THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 23, Issue of June 9, pp. 17338 –17343, 2000
Printed in U.S.A.
Glycosylation Efficiency of Asn-Xaa-Thr Sequons Depends Both on
the Distance from the C Terminus and on the Presence of a
Downstream Transmembrane Segment*
Received for publication, March 20, 2000
Published, JBC Papers in Press, March 22, 2000, DOI 10.1074/jbc.M002317200
IngMarie Nilsson and Gunnar von Heijne‡
From the Department of Biochemistry, Stockholm University, S-106 91 Stockholm, Sweden
Statistical studies of N-glycosylated proteins have indicated that the frequency of nonglycosylated Asn-Xaa(Thr/Ser) sequons increases toward the C terminus
(Gavel, Y., and von Heijne, G. (1990) Protein Eng. 3, 433–
442). Using in vitro transcription/translation of a truncated model protein in the presence of dog pancreas
microsomes, we find that glycosylation efficiency of
Asn-Xaa-Thr sequons indeed is reduced when the sequon is within ⬃60 residues of the C terminus. Surprisingly, the presence of a hydrophobic stop transfer sequence between the Asn-Xaa-Thr sequon and the C
terminus results in a very different dependence of glycosylation efficiency on the distance to the C terminus,
where the presence of the stop transfer segment inside
the ribosome appears to cause a drastic drop in the level
of glycosylation. We speculate that this may reflect a
change in the structure of the ribosome/translocon complex induced by the stop transfer segment.
In mammalian cells, nascent secretory and membrane proteins are co-translationally glycosylated on Asn-Xaa-(Thr/Ser)
sequons by the endoplasmic reticulum (ER)1 enzyme oligosaccharyl transferase (OST) (1). The OST complex is associated
with the Sec61 translocon, through which proteins are translocated into the lumen of the ER or inserted into the ER
membrane (2). A highly regulated mechanism has been observed during synthesis of transmembrane proteins, where the
emergence of a hydrophobic stop transfer (ST) sequence inside
the ribosome triggers the closing of the lumenal end of the
translocon pore and the almost simultaneous opening of the
cytosolic ribosome/translocon seal (3, 4). Structural changes on
both sides of the translocon are thus effected by the ST sequence from inside the ribosome, but whether these changes
are also transmitted to the OST or otherwise influence the
glycosylation reaction is unknown.
Previously, we have found that the lumenally oriented active
site of the OST is positioned at a well defined distance from the
surface of the ER membrane (5) and can be used as a fixed point
of reference to study structural changes in transmembrane
segments in the ER membrane (6, 7). Here, we have measured
* This work was supported by grants from the Swedish Cancer Foundation and the Swedish Natural and Technical Sciences Research
Councils (to G. V. H.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 46-8-16-25-90;
Fax: 46-8-15-36-79; E-mail: gunnar@biokemi.su.se.
1
The abbreviations used are: ER, endoplasmic reticulum; OST, oligosaccharyl transferase; ST, stop transfer; PCR, polymerase chain
reaction.
the efficiency of N-glycosylation of Asn-Xaa-Thr sequons as a
function of their distance from the C-terminal end of a model
protein, in both the absence and the presence of an intervening
hydrophobic ST sequence. When there is no ST sequence between the glycosylation acceptor sequon and the C terminus,
the glycosylation efficiency increases as the stop codon is
moved further away from the sequon and plateaus at a distance
of ⬃60 residues or more. In stark contrast, with poly-Leu ST
sequences of either 15 or 30 hydrophobic residues placed between the acceptor sequon and the C terminus, we observe a
dramatic drop in glycosylation efficiency when the C-terminal
end of the protein is located immediately downstream of the
transmembrane segment. This effect persists until the N-terminal end of the ST sequence is 40 –50 residues away from the
C-terminal residue, suggesting that the presence of a hydrophobic stretch inside the ribosome when translation terminates
somehow prevents access to the OST of a glycosylation acceptor
site located immediately upstream of the hydrophobic segment.
We speculate that this finding may be related to the previously
observed closing of the lumenal end of the translocation channel induced by the appearance of a hydrophobic segment inside
the ribosome (3).
MATERIALS AND METHODS
Enzymes and Chemicals—Unless otherwise stated, all enzymes as
well as plasmid pGEM1, RiboMAX SP6 RNA polymerase system, and
rabbit reticulocyte lysate were from Promega (Madison, WI). Taq polymerase, T7 DNA polymerase, [35S]Met, 14C-methylated protein standards, deoxyribonucleotides, dideoxyribonucleotides, and the cap analog
m7G(5⬘)ppp(5⬘)G were from Amersham Pharmacia Biotech. The glycosylation acceptor peptide N-benzoyl-Asn-Leu-Thr-N-methylamide was
from Quality Controlled Biochemicals (Hopkinton, MA). The signal
peptidase inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was obtained from Sigma. Oligonucleotides were purchased
from Kebo Lab (Stockholm, Sweden) and from Eurosequence (Groningen, the Netherlands). The PCR purification kit and RNeasy RNA clean
up were from Qiagen (Hilden, Germany). Oligonucleotides were from
Cybergene (Stockholm, Sweden).
DNA Manipulations—Insertion of the stop transfer sequences
QQQL14VKKKK and QQQL29VKKKK into the P2 domain of Lep was
performed by introducing BclI and NdeI restriction sites in codon 214
and 220, respectively. Double-stranded oligonucleotides coding for the
above amino acid sequences were then cloned between the BclI and
NdeI restriction sites. Site-specific mutagenesis of the lepB gene used to
make a Asn214 3 Gln mutation (to remove a potential glycosylation
acceptor site), to introduce new Asn-Ser-Thr glycosylation acceptor
sites, and to introduce restriction enzyme cleavage sites was performed
in phage M13 according to the method of Kunkel (8, 9).
The Lep-ST29L(CC/50) construct was made by first cloning a PCRamplified fragment from the P2 domain of Lep into the Lep-ST29L
construct to obtain Lep-ST29L (50). The 5⬘ primer used in the amplification of the Lep fragment had a EcoRV site in codon 150, and the 3⬘
primer had a BssHII site in codon 213 in the lepB gene. The amplified
EcoRV-BssHII fragment was cloned into the Lep-ST29L construct between the natural EcoRV and BssHII restriction sites in codons 130 and
145 in the P2 domain. The resulting fusion had the sequence . . .
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This paper is available on line at http://www.jbc.org
Glycosylation of C-terminal Asn-Xaa-Thr Sequons
130
150
213
17339
145
RGDI L P. . . . PK K RAVGL . . . (numbers refer to the wild
type Lep sequence), increasing the size of the P2 domain by 50 residues.
In a second step, the Lep-ST29L(CC/50) construct was made by ligating
PCR-amplified fragments from a previously constructed Lep-derivative
with a signal peptidase cleavage cassette inserted at the C-terminal end
of a H2 transmembrane segment composed of 14 leucine residues (10)
and from the Lep-ST29L (50) construct. The 5⬘ primer used for amplification of the cleavage cassette containing Lep fragment was situated
210 bases upstream of the translational start site, and the 3⬘ primer
had a BglII site in codon 84 in the lepB gene. The 5⬘ primer used in the
amplification of the Lep-ST29L (50) fragment had a BglII site in codon
88 in the lepB gene. The newly created fusion joint had the amino acid
sequence . . . PFQI86SS88 . . . (numbers refer to the wild type Lep sequence). The amplified fragments were ligated in the gel after agarose
gel purification, and mRNA templates were prepared by PCR amplification of the ligated fragments using 5⬘ and 3⬘ primers as described
below.
For cloning into and in vitro expression from the pGEM1 plasmid,
the 5⬘ end of the lepB gene was modified first by the introduction of a
XbaI site and second by changing the context 5⬘ to the initiator ATG
codon to a “Kozak consensus” sequence (11). Thus, the 5⬘ region of the
gene was modified to: . . .ATAACCCTCTAGAGCCACCATGGCGAAT . . . (XbaI site and initiator codon underlined). Mutants of Lep
made in phage M13 were cloned into pGEM1 behind the SP6 promoter
as a XbaI-SmaI fragment. All mutants were confirmed by DNA sequencing of plasmid and single-stranded M13 DNA using T7 DNA
polymerase.
Templates for in vitro transcription of truncated mRNA with a 3⬘
stop codon were prepared using PCR to amplify fragments from pGEM1
plasmids containing the relevant Lep constructs. The 5⬘ primer was the
same for all PCR reactions and had the sequence 5⬘-TTCGTCCAACCAAACCGACTC-3⬘. This primer is situated 210 bases upstream of the
translational start, and all amplified fragments contained the SP6
transcriptional promoter from pGEM1. The 3⬘ primers were designed
according to the desired positions of the stop codons. All 3⬘ primers
contained the tandem translational stop codons TAG TAA and were
designed to have approximately the same annealing temperature as the
5⬘ primer. PCR amplification was performed with a total of 30 cycles
using an annealing temperature of 52 °C. The amplified DNA products
were purified using the Qiagen PCR purification kit as described in the
manufacturer’s protocol and verified on a 1.2% agarose gel.
In Vitro Transcription—Truncated mRNAs with stop codons were
transcribed from the SP6 promoter using the RiboMAX SP6 RNA polymerase system. Transcriptions were carried out at 30 °C for 12 h. The
mRNAs were purified using Qiagen RNeasy clean up kit and verified on
a 1.2% agarose gel.
In Vitro Translation—Translation in reticulocyte lysate in the presence of dog pancreas microsomes was performed as described in Ref. 12,
and alkaline extraction was performed as described in Ref. 13. Proteins
were analyzed by SDS-polyacrylamide gel electrophoresis, and gels
were quantitated on a Fuji BAS1000 phosphoimager using the MacBAS
2.31 software. The extent of glycosylation of a given mutant was calculated as the quotient between the intensity of the glycosylated band
divided by the summed intensities of the glycosylated and nonglycosylated bands. For some constructs, the assay was repeated in the presence of a glycosylation acceptor tripeptide as described (5) or in the
presence of a signal peptidase inhibitor (14).
RESULTS
We used the well characterized Escherichia coli inner membrane protein leader peptidase (Lep) (Fig. 1A) to measure the
glycosylation efficiency of Asn-Xaa-Thr sequons in C-terminally truncated proteins. When Lep is expressed in vitro in the
presence of dog pancreas microsomes, both the N-terminal tail
and C-terminal P2 domain are translocated to the lumenal
side, the same orientation as in E. coli (5). A cryptic glycosylation site in the P2 domain, Asn214-Glu-Thr, is efficiently glycosylated upon translocation into the lumen.
Glycosylation Efficiency Increases with the Distance from the
C Terminus—To measure glycosylation efficiency as a function
of the position of the glycosylation acceptor site relative to the
C terminus, proteins truncated at various positions in the P2
domain, C-terminal to the naturally occurring, unique Asn214Glu-Thr sequon and to two engineered unique glycosylation
acceptor sites (Asn201-Ser-Thr, Asn203-Ser-Thr), were gener-
FIG. 1. A, orientation of Lep in the microsomal membrane. A cryptic,
naturally occurring glycosylation site at Asn214 is indicated. B, the two
kinds of constructs analyzed in Fig. 2, where either the protein is
truncated at different distances downstream of a fixed glycosylation site
(Asn201 or Asn203; top) or the glycosylation site is placed in different
positions relative to the C terminus of the full-length protein (bottom).
ated by PCR (Fig. 1B) using primers encoding tandem stop
codons and made by in vitro transcription/translation in reticulocyte lysate in the absence and presence of dog pancreas
microsomes. In addition, unique Asn-Ser-Thr acceptor sequons
were placed in different positions relative to the C terminus in
a variant of the full-length Lep protein where the Asn214 sequon had been removed by an Asn214 3 Gln change.
As shown in Fig. 2, although there is some variation depending on the exact location of the stop codon, the glycosylation
efficiency is reduced by more than 50% when the glycosylation
acceptor site is very close to the C terminus, and the efficiency
increases gradually with the distance between the acceptor site
and the C terminus. Full glycosylation, which is 70 – 80% in
this system (5), is observed when the distance between the
acceptor site and the C terminus is ⬃60 residues or more. For
efficient modification, it thus appears that the acceptor site has
to reach or at least be close to the OST active site, which is ⬃65
residues away from the ribosomal P-site (15), before chain
termination. These results are consistent with an earlier study
of the rabies virus glycoprotein, where a reduced glycosylation
efficiency of two mutants truncated, respectively, 27 and 69
residues downstream of an acceptor Asn was noted (16).
A Hydrophobic Stop Transfer Sequence between the Acceptor
Site and the Stop Codon Affects Glycosylation Efficiency—Because the results reported above indicated that glycosylation
efficiency depends on the location of the acceptor site relative to
the OST active site at the time of chain termination, we considered the possibility that glycosylation efficiency may also be
influenced by the presence of a hydrophobic ST sequence between the acceptor site and the C terminus.
We thus constructed C-terminally truncated versions of two
previously studied model proteins (Lep-ST14L and Lep-ST29L)
(6) (Fig. 3A) that contain an engineered glycosylation site
(Asn201-Ser-Thr or Asn203-Ser-Thr) 16 or 18 residues upstream
of a poly-Leu ST sequence composed of either 15 or 30 hydrophobic residues (QQQL14VKKKK or QQQL 29VKKKK). When
expressed as full-length proteins, Lep-ST14L and Lep-ST29L
are efficiently glycosylated on Asn201 and Asn203, respectively
(Fig. 3B).
17340
Glycosylation of C-terminal Asn-Xaa-Thr Sequons
FIG. 2. Glycosylation efficiency increases with the distance
from the C terminus. A, in vitro translation of the Asn201 full-length
construct (lanes 3 and 4) and the Asn201 construct truncated 6 residues
downstream of the acceptor Asn in the Asn201-Ser-Thr glycosylation
sequon (lanes 1 and 2) in the absence (⫺) and presence (⫹) of dog
pancreas rough microsomes (RM). Glycosylated and nonglycosylated
products are shown by black and white dots, respectively. B, the glycosylation efficiency of four different Lep constructs with unique glycosylation acceptor sites in position 201 (black dots), 203 (white dots), 214
(light gray dots), and in different positions relative to the C terminus of
the full-length protein (dark gray dots) is shown as a function of the
number of residues between the acceptor Asn and the C terminus. A
rough fit to the experimental points is shown by the gray area. Truncated proteins were made by PCR amplification of the desired part of
the gene using a 3⬘ primer containing tandem stop codons, followed by
in vitro transcription/translation in the presence of dog pancreas
microsomes.
C-terminal truncations were made by introducing tandem
stop codons at different sites downstream of and inside the ST
segments, and glycosylation was assayed by in vitro transcription/translation in the absence and presence of microsomes. As
shown in Fig. 3C, for both constructs the glycosylation efficiency remains at ⬃35% until the stop codon is placed ⬃10
residues into the hydrophobic stretch and then increases to the
level seen without a hydrophobic segment. Strikingly, the glycosylation efficiency drops sharply as soon as the stop codon is
placed downstream of the ST segment and remains low until
the C terminus of the truncated protein is ⬃70 residues away
from the glycosylation site. Thus, the glycosylation efficiency
appears to depend both on the position of the C terminus
relative to the ST sequence and to the glycosylation acceptor
site.
To determine whether the proteins truncated in or near the
ST sequence were retained in the membrane, we tested two
additional series of constructs. First, we repeated the above
experiments using a Lep-ST29L construct with the glycosylation site placed in position 208, only 11 residues upstream of
the ST segment. We have shown previously that a glycosylation
acceptor site must be at least 13–15 residues upstream of a
membrane-anchored ST segment to be a substrate for OST (5,
6), and indeed, no glycosylation was seen for the LepST29L(Asn208-Ser-Thr) construct, Fig. 4 (lane 1). Similarly, no
glycosylation was seen when this construct was truncated by
placing tandem stop codons 1, 5, and 15 positions downstream
of the ST29L segment (data not shown) or in a position leaving
15 leucine residues in the ST segment (lane 2). However, when
only 10 leucines were left in the ST segment, the glycosylation
efficiency increased to 20% (lane 3), and with only 5 leucines
left, it increased to 38% (lane 4). In the latter construct, the
protein was truncated 16 residues downstream of Asn208, and
the glycosylation efficiency was thus similar to what was found
for the short Lep constructs lacking an ST segment (Fig. 2).
As a second control, we introduced a signal peptidase cleavage cassette (10) at the C-terminal end of the H2 transmembrane segment (Fig. 5A) and assayed membrane integration of
constructs truncated at different positions in the ST segment
by alkaline extraction (17). For this, Lep-ST29L with the acceptor site at Asn203 was modified by (i) exchanging H2 for a
14-leucine-long hydrophobic segment ending with a signal peptidase cleavage cassette and (ii) adding an extra 50 residues to
the P2 domain. The lengthening of the P2 domain was necessary to avoid that the P2-ST fragment remaining after signal
peptidase cleavage overlapped in the gel with nonspecific background bands from the in vitro translation. As seen in Fig. 5B,
the resulting full-length Lep-ST29L(CC/50) construct was
cleaved by signal peptidase and glycosylated when expressed in
the presence of rough microsomes. Significantly, as seen in Fig.
5C, a truncated form of Lep-ST29L(CC/50) with stop codons
placed one residue downstream of the ST segment was not
extracted from the membrane pellet by alkali (bottom panel,
lanes 5 and 6), whereas a truncated form with only 5 leucine
residues left in the ST segment was extractable (top panel,
lanes 5 and 6). Both truncated forms were inefficiently glycosylated, as expected from Fig. 3C, and signal peptidase-mediated cleavage of the constructs was inhibited by the signal
peptidase inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (lane 3).
From these two sets of control experiments, we conclude that
truncated ST segments retaining ⬃15 leucine residues or more
are efficiently anchored in the membrane (presumably in a
transmembrane disposition), whereas those with ⬃10 or fewer
leucine residues are largely released into the lumen of the
microsomes. Thus, the drop in glycosylation efficiency seen in
Fig. 3 for proteins truncated after the ST segment cannot be
explained by a lack of proper membrane integration of the ST
segment.
To rule out that the identity of the codon next to the stop
codon had an influence on the results, we made two further
controls. First, we changed the valine codon GTT at the end of
the ST segment in Lep-ST14L to one (GTG) more frequently
used in mammalian mRNAs, and placed a stop codon in the
next position. Second, we changed the valine codon to one
coding for leucine (CTG), again placing the stop codon in the
next position. In neither case did we observe an effect on
Glycosylation of C-terminal Asn-Xaa-Thr Sequons
17341
FIG. 4. Translation in the presence of dog pancreas rough
microsomes of the full-length Lep-ST29L(Asn208-Ser-Thr) construct (lane 1) and of truncated Lep-ST29L(Asn208-Ser-Thr) constructs with 15 (lane 2), 10 (lane 3), and 5 (lane 4) leucine residue
left in the ST segment. Glycosylated and nonglycosylated products
are shown by black and white dots, respectively.
glycosylation, which remained at ⬃60% (data not shown). Finally, to rule out the possibility that the low level of glycosylation seen when the stop codon was placed immediately downstream of the ST sequence was caused by the four flanking
lysine residues, we changed the C-terminal sequence . . .
LVKKKKHM(stop) to . . . LVDPNQHM(stop) for both the
ST14L and ST29L constructs; again no change in glycosylation
efficiency was seen (data not shown).
DISCUSSION
Statistical studies of the location of glycosylated and nonglycosylated potential acceptor sites in secreted proteins have
indicated that the frequency of nonglycosylated Asn-Xaa-(Thr/
Ser) sequons increases toward the C-terminal end of glycoproteins (18). We now find a similar trend when we successively
increase the number of residues between an acceptor site and
the C terminus of truncated Lep constructs (Fig. 2), suggesting
that glycosylation is inefficient when chain termination happens before the acceptor site reaches the OST active site. Previous measurements have shown that ⬃65 residues in a nascent polypeptide chain are required to span the distance
between the ribosomal P-site and the OST active site (15), and
we indeed see fully efficient glycosylation only when ⬃60 residues or more separate the glycosylation acceptor site from the
C terminus.
The rate of chain elongation in vitro is quite slow (⬃1 residue/s (19)), and it is possible that the reduction in glycosylation
efficiency seen when chain termination precedes glycosylation
is the result of a more rapid translocation of the nascent chain
relative to the OST once it has been detached from the ribosome. Although we have not tested the truncated Lep constructs in vivo, the observation that more nonglycosylated Asn-
FIG. 3. Glycosylation efficiency is influenced by a C-terminal
stop transfer sequence. A, Lep constructs used. The sequences of the
ST14L and ST29L inserts are shown, and the positions of the Asn201
and Asn203 glycosylation acceptor sites are indicated. B, translation of
the full-length Asn201-ST14L (lanes 1 and 2) and Asn203-ST29L (lanes 3
and 4) constructs in the absence (⫺RM) and presence (⫹RM) of dog
pancreas rough microsomes. Glycosylated and nonglycosylated prod-
ucts are shown by black and white dots, respectively. C, glycosylation
efficiency of truncated Asn201-ST14L (E) and Asn203-ST29L (●) constructs as a function of the number of residues between the acceptor
Asn and the C terminus. The locations of the 14L and 29L hydrophobic
ST segments relative to the glycosylation acceptor site are shown as
hatched bars, the C-terminal sequences of a few selected constructs are
given, and the relation between glycosylation efficiency and distance to
the C terminus from Fig. 2 is indicated by the gray area. Note that the
gradation of the x axis is different from that in Fig. 2.
17342
Glycosylation of C-terminal Asn-Xaa-Thr Sequons
Xaa-(Thr/Ser) sequons are found near the C-terminal end of
secreted glycoproteins (18) suggest that also in vivo chain termination can influence glycosylation efficiency. We also note
that the reduction in glycosylation efficiency seen when the
glycosylation site is close to the C-terminal end of the protein
needs to be taken into account when mapping the topology of
integral membrane proteins by glycosylation scanning (14, 20 –
22) and points to the necessity to use sufficiently large Cterminal reporter domains.
When a hydrophobic ST sequence is present between the
acceptor site and the C terminus, a very different behavior is
observed. In particular, for constructs where the C-terminal
end of the protein is located in a region immediately downstream of the hydrophobic segment, very low levels of glycosylation are seen (Fig. 3C), even though the separation between
the acceptor site and the C terminus is ⬃50 residues or more.
The level of glycosylation returns to “normal” only when the C
terminus is ⬃70 residues or more away from the acceptor site,
i.e. at a point where the acceptor site has just emerged in the
vicinity of the OST active site when the stop codon is reached
(15). The reduction in the glycosylation efficiency thus corresponds to a situation where the ST sequence has been fully
polymerized but is still largely inside the ribosome at the time
of chain termination.
The dramatic effect on glycosylation caused by the ST sequence suggests that the hydrophobic segment may in some
way affect the structure of the ribosome-translocon-OST complex from inside the ribosome. Indeed, recent experiments have
shown that the translocon pore is gated during membrane
protein integration; whereas the translocon pore is normally
open toward the lumen and closed by the ribosome on the
cytosolic side during chain translocation, this situation is rapidly reversed when an ST sequence appears inside the ribosome
(3, 4). The conversion to a state open toward the cytosol and
closed toward the lumen happens already when 4 –9 residues
have been polymerized in addition to the ST sequence. Similarly, the drop in glycosylation efficiency seen in Fig. 3C happens when the stop codon is placed only a couple of residues
downstream of the hydrophobic stretch, both for ST14L and for
ST29L. We thus speculate that the closing of the lumenal end
of the translocon induced by the presence of an ST sequence in
the ribosome also affects the way in which the nascent chain is
exposed to the OST and thus indirectly the efficiency with
which glycosylation acceptor sites near the C-terminal end of
the nascent chain are modified.
Acknowledgment—We thank M. O. Lively for sharing unpublished
data on signal peptidase inhibitors.
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FIG. 5. A, the general design of the Lep-ST29L(CC/50) construct. The
amino acid sequence of the region around the cleavage cassette in H2 is
shown, with the designed signal peptidase cleavage site indicated by
the arrow. P83 is residue 83 in Lep. B, translation of full-length LepST29L(CC/50) in the absence (⫺) and presence (⫹) of rough microsomes
(RM) and a glycosylation-inhibiting acceptor peptide (AP). The fragments resulting from signal peptidase cleavage are indicated by arrows,
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(SPI), and a glycosylation-inhibiting acceptor peptide (AP) are indicated. Lanes 5 and 6 show the results of an alkaline extraction experiment. P, membrane pellet; S, supernatant. The fragments resulting
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