cells expressing this protein are presented inFig. 2, one cell being surface positive and the other havingundetectable levels of protein on the cell surface. All cellsexpressing these G proteins could be detected by staining forintracellular G protein after permeabilization with nonionicdetergent. Thus, the nonglycosylated Orsay G protein intransfected cells could be transported to the plasma mem-brane, as found for Orsay G protein in VSV-infected tuni-camycin-treated cells (8).The amino acid differences in the N-terminal half of Orsay
FIG. 2. Detection of mutant G proteins by indirect immunofluorescence microscopy. Transfected COS-1 cells grown on cover slips at 37°Cwere fixed 44 h after transfection and stained for surface and internal G proteins as described in Materials and Methods. Each set ofmicrographs shows the same cell photographed with the rhodamine (surface G) and fluorescein (internal G) filters. Two examples of cellsexpressing the nonglycosylated protein O-TA1,2 are shown.
G protein are responsible for the less stringent carbohydraterequirement in transport. Comparison of the nucleotidesequences of the G proteins encoded by the two strains ofVSV (6) predicts only 10 amino acid differences between theproteins (Fig. 3). One of the changes is in the cytoplasmicdomain, and many of the others are conservative. The leastconservative of the changes are in the N-terminal half of thepolypeptide near the first glycosylation site, at residues 172(tyrosine in S-G, aspartic acid in O-G) and 231 (glycine inS-G, aspartic acid in O-G). To determine whether the aminoacids responsible for the differing carbohydrate require-ments of the two proteins resided in this portion of theprotein, we first constructed recombinants encoding theN-terminal half of one polypeptide and the C-terminal half of
the other. DNAs encoding both nonglycosylated proteins(S-TA1,2 and 0-TA1,2) were recombined at a central restric-tion site (at the codon for amino acid 262) and subclonedback into the expression vector (Fig. 3). The proteinsencoded by these DNAs are termed SO-TA1,2 (with theN-terminal portion of S-TA1,2 and the C-terminal portion ofO-TA1,2) and OS-TA1,2 (with the reciprocal portions ofeach protein). OS-TA1,2 has both of the aspartic acidsubstitutions mentioned above, as well as three others.The intracellular distribution of these recombinant pro-
teins was analyzed by indirect immunofluorescence micros-copy as described above. Figure 4 shows that OS-TA1,2 wasdetected at the cell surface, whereas SO-TA1,2 was not.Thus, the less stringent carbohydrate requirement for intra-
FIG. 3. Recombinant nonglycosylated G proteins. Schematic representation of the nonglycosylated San Juan (S-TA1,2, solid bar) andOrsay (O-TA1,2, hatched bar) G proteins showing the amino acid differences and the two recombinant G proteins generated by exchangingthe coding sequences at a central PstI restriction site. The numbers on the top line show the residue numbers at which differences occur, andthe x's show the positions of the mutated wild-type glycosylation sites. The first box represents the signal sequence, and the second boxrepresents the membrane-spanning domain.
INTERNAL SURFACE
S-TA 1,2
0-TA 1,2
SO-TA 1,2
OS-TA 1.2
FIG. 4. Detection of recombinant nonglycosylated G proteins by indirect immunofluorescence microscopy. Transfected COS-1 cellsgrown on cover slips were fixed and stained as described in the legend to Fig. 2. SO-TA1,2 is the protein with the N-terminal half of S-TA1,2and the C-terminal half of O-TA1,2, and OS-TA1,2 is the reciprocal protein.
FIG. 5. Lactoperoxidase-catalyzed iodination of cell surface Gproteins. Parallel dishes of transfected COS-1 cells were eithersurface labeled with 1251 as described in Materials and Methods or
labeled with 100 p.Ci of L-[35S]methionine per ml for 1 h, anddetergent lysates of the cells were immunoprecipitated with anti-VSV antiserum and electrophoresed in an SDS-polyacrylamide gel.
cellular transport of Orsay G protein is due to amino aciddifferences in the N-terminal half of the polypeptide.The level of expression of these proteins on the cell
surface was analyzed in a more quantitative fashion withlactoperoxidase-catalyzed iodination of intact cells. A par-allel set of transfected cells was biosynthetically labeled withL-[35S]methionine. The O-TA1,2 and OS-TA1,2 proteins canbe detected in immunoprecipitates from surface-iodinatedcells at a reduced level as compared with the wild-type SanJuan and Orsay G proteins (Fig. 5). Densitometry of theautoradiogram indicates that these proteins are detected atabout a 10- to 20-fold-lower level than the wild-type Orsay Gprotein (when corrected for the level of expression asdetermined by L-[35S]methionine labeling). These numberscannot be considered absolutely quantitative since the ty-rosine residues of the mutant and wild-type proteins may notbe equally accessible to iodination. S-TA1,2 and SO-TA1,2cannot be detected in immunoprecipitates of surface-iodi-nated cells, as predicted from the results obtained withindirect immunofluorescence microscopy.A characteristic difference in relative mobility on poly-
acrylamide gels containing sodium dodecyl sulfate (SDS)between the San Juan and Orsay G proteins can be observedin Fig. 5, with the Orsay G protein migrating more slowly.The difference is not due to differential processing of thecarbohydrate, as it is also observed in the nonglycosylatedmutant proteins. Note that the mobility difference can belocalized to the N-terminal half of the polypeptide, since
SO-TA1,2 comigrates with S-TA1,2 and OS-TA1,2 comi-grates with O-TA1,2.Can a single-amino-acid change substitute for the stringent
carbohydrate requirement for transport of S-TA1,2? To de-termine whether a single-amino-acid substitution might beresponsible for the less stringent carbohydrate requirementobserved for O-TA1,2 as compared with S-TA1,2, we con-
centrated first on the two least conservative changes in theN-terminal half of the protein. As mentioned above, thereare two aspartic acid residues in the Orsay G protein (atpositions 172 and 231), which in the San Juan G protein are
tyrosine and glycine, respectively (Fig. 3). The codon fortyrosine 172 in S-TA1,2 was changed to that for asparticacid, using an oligonucleotide to change a single nucleotide,as described in Materials and Methods. The mutant S-TA1,2protein encoding an aspartic acid at residue 231 was con-
structed by recombining coding sequences for S-TA1,2 withthose for the Orsay G protein, also described in Materialsand Methods. The mutant proteins are termed S-TA1,2-YD(tyrosine changed to aspartic acid at residue 172) and S-TA1,2-GD (glycine changed to aspartic acid at residue 231).DNAs encoding these mutant proteins were transfected
onto COS-1 cells, and the localization of proteins was
determined by indirect immunofluorescence microscopy.Figure 6 shows that the single-amino-acid substitution inS-TA1,2-YD results in transport of the protein to the plasmamembrane and that the immunofluorescence staining patternis indistinguishable from that of O-TA1,2. Thus, the singletyrosine-to-aspartic acid substitution was sufficient to allowtransport of the nonglycosylated San Juan G protein to thecell surface. Most cells expressing S-TA1,2-GD (with theglycine-to-aspartic acid substitution) possessed a stainingpattern similar to that of S-TA1,2, with undetectable levelsof protein on the cell surface. However, an occasional cellhad low levels of protein on the plasma membrane (notshown), suggesting that this amino acid change might alsopartially substitute for the stringent carbohydrate require-ment of S-TA1,2 in intracellular transport.To obtain more quantitative data, transfected cells were
labeled by lactoperoxidase-catalyzed iodination. Paralleldishes of transfected cells were biosynthetically labeled withL-[35S]methionine, and G proteins were isolated by immu-noprecipitation and electrophoresed on a polyacrylamide gel(Fig. 7). As predicted by the immunofluorescence results,S-TA1,2-YD could be detected in immunoprecipitates fromsurface-iodinated cells. The amount of protein detected bythis procedure was similar to that with O-TA1,2 when thedifference in level of expression (determined by L-
[35S]methionine labeling) is taken into account. The amountdetected at the cell surface is about 10- to 20-fold lower thanthat for the wild-type Orsay protein O-G. Although themajority of cells expressing S-TA1,2-GD did not have de-tectable levels of protein at the cell surface by immunofluo-rescent staining, a low level could be observed in immuno-precipitates from iodinated cells. These data suggest that,although the major contribution to the less stringent carbo-hydrate requirement of Orsay G protein may come fromaspartic acid 172, a smaller contribution may come fromaspartic acid 231. Although densitometry of the autoradio-gram shown in Fig. 7 suggests that aspartic acid 172 can fullysubstitute for the stringent carbohydrate requirement ofS-TA1,2, absolute quantitation by this method is subject tothe assumption that the tyrosine residues in the wild-typeand mutant proteins are all equally accessible to iodination.Thus, as mentioned above, the level of protein expressed at
GLYCOSYLATION REQUIREMENT FOR INTRACELLULAR TRANSPORT 3807
INTERNAL
S-TA 1,2
0-TA 1.2
S-TA ,2-YD
S-TA 1.2-GD
FIG. 6. Indirect immunofluorescence microscopy of cells expressing the nonglycosylated San Juan G protein with single-amino-acidsubstitutions. Transfected COS-1 cells grown on cover slips were fixed and stained for surface and internal G proteins as described in thelegend to Fig. 2. S-TA1,2-YD is the nonglycosylated San Juan G protein with Tyr-172 changed to Asp, and S-TA1,2-GD is that with Gly-231changed to Asp.
the surface of transfected cells as determined by this proce-dure can only be considered an estimate.The characteristic mobility difference between the San
Juan and Orsay G proteins is determined by a single aminoacid at residue 172 (Fig. 7). The presence of an aspartic acidresidue instead of a tyrosine at this position in S-TA1,2(S-TA1,2-YD) results in the same decreased mobility of theprotein that is observed for O-TA1,2. Thus, the presence ofthis aspartic acid not only eliminates the stringent require-ment for N-linked oligosaccharides in intracellular transportof the San Juan G protein but also affects SDS binding or theability of SDS to completely denature the polypeptide.
Rates of transport of the wild-type and nonglycosylatedOrsay G proteins to the plasma membrane. To address thepossibility that N-linked carbohydrate was enhancing therate of transport as well as promoting a conformation com-patible with transport, we analyzed the rates at which thenewly synthesized Orsay wild-type G protein and its non-glycosylated counterpart, O-TA1,2, reached the cell surface.We used a surface immunoprecipitation assay which mea-sures the rate at which newly synthesized proteins arrive atthe plasma membrane. Replicate wells of transfected cellswere pulse-labeled with L-[35S]methionine for 15 min andthen harvested immediately or after incubation with excess
unlabeled methionine for various times. Surface and intrac-ellular G proteins were isolated as described in Materials andMethods. To quantitate the results, fluorographs were sub-jected to densitometry, and the percent surface G proteinwas determined from the total recovered (surface plus intra-cellular). The graph in Fig. 8 shows the percentage of total Gprotein detected on the cell surface at each time pointcompared with the maximum surface expression obtainedfor each protein. Although the maximum O-G expressed atthe cell surface is greater than that of O-TA1,2, the propor-tion of O-TA1,2 molecules which do reach the cell surfaceappear to be transported at the same rate at which theglycosylated wild-type protein is transported. This findingsuggests that the oligosaccharides do not function as a directsignal in promoting transport of the Orsay G protein to thecell surface but function indirectly, by inducing or maintain-ing polypeptide conformation. The aspartic acid residue atposition 172 probably functions similarly, although lessefficiently.The maximum amount of wild-type Orsay G protein
accessible to antibody was only about 60%, even after longperiods of chase. This was consistent in three experimentsand may reflect the fact that the antibody cannot reach theentire surface of the cell (that surface in contact with other
FIG. 8. Evidence showing that the rates at which glycosylatedand nonglycosylated Orsay G proteins reach the cell surface aresimilar. Transfected COS-1 cells in 16-mm wells were pulse-labeledwith L-[5S]methionine for 15 min and chased in the presence ofexcess unlabeled methionine for the times indicated. Surface andintracellular G proteins were immunoprecipitated as described inMaterials and Methods. The percentage of total protein recovered ateach time point is expressed as the percentage of the maximumwhich reaches the cell surface. For O-G, the maximum at the cellsurface was 60%; for O-TA1,2, it was 47%.
FIG. 7. Lactoperoxidase-catalyzed iodination of cell surface S-Gproteins with single-amino-acid substitutions. Transfected COS-1cells were labeled with 1251 or L-[35S]methionine, immunoprecipi-tated, and electrophoresed as described in the legend to Fig. 5.Markers are the VSV G, N, and M proteins.
cells or the culture dish). Although the level of O-TA1,2which can be detected at the cell surface is lower than thewild-type protein (40 to 50% of the total), it is higher than the10% expected from lactoperoxidase-catalyzed iodinations(Fig. 5 and 7). This probably reflects the technical differ-ences between the two assays. It is possible that the anti-body binds more efficiently to the nonglycosylated protein atthe surface of intact cells than to the glycosylated proteindue to steric hindrance by the oligosaccharides. This wouldexplain the greater proportion of 0-TA1,2 detected at thecell surface by surface immunoprecipitation as comparedwith immunoprecipitates from surface-iodinated cells.
DISCUSSION
In this report, we have shown that a single-amino-aciddifference between two closely related VSV G proteins candetermine the stringency of the requirement for N-linkedcarbohydrate for transport to the cell surface. Thus, thesubstitution of an aspartic acid residue for a tyrosine atposition 172 in the San Juan G protein allows transport of thenonglycosylated protein to the cell surface at 37°C. The sameamino acid substitution is responsible for the mobility differ-ence observed for the San Juan and Orsay G proteins inpolyacrylamide gels (Fig. 7). The other amino acid substitu-tion that we analyzed, aspartic acid in place of glycine atposition 231, could also substitute for the carbohydraterequirement of San Juan G protein, although less efficientlythan aspartic acid 172.The studies by Gibson et al. (8) showed that the nongly-
cosylated Orsay G protein from VSV-infected cells treatedwith tunicamycin could be detected at the cell surface when
the cells were grown at reduced temperature (30°C), whereasthe nonglycosylated San Juan G protein could not. How-ever, neither of the nonglycosylated proteins could be de-tected on the plasma membrane in cells grown at 37°C. Incontrast, our experiments were performed at 37°C, since wecould detect the nonglycosylated Orsay G protein on thesurface of cells grown at this temperature. We have reportedrecently that the nonglycosylated San Juan G protein can bedetected at a low level on the plasma membrane of trans-fected cells grown at 30°C (17). It is possible that a greaterlevel of sensitivity can be achieved by using transfected cellsas compared with virus-infected cells, since G protein isallowed to accumulate over a period of several days ratherthan hours. Alternatively, the two threonine-to-alanine sub-stitutions in the nonglycosylated Orsay and San Juan Gproteins might allow more efficient transport of these pro-teins than in tunicamycin-treated cells.The finding that one amino acid difference can alter the
carbohydrate requirement for intracellular transport impliesthat the polypeptide folding in this region must be critical forthis process. A single-amino-acid substitution at position 204in the Orsay G protein (a serine in place of a phenylalanine)results in temperature-sensitive intracellular transport of theG protein from tsO45 (6). The proximity of the two asparticacid substitutions in the Orsay G protein to the first glyco-sylation site at asparagine 179 may indicate that the presenceof one or more acidic residues in this region can induce thesame critical polypeptide conformation induced by the hy-drophilic oligosaccharide. The role that the acidic aminoacids or oligosaccharides might play in inducing a criticalconformation could either be at the level of initial polypep-tide folding during biosynthesis or posttranslationally, per-haps at an oligomerization step. G protein has been shownrecently to form a trimer before its transport out of theendoplasmic reticulum (4, 12). The constraints on polypep-tide folding patterns induced by a large, hydrophilic oli-gosaccharide could easily be imagined to limit the interfacebetween monomers when trimerization occurs.Our earlier studies demonstrated that an oligosaccharide
at some new sites in the San Juan G protein lacking the
GLYCOSYLATION REQUIREMENT FOR INTRACELLULAR TRANSPORT 3809
normal sites promoted transport of the protein to the cellsurface, and at other new sites transport was inhibited (16).We concluded that the oligosaccharides were most likelyinvolved in promoting a folding step required for transport.Some of these mutant G proteins with altered glycosylationsites which are not transported were recently shown to formaggregates in the endoplasmic reticulum rather than trimers(5). Here we have shown that the rates at which newlysynthesized wild-type and nonglycosylated Orsay G proteinsreach the cell surface are similar (Fig. 8). It thus appears thatthe carbohydrate does not promote the intracellular trans-port of the protein directly, but indirectly as suggestedpreviously by Gibson et al. (8). The aspartic acid residue atposition 172 in the Orsay G protein could influence theconformation of the polypeptide in a manner similar, al-though less efficient, to that of an N-linked oligosaccharide,such that only a fraction of the molecules attains the properconformation. A larger fraction of the molecules may attainthe proper conformation at reduced temperature, whichwould explain the enhanced transport of the nonglycosylatedprotein at 30°C (17).
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants A124345and GM37908 from the National Institutes of Health.
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