From the Department of Pharmacology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
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ABSTRACT |
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Dilysine signals confer localization of type I
membrane proteins to the endoplasmic reticulum (ER). According to the
prevailing model these signals target proteins to the ER by COP
I-mediated retrieval from post-ER compartments, whereas the actual
retention mechanism in the ER is unknown. We expressed chimeric
membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or
-Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF
constructs, which had access to post-ER compartments, the KKAA chimeras
were localized to the ER by confocal microscopy and were neither
processed by cis-Golgi-specific enzymes in vivo
nor included into ER-derived transport vesicles in an in
vitro budding assay, suggesting that KKAA-bearing proteins are
permanently retained in the ER. The ER localization was nonsaturable
and exclusively mediated by the dilysine signal because mutating the
lysines to alanines led to cell surface expression of the chimeras.
Although the KKAA signal avidly binds COP I in vitro, the
ER retention by this signal does not depend on intact COP I in
vivo because it was not affected in an Many type I membrane proteins carry a dilysine signal in their
cytosolic domain that confers localization to the ER
(1).1 This signal has been
defined as two lysine residues at positions The ERGIC marker protein ERGIC-53 is a type I membrane protein carrying
a dilysine signal with the sequence -Lys-Lys-Phe-Phe (KKFF) (14).
ERGIC-53 is a mannose lectin continuously recycling in the early
secretory pathway, suggesting that it may operate as a transport
receptor for glycoproteins (15, 16). In support of this notion,
antibodies to the cytoplasmic tail of ERGIC-53 inhibit transport of
vesicular stomatitis virus G protein (17), mistargeting of ERGIC-53 to
the ER impairs the secretion of cathepsin C (18), and mutations in
ERGIC-53 can lead to combined factor V/VIII deficiency in humans
presumably due to inefficient secretion of these coagulation factors
(19). Targeting of ERGIC-53 is controlled by at least three signals. In
addition to the dilysine signal the two C-terminal phenylalanines
operate as an ER exit determinant (20). Moreover, the luminal and
transmembrane domains of ERGIC-53 in combination contain information
for intracellular retention that is abolished by exchanging the
transmembrane domain of ERGIC-53 by that of the plasma membrane protein
CD4 (20). If the two C-terminal phenylalanines are changed to alanines, resulting in the C-terminal sequence KKAA, ERGIC-53 is mislocalized to
the ER (20, 21). Although the most straightforward interpretation of
this finding is that the replacement of the two phenylalanines inactivates the ER exit signal, an alternative possibility is that the
dilysine signal KKAA mediates direct retention in the ER. To test for
retention, we have transferred the KKAA dilysine signal to a plasma
membrane reporter protein, CD4, that is known not to carry
intracellular retention information. Here we show that chimeric
proteins possessing the C-terminal dilysine signal KKAA are permanently
retained in the ER, in contrast to KKFF chimeras that can leave the ER.
The results suggest that dilysine signals can operate in both ER
retention and retrieval.
Recombinant DNA--
The cytosolic tails of the CD4 chimeras
were generated by polymerase chain reaction and fused to the luminal
and transmembrane domain of the CD4 molecule after codon 395 using a
Bsu36I site as described (21). A construct encompassing the
luminal and transmembrane domain of CD4 fused to a cytosolic tail with
the sequence RRAAAASAKKFF was used as a template. This construct was generated according to Itin et al. (21). The CD4 chimeras
were cloned into the pcDNA3 vector under the CMV promoter. The GM
construct was as reported (21). The L53T4KKAA mutant was constructed by polymerase chain reaction using the L53T4C53 construct (21), as a
template. All constructs were confirmed by DNA sequencing.
Antibodies--
Antibodies used were: mAb G1/93 (IgG1) against
human ERGIC-53 (22), mAb 9E10.2 (IgG1) against a c-Myc epitope (ATCC
CRL 1729), mAbs HP2/6.1 (IgG2A; kindly provided by Francisco
Sanchez-Madrid (23)) and RFT4 (IgG1; kindly provided by George Janossy
through the MRC AIDS reagent project (24)) against CD4. mAb A1/59
(IgG1) against the ER protein p63 was produced by the hybridoma
technique (22) using an ERGIC fraction of Vero cells (25) as an antigen.
Cell Culture and Transfection--
Chinese hamster ovary Lec-1
cells (ATCC CRL 1735; Ref. 27) were grown in Metabolic Labeling, Immunoprecipitation, and endo-D
Treatment--
Cells of a 60-mm dish were metabolically labeled with
50-100 µCi of [35S]methionine in a total volume of 1 ml of medium (minimum essential medium, 10% dialyzed fetal calf serum,
1% nonessential amino acids). After the chase, the cells were lysed in
1 ml of lysis buffer (100 mM sodium phosphate, 1% Triton
X-100, pH 8, and protease inhibitors). Lysates were cleared by
centrifugation at 100,000 × g for 1 h. Each
supernatant was added to 40 µl of protein A-Sepharose (Pharmacia) to
which 1 µl of mAb HP2/6.1 had been prebound. After incubation
overnight, samples were split in two, and proteins were eluted from the
protein A beads by boiling for 3 min in 30 µl of endo-D buffer (50 mM Na2HPO4, pH 6.5 (sodium
citrate), 2% Triton X-100, 0.1% SDS, 5 mM EDTA, and
protease inhibitors). Another 30 µl of endo-D buffer was added to
each sample, and 3 milliunits of endo-D (Roche Molecular Biochemicals)
was added to every second sample followed by incubation at 37 °C for
20 h. Samples were then boiled in sample buffer and analyzed by
SDS-PAGE followed by fluorography. Endoglycosidase H was used as
described (20).
Immunofluorescence Microscopy--
48 h posttransfection Lec-1
cells were fixed with 3% paraformaldehyde, permeabilized with 0.1%
saponin, and processed for indirect immunofluorescence as described
(21). AlF4 In Vitro Budding Assay--
The in vitro budding was
performed as described (30) with the following modifications.
Microsomes were prepared from eight 15-cm dishes of confluent Lec-1
cells stably transfected with either the GM or the L53T4KKAA construct.
The cells were homogenized by passing them 10 times through a
ball-bearing homogenizer with a clearance of 20 µm. The final pellet
was resuspended in 80 µl of transport buffer giving a protein
concentration between 5-15 mg/ml and used directly in a single budding
assay. Samples were analyzed by SDS-PAGE followed by Western blotting
(ECL). Blots were quantified by densitometry. The Sar1 (T39N) mutant
protein was purified according to Rowe and Balch (26) and used at 3 µM final concentration in the assay.
A KKAA Chimera Is Localized to the ER--
We have previously
shown that a construct of ERGIC-53 containing the dilysine signal KKAA
is retained in the ER (20, 21). Because the combined presence of the
luminal and transmembrane domains of ERGIC-53 already confer some
retention, the contribution of the KKAA signal to ER retention could
not be evaluated using this reporter protein.
To elucidate whether KKAA can mediate ER retention, it was fused to the
transmembrane and luminal domain of CD4. CD4 is a cell surface protein
known not to contain intracellular retention information. The CD4
luminal domain has two N-linked glycosylation sites, which
are useful for determining the intracellular location of CD4 chimeras
biochemically. The chimeras CD4KKAA, CD4KKFF, and CD4R2A10, depicted in
Fig. 1, were transiently expressed in Lec-1 cells, and their expression was analyzed by immunofluorescence microscopy. CD4KKAA exhibited typical ER staining and could not be
detected at the cell surface of nonpermeabilized cells (Fig. 2, a and b). The
CD4KKFF construct gave an ER pattern but was also detectable in the
Golgi area (Fig. 2c) and at the cell surface (Fig. 2,
c and d). The inability of the KKFF signal to
mediate complete intracellular retention is likely due to saturation of the retention mechanism by overexpression. Such a leakage has already
been observed for wild type ERGIC-53 (21, 33), and suggests that the
dilysine-dependent retrieval mechanism is saturable. The
CD4R2A10 construct showed cell surface staining as expected for a
construct without retention signals. These results indicate that the
targeting mechanisms mediated by KKAA and KKFF are different or that
the two signals have different affinities for the same sorting
machinery.
The KKAA Signal Prevents Transport to cis-Golgi--
To
investigate whether the CD4KKAA protein had access to the
cis-Golgi and would be localized to the ER by retrieval from the Golgi, its carbohydrate modifications were studied in Lec-1 cells.
Because these cells lack the cis-Golgi enzyme
N-acetylglucosaminyltransferase, glycoproteins passing
through the cis-Golgi acquire permanent endo-D sensitivity
(27). Lec-1 cells transiently transfected with either CD4KKAA, CD4KKFF,
or CD4R2A10 were labeled with [35S]methionine for 30 min
and chased for 6 h. Cell lysates were immunoprecipitated with a
mAb against CD4, and the precipitates were incubated with or without
endo-D prior to SDS-PAGE analysis. In the absence of endo-D (Fig.
3, upper panel) CD4KKFF
(lane 4 versus lane 3) and CD4R2A10 (lane 6 versus lane 5) showed increased electrophoretic
mobility after the 6-h chase, indicating posttranslational trimming. By
contrast, the mobility of CD4KKAA changed only minimally (lane
2 versus lane 1), suggesting that this construct did
not have access to the compartment(s) containing the trimming enzymes that processed CD4KKFF and CD4R2A10. Because the CD4 chimeras contain
two N-linked glycosylation sites, the small shift of CD4KKAA can be explained by The KKAA Signal Prevents Transport to the ERGIC--
The inability
of CD4KKAA to reach the cis-Golgi may reflect efficient
retrieval from the ERGIC. AlF4 A KKAA Chimera Is Excluded from ER Transport Vesicles--
To
investigate in a more direct way whether a KKAA signal prevents exit
from the ER, we isolated ER transport vesicles using a recently
described in vitro budding assay (26, 30). Microsomes were
isolated from Lec-1 cells permanently expressing a fusion protein
consisting of the luminal domain of ERGIC-53, the transmembrane domain
of CD4 and a RSQQEAAAKKAA cytosolic tail (construct L53T4KKAA; Fig. 1).
The reason for using this construct was that attempts to obtain a
stable cell line expressing high amounts of CD4KKAA used above were
unsuccessful. We have previously shown that the luminal and
transmembrane domains of ERGIC-53 in combination contain pre-Golgi
retention information (20). By exchanging the transmembrane domain of
ERGIC-53 with that of CD4, this retention is abolished and any
remaining retention is exclusively confined to the KKAA signal of the
cytosolic tail (20, 21).
Transport vesicles were generated by incubating the microsomes with
cytosol and an ATP-regenerating system at 32 °C for up to 40 min. As
a positive control, microsomes were isolated from Lec-1 cells stably
expressing GM. After incubation, the transport vesicles were separated
from the microsomes by differential centrifugation. The medium speed
pellet containing the microsomes and the high speed pellet (HSP)
containing the vesicles were analyzed by SDS-PAGE followed by Western
blotting. GM and L53T4KKAA were detected with anti-Myc antibodies. At
no time point could the L53T4KKAA protein be detected in the HSP,
indicating that the protein was not incorporated into transport
vesicles (Fig. 5a, lower
panel; Fig. 5b, quantification). In contrast, GM, which
continuously recycles between the ER, ERGIC, and cis-Golgi,
was detected in the HSP after a 20-min incubation (Fig. 5a,
upper panel). The appearance of GM in HSP was not due to ER
fragmentation because the ER resident protein p63 was absent from HSP
(Fig. 5c). It was entirely inhibited in the absence of either an ATP regenerating system or cytosol in the budding reaction (Fig. 5d) or inhibited by >80% by a dominant negative
mutant Sar1 protein, Sar1T39N (Fig. 5e), confirming that the
appearance of GM in the HSP was due to vesicle budding.
KKAA-mediated ER Retention Does Not Depend on Intact
Coatomer--
Previous studies have shown that peptides terminating in
KKAA avidly bind coatomer (17, 20). To test whether KKAA-mediated ER
retention requires coatomer, we used a mutant CHO cell line (ldlF) that
expresses a temperature-sensitive point mutation in the coatomer
subunit It is currently assumed that dilysine targeting signals operate by
retrieval rather than retention (1). This notion is based on the
findings that chimeras bearing some dilysine signals carry
Golgi-specific carbohydrate modifications despite their localization to
the ER both in mammalian and yeast cells (2-4).
Surprisingly, we were unable to find evidence for recycling of chimeric
proteins with a C-terminal KKAA dilysine signal. These chimeras
localized to the ER, did not acquire Golgi-dependent endo-D
sensitivity in Lec1 cells, were not concentrated in the ERGIC by
AlF4 What is the mechanism by which KKAA mediates ER retention? Previous
transfection experiments in COS cells have shown that the intracellular
retention of ERGIC-53 and CD4 chimeras carrying the wild type KKFF
signal is saturable by overexpression very much in contrast to that of
ERGIC-53 terminating in KKAA (21, 33). It was of interest therefore to
determine whether COP I known to specifically recognize dilysine
signals plays a role in the ER retention mediated by the KKAA signal.
Although the KKAA signal avidly binds COP I in vitro (20)
and COP I has be localized to some areas of the ER (34), our results
with the In conclusion, we have shown that the dilysine signal KKAA mediates
permanent retention of type I membrane proteins in the ER. We propose
that dilysine ER targeting signals can mediate retention in addition to
retrieval. Thus protein targeting to the ER by dilysine signals can be
mediated by at least two mechanisms that are not mutually exclusive:
signal-mediated retention and signal-mediated retrieval.
-COP-deficient cell
line. We propose that dilysine ER targeting signals can mediate ER
retention in addition to retrieval.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 and
4
(KKXX) or
3 and
5 (KXKXX) from the
C terminus exposed on the cytosolic side of the ER membrane (2).
Chimeric proteins bearing a dilysine signal are localized to the ER but
can acquire Golgi-specific carbohydrate modifications, suggesting that
ER localization involves retrieval from the Golgi (3-5). Recent evidence implicates COP I (coat protein type
I) in the recognition of the dilysine signal. COP I
(coatomer) is a complex of seven proteins termed
-,
-,
'-,
-,
-,
-, and
-COP (6-8) mainly localized to the ER/Golgi
intermediate compartment (ERGIC) and the Golgi (9). Functional dilysine
signals bind to coatomer in vitro (10), and yeast mutants of
COP subunits fail to localize reporter proteins to the ER (11-13).
Some of these mutants are unable to bind to functional dilysine signals
in vitro, supporting the notion that coatomer plays an
essential role in retrograde Golgi-to-ER transport of dilysine-bearing proteins.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum essential
medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin,
100 µg/ml streptomycin, and 1 mg/ml fungizone (Life Technologies,
Inc.). ldlF cells (kindly provided by Monty Krieger) were grown in
Ham's F-12 medium supplemented with 5% fetal calf serum, antibiotics,
and fungizone. Stable cell lines expressing the GM and L53T4KKAA
constructs were generated as described in Kappeler et al.
(20). For transient expression, Lec-1 cells were transfected by the
calcium phosphate precipitation method and ldlF cells using
Transfectam® (Promega, Madison, WI). For pulse-chase experiments 60-mm
dishes were each transfected with 5-8 µg of DNA. For
immunofluorescence microscopy cells were grown in eight-well
multichamber Permanox slides (Nunc, Life Technologies, Inc.) and
transfected with 0.6 µg of DNA/well. For cotransfections, 0.3 µg of
DNA of each construct was used.
was used at the following
concentrations: 30 mM NaF and 50 µM AlCl3. Cells were analyzed with a Reichert Polyvar
immunofluorescence microscope or a Leica confocal laser scanning
microscope (0.25-µm optical sections).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of ERGIC-53 and CD4
constructs. The amino acid sequences of the cytosolic tails are
indicated in single-letter codes. CD4 domains are indicated in
white, and ERGIC-53 domains are in gray. The
c-Myc epitope tag present after the signal sequence cleavage site in
the GM and L53T4KKAA constructs is marked in black. In the
L53T4KKAA construct, L53 stands for luminal domain of
ERGIC-53; T4 stands for transmembrane domain of
CD4; KKAA stands for cytosolic tail containing the
KKAA dilysine signal.
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Fig. 2.
The KKAA construct localizes to the ER.
Immunofluorescence localization of the CD4 chimeras. Lec-1 cells were
transiently transfected with the CD4KKAA (a and
b), CD4KKFF (c and d), or CD4R2A10
(e and f) construct. 48 h posttransfection
the cells were stained before fixation and permeabilization
(b, d, and f) or after fixation and
permeabilization (a, c, and e).
Transfected proteins were detected with mAb RFT4 against the CD4
protein and a fluorescein-labeled goat anti-mouse secondary antibody.
Bar, 18 µm.
-1,2-mannosidase-mediated trimming in the ER,
whereas CD4KKFF and CD4R2A10 would be further trimmed by Golgi mannosidase I (28). Fig. 3 (lower panel) indeed shows that a fraction of CD4KKFF (lane 4) and CD4R2A10 (lane
6) was endo-D-sensitive after the 6-h chase, whereas CD4KKAA
remained entirely endo-D-resistant (lane 2). The apparent
incomplete endo-D sensitivity of CD4KKFF and CD4R2A10 after 6 h is
likely due to incomplete digestion with endo-D, a well known
phenomenon, and not to incomplete trimming to mannose 5. The slight
shift of CD4KKAA seen both in the absence and presence of endo-D was
prevented by the mannosidase inhibitor deoxymannojirimycin (not shown),
which, together with the endo-D insensitivity, corroborates the notion
of a mannose trimming event in the ER. These results suggest that
CD4KKFF and CD4R2A10 had access to the cis-Golgi, whereas
CD4KKAA did not.
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Fig. 3.
The KKAA construct remains
endo-D-resistant. Lec-1 cells were transiently transfected with
the CD4KKAA, CD4KKFF, and CD4R2A10 constructs. 48 h
posttransfection the cells were labeled with
[35S]methionine for 30 min followed by a chase for 0 or
6 h. The cells were lysed, and the lysates immunoprecipitated with
mAb HP2/6.1 against CD4. The precipitates were split in two and
incubated with or without 3 milliunits of endo-D. After incubation,
samples were analyzed by SDS-PAGE followed by fluorography. The
constructs are denoted KKAA, KKFF, and
R2A10 for CD4KKAA, CD4KKFF, and CD4R2A10, respectively.
S, endo-D-sensitive protein.
was
used to test this possibility. AlF4
,
an activator of trimeric G proteins, blocks anterograde and retrograde
transport of proteins from the ERGIC (16, 20, 29), presumably by
locking coatomer to membranes. If CD4KKAA would be transported from the
ER to the ERGIC and rapidly retrieved from there, it should accumulate
in the ERGIC in AlF4
treated cells. To
this end, Lec-1 cells co-expressing CD4KKAA and GM were treated with
AlF4
for 30 min, double stained for
CD4 and ERGIC-53, and analyzed by confocal laser scanning microscopy.
In the absence of AlF4
, CD4KKAA was
exclusively confined to the ER, whereas GM, in addition to the ER, also
localized to a juxtanuclear area in the Golgi region (Fig.
4, left panel). Upon
AlF4
treatment, the staining pattern
for CD4KKAA remained unchanged, very much in contrast to GM, which was
depleted from the ER and concentrated in the juxtanuclear area (Fig. 4,
right panel). We conclude that CD4KKAA is unable to recycle
via the ERGIC.
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Fig. 4.
CD4KKAA does not accumulate in the ERGIC in
the presence of AlF4 . Lec-1 cells
were cotransfected with the GM and CD4KKAA constructs. 48 h
posttransfection, the cells were incubated with or without
AlF4
for 30 min and processed for
confocal double immunofluorescent microscopy. GM protein was visualized
with mAb G1/93 followed by fluorescein isothiocyanate-conjugated goat
anti-IgG1 mouse antibodies and CD4KKAA with mAb HP2/6.1 followed by
tetramethyl rhodamine isothiocyanate-conjugated goat anti-IgG2A mouse
antibodies. The cells were examined using a Leica confocal laser
scanning microscope. Bar, 20 µm.
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Fig. 5.
A KKAA construct does not enter ER-derived
vesicles. Microsomes of Lec1 cells stably transfected with GM or
L53T4KKAA were incubated in an in vitro budding assay
mixture at 32 °C for 0, 20, and 40 min. ER transport vesicles were
separated from the microsomes by differential centrifugation and
analyzed by SDS-PAGE followed by Western blotting. 20% of the medium
speed pellet and 100% of the high speed pellet were applied per lane.
a, budding assay with microsomes from GM cells (upper
panel) and L53T4KKAA cells (lower panel). b,
quantification of the amount of GM and L53T4KKAA appearing in the high
speed pellet after a 20-min incubation at 32 °C. Shown are means of
four independent experiments ± S.D. c, distribution of
the p63 ER protein in a budding assay performed with GM microsomes for
20 min. First the GM protein was detected (left two lanes),
then the blot was stripped and probed for p63 (right two
lanes). d, cytosol and ATP dependence of the budding
assay with GM microsomes. The reaction mixture was incubated for 20 min
in the presence (normal) or absence of either ATP ( ATP) or
cytosol (
cytosol). The constructs were visualized with anti-Myc (mAb
9E.10.2). e, Sar1 dependence of the budding assay. A
dominant-negative mutant of Sar1, Sar1T39N (final concentration, 3 µM), inhibits vesicle formation from GM microsomes.
Sar1T39N was added to the complete budding mixture for 20 min.
M, medium speed pellet containing the microsomes;
H, high speed pellet containing the transport
vesicles.
-COP (31). At the nonpermissive temperature (39.5 °C)
ldlF cells have a defect in transport of newly synthesized glycoproteins through the Golgi as indicated by delayed or incomplete oligosaccharide processing. If COP I was to play a role in
KKAA-mediated ER retention, CD4KKAA can be expected to leave the ER and
undergo cis-Golgi-dependent oligosaccharide
processing in
-COP-defective cells. ldlF cells grown at 34 °C
(permissive temperature) were transfected with CD4KKAA or CD4R2A10
cDNA. 38 h posttransfection, the cells were transferred to a
39.5 °C incubator for 10 h. Then the cells were pulse-labeled
with [35S]methionine for 30 min, chased for 3 h with
methionine in excess, and subjected to immunoprecipitation with
antibodies against CD4. The precipitates were analyzed by SDS-PAGE
followed by fluorography (Fig. 6).
CD4R2A10 remained entirely endoglycosidase H-sensitive after the 3-h
chase (not shown), indicating that transport through the Golgi was
impaired at 39.5 °C as expected (31). However, during the chase
CD4R2A10 underwent an apparent size reduction characteristic for the
cis-Golgi form (Fig. 6, lane 4; see also Fig. 3).
By contrast, CD4KKAA did not show the Golgi-specific Mr transition at 3 h (lane 3),
and by immunofluorescence microscopy the localization of this construct
was unchanged (not shown), strongly suggesting that CD4KKAA does not
leave the ER when COP I is impaired. We conclude that intact coatomer
is not required for ER retention by the KKAA signal.
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Fig. 6.
R2A10 but not KKAA allows transport to the
Golgi in -COP deficient ldlF cells. ldlF
cells were transiently transfected with either the CD4KKAA or CD4R2A10
construct. 38 h posttransfection the cells were transferred to
39.5 °C and incubated for 10 h prior to labeling. Cells were
labeled with [35S]methionine for 30 min, chased for
3 h, lysed, and immunoprecipitated with mAb HP2/6.1 against CD4.
Precipitates were analyzed by SDS-PAGE followed by fluorography. The
constructs are denoted KKAA and R2A10 for CD4KKAA
and CD4R2A10, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and were excluded from
ER-derived transport vesicles. These data strongly support the notion
that KKAA is a true ER retention signal. ER retention is not simply due
to the lack of an ER-export signal because chimeras with a polyalanine
tail lacking transport information were not retained. Moreover, the
inactivation of an ER exit signal does not entirely block ER exit (20,
32). It has previously been noted that the rate at which various
dilysine signal-tagged reporter proteins acquired Golgi modifications
greatly varied, as did the degree to which they co-localized to post-ER compartments (4). It was therefore proposed that the sequence context
of a dilysine motif may affect the efficiency of retrieval (1). Our
data with KKAA suggest retention as an additional function of dilysine
signals, whereby the sequence context of dilysine signals determines
the efficiency of ER retention. Although it is likely that the sequence
context also affects the efficiency of protein retrieval, this issue
remains to be reinvestigated in light of our present findings.
-COP-deficient ldlF cells suggest that intact coatomer is
not required for ER retention. Unlike CD4R2A10 the KKAA protein did not
acquire cis-Golgi-specific carbohydrate modifications in
these cells at the nonpermissive temperature. Under these conditions coatomer of ldlF cells is unable to bind to the dilysine
signal.2 These data are in
line with the notion that COP I is primarily involved in retrograde
transport (11), although other traffic routes may also depend on
coatomer. Another possibility for retention would be tight interaction
of the KKAA signal with microtubules. Studies with a synthetic peptide
containing the dilysine signal of the adenovirus E19 protein indicate
that this peptide can bind
-tubulin and promote tubulin
polymerization in vitro (35). However, if microtubules were
destroyed by nocodazole, KKAA-mediated ER retention was not
affected,3 rendering such a
mechanism less likely. Yet another notion for protein retention in a
membrane organelle is the formation of oligomers too large to be
included into transport vesicles (36). Such a mechanism appears
unlikely because CD4 is a monomer and KKAA-bearing ERGIC-53
oligomerizes with normal kinetics and no such large aggregates were
observed (Ref. 21 and this study). Interaction with an ER resident
protein appears a likely possibility for explaining the ER retention,
although our attempts to identify such a partner protein were
unsuccessful. Interaction with ER-specific lipids is another
possibility that remains to be tested.
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ACKNOWLEDGEMENTS |
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We thank Käthy Bucher for excellent technical assistance, William Balch for the Sar1T39N cDNA and test reagents for the in vitro budding assay, Monty Krieger for providing ldlF cells, Francisco Sanchez-Madrid and George Janoussy for antibodies against CD4, and members of the Hauri lab for helpful suggestions.
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FOOTNOTES |
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* This work was supported by grants from the Swiss National Science Foundation and the Kantons of Basel and an EMBO long term fellowship (to H. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Dept. of Pharmacology,
Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel,
Switzerland. Tel.: 41-61-267-22-22; Fax: 41-61-267-22-08; E-mail:
Hauri{at}ubaclu.unibas.ch.
2 M. Gomez, personal communication.
3 H. Andersson and H.-P. Hauri, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; endo-D, endoglycosidase D; ERGIC, ER/Golgi intermediate compartment; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; HSP, high speed pellet.
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