(Received for publication, November 27, 1996, and in revised form, March 31, 1997)
From the Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom
TGN38 is a type I integral membrane protein that cycles between the trans-Golgi network and the plasma membrane. Internalization at the cell surface and targeting back to the trans-Golgi network is dependent on a hexapeptide motif, SDYQRL, in the cytosolic tail of the protein. It was recently demonstrated that this motif specifically interacts with the µ2 subunit of the AP2 adaptor complex. We have studied the interaction between the entire cytosolic domain of TGN38 and µ2 using the yeast two hybrid system, in vitro binding of recombinant fusion proteins and IAsys optical biosensor technology. A specific interaction has been demonstrated in each of the systems we have employed. We have shown an absolute requirement for Tyr-333 of TGN38 in binding to µ2. In addition we found that mutation of Ser-331 to alanine reduces the affinity of the interaction. By measuring tryptophan fluorescence at equilibrium, we have also determined the dissociation constant for the interaction between the entire cytosolic tail of TGN38 and µ2 as 58 nM. In contrast to previously published work, our data suggest that not only Tyr-333 but also its context is important in determining the specificity of binding of TGN38 to µ2.
Efficient cell surface internalization of many proteins is mediated by their sequestration into endocytic vesicles. The hallmark feature of the process is the localization of endocytic proteins to clathrin-coated pits at the plasma membrane and their subsequent internalization in coated vesicles. Formation of the coated vesicle depends on recruitment of the vesicle coat to the plasma membrane. The coat of clathrin-coated vesicles involves binding of adaptor complexes onto the membrane and their subsequent binding of soluble clathrin. The adaptor complexes are also believed to be involved in the selective concentration of cargo proteins into coated pits.
The high efficiency internalization of many different proteins at the
cell surface has been shown to be mediated through targeting signals
within the cytosolic domain. Many proteins including the transferrin
receptor, mannose-6-phosphate receptor, and TGN38 have tyrosine-based
internalization motifs conforming to the consensus, YXX,
where
represents a hydrophobic amino acid within their cytosolic
domains (1). TGN38 is a type I transmembrane glycoprotein that cycles
between the trans-Golgi network
(TGN)1 and the plasma membrane (2-6). The
tyrosine-based motif of TGN38 is located within the hexapeptide SDYQRL.
This sequence has been shown to be responsible for the high efficiency
internalization of the protein at the cell surface and its subsequent
targeting back to the trans-Golgi network (3-5). Various
roles have been proposed for TGN38 (7), but its precise function
remains to be elucidated.
The best characterized of the adaptor complexes are AP1 and AP2, which
bind to TGN membranes and the plasma membrane, respectively (8, 9).
Both are composed of two large (>100 kDa) adaptin subunits ( and
for AP1 and
and
for AP2), a medium chain (µ) subunit, and
a small (
) chain subunit. The
subunits of both AP1 and AP2
complexes have been shown to bind to clathrin (10, 11). Several
proteins are known to bind directly to the AP2 adaptor complex. For
example, epidermal growth factor receptors can be co-immunoprecipitated
with AP2 complexes in stoichiometric amounts (12, 13). Purified adaptor
complexes have also been shown to bind synthetic peptides corresponding
to regions within the cytosolic domains of the mannose-6-phosphate
receptor, low density lipoprotein receptor, and lysosomal acid
phosphatase, all of which have tyrosine-based sorting signals in their
cytosolic domains (14-16). It was recently shown that the
tyrosine-based internalization signal of TGN38, SDYQRL (Fig.
1), interacts with the medium chain subunit (µ2) of
the plasma membrane adaptor complex, AP2 (17) which suggests a role for
the µ subunits in mediating interaction with, at least some,
tyrosine-based targeting signals. The tyrosine motif of Lamp-1
(lysosomal associated membrane protein-1, GYQTI) has also recently been
shown to bind to both AP1 and AP2 in vitro (18).
The interaction between TGN38 and the µ2 subunit was initially detected by screening a yeast two hybrid library with a triplet repeat of the hexapeptide motif SDYQRL (17). The system was also used to show that the triplet repeat (SDYQRL)3 could interact with the µ1 subunit of AP1, the TGN adaptor complex. A single copy of the repeat sequence as well as the complete cytosolic domain of TGN38 were also shown to interact with µ2. The specificity of interaction was confirmed from binding assays using in vitro translated µ2 and a GST-(SDYQRL)3 fusion protein. The data obtained from these experiments showed an absolute requirement for the tyrosine residue of the motif, in agreement with previously published data (3-5). Binding could also be competed using a synthetic peptide corresponding to the same sequence.
We decided to examine the interaction between the complete cytosolic tail of TGN38 and µ2 in more detail. Apart from demonstrating an interaction between the two proteins in the two hybrid system, Ohno et al. (17) presented no further work using the entire TGN38 tail sequence. As well as confirming the importance of Tyr-333 of TGN38, we have examined the effect of mutating Ser-331 of the SDYQRL motif to alanine with regard to µ2 binding. This residue has previously been shown to be important for both internalization of TGN38 at the cell surface and targeting back to the TGN (5). We have used the two hybrid system, interaction of recombinant fusion proteins, and optical biosensor technology to study the interaction. In addition, the equilibrium dissociation constant for the interaction has been determined by fluorescence quenching. Our data support previous observations (17) with the exception that we show a clear role for the serine residue of the SDYQRL motif in determining the specificity of binding. This suggests that the context of the sequence and not only the presence of the tyrosine motif is important in determining molecular recognition by the adaptor complexes.
All reagents were purchased from Sigma (Poole, UK) unless otherwise stated. All plasmid manipulations were carried out according to standard methodologies (19).
Mutagenesis of TGN38
Mutations were introduced into TGN38 by PCR using primers
incorporating the mutated codons. Mutation S331A was initially
incorporated into the full TGN38 coding sequence, which was then used
as a PCR template to amplify the cytosolic domain. The mutation Y333A was incorporated into a construct lacking the four carboxyl-terminal residues of TGN38 that were added back during subsequent amplification of the cytosolic domain alone. Primer sequences were as follows: S331A, 5-AAGCTTTAGGTTCAAACGTTGGTAGTCAGCGGCCTTTGG-3
;
Y333A, 5
-CAAACGTTGGGCGTCAGCGGCCTTTGG-3
. Each primer was used in
conjunction with a sense TGN38 primer incorporating an
Asp718I site at the extreme 5
end of the TGN38 coding
region: 5
-GGTACCAGACTACAGGATGCAGTTCCTGG-3
. All oligonucleotides were
synthesized within the Biotechnology and Biological Sciences Research
Council funded Molecular Recognition Center of the University of
Bristol.
Plasmid Manipulation
All sequences were amplified for subcloning by 10 cycles of PCR using 2 units of Taq DNA polymerase (Boehringer Mannheim, Lewes, UK), 0.2 mM dNTPs, 1.5 mM MgCl2, and 5 pmol of each primer. PCR products were gel purified and subcloned via pGEM-T (Promega, Southampton, UK) to the target vector using standard methods (19). All sequences amplified by PCR were confirmed by automated DNA sequencing.
µ1 and µ2cDNAs encoding rat µ1 and µ2 were
kindly provided by Dr. M. S. Robinson (University of Cambridge, UK).
The full coding sequence of each was amplified by PCR and subcloned, in
frame, into (i) the two hybrid activation domain vector, pVP16 (20),
creating pVP16-µ1 and pVP16-µ2, respectively, and (ii) the
plasmid-based prokaryotic expression vector pET32a (Novagen, Abingdon,
UK) for bacterial expression. Amino-terminally truncated µ2
(corresponding to amino acids 121-435 of µ2, generating µ2N)
was also made and subcloned into pET32a.
Wild-type and mutant versions of the cytosolic tail of TGN38 were amplified by PCR and subcloned into (i) pGEX4T (Pharmacia Biotech Inc., St. Albans, UK) for expression as GST fusion proteins, (ii) pET32a (Novagen), for expression as thioredoxin fusion proteins, and (iii) the two hybrid DNA binding domain vector, pBTM116 (20).
Two Hybrid Analysis
Saccharomyces cerevisiae strain L40 (MATa his3D200
trp1-901 leu2-3, 112 ade2 LYS2::(lexAop)4-HIS3
URA3::(lexAop)8-lacZ gal4 gal80 (20)) was
co-transformed with plasmids to express TGN38 cytosolic tail variants
as LexA fusions and µ1 or µ2 as VP16 fusions, according to
previously published methods (21). Cells were also transformed with
pVP16-µ1/µ2 and pLexA-Lamin as a negative control for interaction.
Cells were then washed in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and plated onto selective medium (lacking Trp and
Leu). After 3-4 days, colonies were streaked onto selective medium
lacking Trp, Leu, Lys, and Ura and grown at 30 °C for a further 3 days. Colonies were then assayed for -galactosidase activity.
Colonies were lifted onto a Whatman 1 filter and frozen by submerging
in liquid nitrogen for 10 s. The filter was then thawed at room
temperature and layered over another filter, presoaked in Z buffer (60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0) containing 0.4 mg·ml
1 X-gal. Filters were then incubated overnight at
30 °C in humidified Petri dishes.
Expression of Recombinant Proteins
TGN38 cytosolic domains were expressed as GST fusion proteins in
Escherichia coli strain BL21DE3 and purified on
glutathione-agarose according to recommended protocols (Pharmacia).
Briefly, cultures were inoculated with 1% (v/v) of saturated overnight
culture, grown to A600 = 0.4 and induced for
1 h with 0.1 mM
isopropyl--D-thiogalactoside. Following sonication in 50 mM sodium phosphate, pH 7.4, supplemented with protease
inhibitors (1 µg·ml
1 leupeptin, 2 µg·ml
1 antipain, 10 µg·ml
1
benzamidine, 10 units·ml
1 aprotinin, 1 µg·ml
1 chymostatin, 1 µg·ml
1
pepstatin A), purification was achieved using glutathione-agarose with
washes in phosphate-buffered saline. Thioredoxin fusions were expressed
in an identical manner and purified using TalonTM purification resin
(CLONTECH) according to the manufacturer's instructions. All proteins were dialyzed into 50 mM sodium
phosphate, pH 7.4, and stored at
70 °C.
Full-length µ2 and µ2N were also expressed in BL21DE3 as
follows. Cultures were inoculated with 10% (v/v) overnight culture, grown for a further 2 h at 37 °C with shaking, and induced for 30 min with 1 mM
isopropyl-
-D-thiogalactoside. Cells were pelleted and
lysed by sonication; the insoluble pellet was washed twice in 100 mM sodium phosphate containing 1% (w/v) Triton X-100
before solubilization in 50 mM sodium phosphate containing
8 M urea and 10 mM dithiothreitol for 1 h
at room temperature. Proteins were refolded by stepwise dialysis into
lower concentrations of urea at 4 °C. Buffers for dialysis down to
0.5 M urea contained 2 mM dithiothreitol; for
lower urea concentrations (less than 0.5 M), dithiothreitol
was omitted and 10% glycerol was included. Finally, µ2
N and µ2
proteins were dialyzed into 50 mM sodium phosphate containing 10% glycerol and stored at
70 °C.
In Vitro Binding of Fusion Proteins
GST-TGN38 tail fusions (30 µg each) and Trx-µ2N (10 µg)
were incubated together for 1 h at room temperature in a total
volume of 0.2 ml of 50 mM sodium phosphate, pH 7.4, containing 10% glycerol and supplemented with protease inhibitors (5 µg·ml
1 leupeptin, 10 µg·ml
1
antipain, 50 µg·ml
1 benzamidine, 50 units·ml
1 aprotinin, 5 µg·ml
1
chymostatin, 5 µg·ml
1 pepstatin A). Complexes were
then immunoprecipitated for 1 h with a polyclonal antibody that
specifically recognizes thioredoxin that had been preadsorbed to
protein G-Sepharose (20 µl bed volume). Complexes were washed twice
in binding buffer and separated by SDS-polyacrylamide gel
electrophoresis on 12% gels and stained with Coomassie Blue R-250.
Band intensities were quantitated following scanning of individual gels
on a Apple Macintosh® computer using the public domain NIH Image
program (developed at the U. S. National Institutes of Health and
available on the Internet).2
IAsys Optical Biosensor Measurements
Experiments were performed using an IAsys resonant mirror
optical biosensor (Affinity Sensors, Cambridge, UK). Thioredoxin fusions of TGN38 cytosolic domains were immobilized on
carboxymethyl-dextran cuvette surfaces according to the manufacturer's
instructions. Binding experiments were performed in 50 mM
sodium phosphate, pH 7.4, containing 10% glycerol. µ2N was added
to a final concentration of 0.2 µM in a total volume of
200 µl, and changes in resonant angle were monitored at 1-s intervals
for approximately 300 s. Experiments were performed at 25 °C
with a stirrer speed of 100 rpm. For repeated measurements using the
same cuvette, surfaces were regenerated by washing for 2 min in 200 mM formic acid. For peptide competition experiments,
µ2
N was preincubated with 10 µM peptide
(CKASDYQRLNLKL) for 1 h at room temperature before addition to the
cuvette.
Fluorimetry
Fluorescence emission was monitored between 300 and 450 nm
following excitation at 290 nm using a Perkin-Elmer fluorimeter. A 2-ml
cuvette containing Trx-µ2N at 50 nM was used. 5-µl
aliquots of GST-TGN38 fusion were added sequentially (each addition
increasing the concentration of GST fusion in the cuvette by 30 nM), and fluorescence emission was monitored. A base line
of fluorescence from the buffer alone was subtracted from each data set
prior to analysis. Data were plotted and analyzed using the GraFit
package. Data were analyzed according to the following equations
describing the addition of B (GST-TGN38 fusion) to A (Trx-µ2
N),
and the equilibrium dissociation constant (Kd) was
calculated: Fluorescence, F = Ia·[A] + Ib·[B] + Iab·[AB], where I represents the
fluorescence intensity, [A] represents the concentration of unbound
A, [B] represents the concentration of unbound B, and [AB]
represents the concentration of AB complex; [A] = [A]o·(1
), [A:B] = [A]o·
, [B]o
[A:B], and
= (([B]o + [A]o + Kd)
([B]o + [A]o + Kd)2
4[A]o·[B]o)0.5)/2.[A]o,
where
represents the saturation of A with B, [A]o represents the total concentration of A, [B]o represents
the total concentration of B, and Kd represents the
equilibrium dissociation constant.
Two hybrid screening (22) of a mouse spleen cDNA library with a triplet sequence of the SDYQRL motif found within the cytosolic domain of TGN38 has recently led to the identification of the µ2 subunit of the AP2 adaptor complex as a binding partner (17). Further analyses confirmed the interaction using a single copy of the motif as well as the cytosolic domain of TGN38. We have used an alternative two hybrid system to test this interaction as well as to assess the strength of interaction with µ1 and µ2 of a number of TGN38 constructs mutated in the serine or tyrosine residues of the SDYQRL motif.
In frame fusions of TGN38 cytosolic domain sequences with LexA and
coding sequences of µ1 or µ2 with VP16 were generated and transformed into yeast strain L40. Fig. 2 shows the
result of a two hybrid -galactosidase assay to test for interaction
between the cytosolic domain of TGN38 and µ1 or µ2. No activation
of transcription was detected with any of the constructs individually
nor following co-transformation of yeast with pVP16-µ1 or pVP16-µ2
and with a negative control plasmid encoding a LexA-Lamin fusion. A
positive signal was seen following cotransformation of yeast with µ2
and the wild-type cytosolic tail of TGN38 after a 6-h incubation. No
interaction was observed between any of the other paired constructs even following incubation in the presence of X-gal for up to 24 h.
Mutation of Tyr-333 of TGN38 to Ala (Y333A) abolished the interaction, confirming the requirement for a tyrosine-based signal. Mutation of
Ser-331 to Ala (S331A) also abolished the interaction. This indicates a
requirement for Ser-331 in mediating a high affinity binding of TGN38
to µ2. No interaction was detected between any of the TGN38
constructs and µ1 (the medium chain of the TGN adaptor complex,
AP1).
Interaction of Fusion Proteins
To extend the data from the
two hybrid system, we have used an in vitro system involving
interaction of recombinant fusion proteins of the two binding partners.
The cytosolic domain of TGN38 (and mutants thereof) was expressed as a
GST fusion, and µ2 and µ2N were expressed as thioredoxin
fusions. Proteins were incubated together in solution followed by
immunoprecipitation using a thioredoxin-specific polyclonal antibody.
The strength of interaction was then assessed by Coomassie Blue
staining of polyacrylamide gels. The data of Ohno et al.
(17) suggested that µ2 truncated in the first 120 amino acids bound
with equal efficiency to TGN38. We too observed equivalent binding of
full-length and truncated µ2 to the cytosolic tail of TGN38. No
in vitro data could be obtained for binding of µ1 because
soluble protein could not be generated in sufficient amounts. The
results of these experiments showed, in agreement with the two hybrid
data, that the wild-type cytosolic domain of TGN38 interacts with
µ2
N (Fig. 3A, lane 2). Binding was considerably reduced by mutation of either the tyrosine or
serine residues of the SDYQRL motif to alanine (Fig. 3A,
lanes 3 and 4, respectively). Quantitation of
band intensities using the NIH Image package (Fig. 3B)
showed that there was more than 2-fold greater binding of µ2
N to
the wild-type TGN38 fusion protein compared with either S331A or Y333A
mutants. A possible explanation for the observed binding of both the
S331A and Y333A mutants above that of GST alone is that the GST fusion
proteins used in this part of the study may have the potential to
dimerize (23). Multimeric forms of the cytosolic domain of TGN38 may
mediate a different interaction with µ2 than the LexA and thioredoxin
fusions used for the other analyses described here. These results,
however, do support the observation from the two hybrid analysis that
the S331A mutation disrupts the interaction with µ2
N.
IAsys Optical Biosensor Measurements
The IAsys resonant
mirror optical biosensor was used to analyze the interaction between
the two proteins in real time. TGN38-thioredoxin fusion proteins were
immobilized onto carboxy-methyl dextran cuvettes and binding of
µ2N determined. The thioredoxin fusion of TGN38 was used for this
part of the study due to the increased stability of these constructs
over GST fusions. All TGN38 constructs as well as the thioredoxin
domain alone were immobilized at a concentration of 8 ng·mm2 protein. Change in resonant angle was monitored
continuously over a 5-min period following the addition of µ2
N to
a final concentration of 200 nM in 50 mM sodium
phosphate, pH 7.4, containing 10% glycerol. Binding curves for these
experiments are shown in Fig. 4A. The data
are consistent with those obtained from the two previous approaches. As
anticipated, µ2
N did not bind to Trx-TGN38 fusions in which the
tyrosine residue within the SDYQRL motif had been mutated to alanine. A
significant reduction in binding was observed for the of Ser-331 to
alanine mutant (S331A, Fig. 4A, open squares). No
significant binding to thioredoxin alone was observed. Binding curves
were replicated in each case following regeneration of the cuvette
surface with 200 mM formic acid. Fig. 4B shows
that this treatment did not reduce the bioactivity of the surface with
regard to µ2
N binding (wt 1 and wt 2 in Fig. 4B).
Binding of µ2N was competed by preincubation with a 50-fold molar
excess of synthetic peptide corresponding to part of the cytosolic tail
of TGN38 overlapping the SDYQRL motif. Preincubation of µ2
N with
the peptide followed by subsequent addition to the biosensor cuvette
reduced binding significantly (Fig. 4B, wt + pep). Binding of µ2
N was repeated following regeneration of
the surface as before to remove the peptide and any bound µ2
N. In this case a reduction in binding was observed (wt 3 in Fig.
4B). The level of binding was still above that seen in the
presence of competing peptide but less than that seen initially. The
most plausible explanation for this is that the peptide was not
completely removed from the dextran matrix by formic acid treatment.
The data obtained from the optical biosensor are in complete agreement with that obtained from the other approaches but also show that the
effect of the S331A mutation is not as significant as that of
Y333A.
Equilibrium measurement of
tryptophan fluorescence was used to determine the dissociation constant
for the interaction between TGN38 and µ2N. Sequential addition of
GST-TGN38 to Trx-µ2
N would lead to linear increase in fluorescence
due to the four tryptophan residues of GST-TGN38. Deviation from the
linearity of this increase indicates an interaction between the two
proteins. Change in fluorescence was plotted against concentration of
GST-TGN38 and then subtracted from the linear increase due to
sequential addition of TGN38. The resulting fluorescence quench data
were curve fitted to the equation shown under "Experimental
Procedures" from which the equilibrium dissociation constant
(Kd) was calculated (Fig. 5). Using
this method the Kd for the interaction between
wild-type TGN38 tail and µ2
N was calculated as 58 nM (standard error ± 7). The Kd for the
interaction of the S331A fusion with µ2
N was similarly determined
as 103 nM (standard error ± 8). The dissociation
constant for the binding of the Y333A mutant was found to be 755 nM (standard error ± 100). These data are in entirely
consistent with those obtained from the other approaches described
here. In particular, the lower affinity of the S331A mutant compared
with the wild-type protein for µ2
N reflects the binding curve
obtained using the IAsys optical biosensor, suggesting that the
mutation S331A does indeed affect the binding of the cytosolic domain
of TGN38 to µ2
N.
We have used a number of complementary approaches to study the interaction of a tyrosine-based internalization signal with the medium chain component, µ2, of the adaptor complex, AP2. We have studied the internalization signal of TGN38, SDYQRL, which in isolation has previously been shown to interact with the µ2 protein in a two hybrid system and in an in vitro binding assay (17). The context of critical tyrosine residues within internalization signals is almost certainly important for specificity, for example, the spacing of the tyrosine motif of lamp-1 from the membrane is critical for interaction with the TGN adaptor complex, AP1 (24). The triplet repeats used by Ohno et al. (17) would obviously also be very different in structure to the entire cytosolic domain of TGN38, which has been shown by NMR to form a nascent helix in solution (25). For this reason, we chose to study the interaction in more detail using the entire cytosolic domain of TGN38.
Initial experiments exploited the yeast two hybrid system (22) to study the interaction. Interaction was detected between µ2 and the wild-type TGN38 cytosolic domain but not following mutation of the tyrosine or serine residues of the SDYQRL motif to alanine. No interaction was detected between the cytosolic tail of TGN38 and µ1. This is in conflict with the results of Ohno et al. (17), who readily detected an interaction between a triplet repeat of SDYQRL and µ1. This suggests that the interaction, if any, between the entire cytosolic tail of TGN38 and µ1 is too weak to be detected using the sensitive two hybrid system we employed. It can be inferred from this that the context of the critical tyrosine residue is an important determinant of adaptor binding. Indeed, recent surface plasmon resonance data from the same group showed that the cytosolic domain of TGN38 does bind to AP1 with lower affinity than AP2 (26) in agreement with our two hybrid data. Similarly, the endocytic signal of the epidermal growth factor receptor has been shown to bind to AP1 with lower affinity than to AP2 (27).
The two hybrid experiments reported here also show an importance for
Ser-331 of the SDYQRL motif in determining binding specificity. This
result complements the earlier biological observation that mutation of
serine-331 plays a role in determining the intracellular localization
of TGN38 (5). Ohno et al. demonstrated that mutation of this
residue to alanine did not affect binding of an isolated SDYQRL motif
to µ2 when assayed for growth on histidine-deficient medium (17).
These growth assays are not quantitative, and so it is difficult to
determine the relative importance of this mutation. In our hands the
same mutation, in the context of the entire cytosolic tail sequence,
completely abolished interaction with µ2 as evidenced by lack of
activation of transcription of the -galactosidase reporter gene.
More recently, two hybrid screening of a combinatorial cDNA library
of constructs ending AAYXXX (26) also failed to show any
role for Ser-331 of TGN38 in mediating binding to µ2. Again, this
discrepancy with our data is most easily explained by the context of
the tyrosine motif. Our constructs have the additional four amino acids
(NLKL) of TGN38 present, whereas those used for screening of the
combinatorial library did not. Thus, although the tetrapeptide sequence
of YXX
may be sufficient for directing binding to µ2,
it is clear that the exact sequence context of these motifs affects the
affinity of binding and therefore presumably the efficiency of sorting
into endocytic vesicles.
To complement the two hybrid data, we investigated the interaction
between recombinant fusion proteins of the two binding partners. Fusion
proteins were incubated together, and complexes were immunoprecipitated
and analyzed by SDS-polyacrylamide gel electrophoresis. Using this
approach we were able to detect binding of the wild-type cytosolic
domain of TGN38 to µ2N (a µ2 construct deleted in the first 120 amino acids, a modification that has been shown not to alter binding to
the cytosolic domain of TGN38; Ref. 17). In addition, this result
confirmed the observation apparent from our two hybrid results that
both Ser-331 and Tyr-333 of the cytosolic domain of TGN38 are important
in mediating a high affinity interaction with µ2
N.
In addition to the above approaches, we have utilized the IAsys
resonant mirror optical biosensor to investigate the interaction between TGN38 and µ2N. The biosensor measures changes in resonant angle that occur on binding of, in this case, µ2
N to TGN38
cytosolic domains immobilized on a cuvette surface in real time. No
binding of the thioredoxin fusion domain alone was observed, indicating that any interaction observed is due to the µ2
N domain. We were able to confirm the absolute requirement of Tyr-333 in the interaction because mutation to alanine (Y333A) abolished the interaction. In
addition, binding was also competable using a synthetic peptide containing the SDYQRL motif. The inhibition of binding was significant but not complete, probably due to insufficiently high peptide concentrations (10 µM). Indeed Heilker et al.
(28) found that 20 µM peptide did not significantly
affect binding of internalization motifs to adaptor complexes as
assessed by surface plasmon resonance. Very high concentrations of
competing peptide were also required for competition of binding of
lamp-1 to adaptor complexes (18). This may be a feature of
the different affinities of the free peptide and immobilized fusion
protein for µ2 resulting from the context or presentation of the
tyrosine motif. The peptide used in this study had only four residues
upstream of the SDYQRL motif, and it is possible that this greatly
reduced the affinity of interaction with µ2 compared with the entire
cytosolic domain of TGN38. The fact that binding was at least to some
extent competable suggests specificity of interaction.
Due to the exquisite sensitivity of the system, the IAsys experiments showed an effect of Ser-331 in determining efficiency of binding not apparent from the two hybrid analyses or immunoprecipitation. Mutation of this residue to alanine (S331A) significantly reduced the amount of binding detected as well as the initial rate of the interaction but unlike the Y333A mutation caused only a partial disruption of binding. This suggests that in the context of the complete TGN38 tail sequence, this upstream residue is important in determining the affinity of interaction between TGN38 and the adaptor complex and is consistent with previously published data implicating Ser-331 in internalization and return of TGN38 to the TGN (5). Furthermore, overexpression of S331A TGN38 in COS-7 cells does lead to enhanced expression of TGN38 at the cell surface compared with similar levels of expression of wild-type TGN38.3
To determine the equilibrium dissociation constant for the interaction
between TGN38 and µ2, we studied quenching of tryptophan fluorescence
on binding. On addition of TGN38 fusion proteins, a nonlinear increase
of fluorescence was observed, and from this dissociation constants for
the interactions were calculated. Strong binding of the wild-type
cytosolic domain of TGN38 to µ2N was observed
(Kd = 58 nM) with somewhat weaker
binding for the S331A mutant (Kd = 103 nM). Binding of the Y333A mutant was significantly lower
(Kd>700 nM). Interaction was barely
detectable with this mutant as evidenced by the low confidence limits
of the calculated dissociation constant (standard error = ±100
nM). This quantitative approach confirms the observations from the other techniques described here. The data confirm that the
tyrosine residue within the SDYQRL motif of TGN38 is essential for
binding to the µ2 chain of the AP2 complex and in addition indicate
that the serine residue within this motif is also important in
mediating high affinity binding to the complex.
Our data are somewhat in conflict with previously published results
(17) because they indicate an important role for residues other than
the tyrosine and bulky hydrophobic residue of the YXX consensus in determining specificity of binding of tyrosine-based targeting motifs to adaptors. This additional specificity may have a
role in determining the subcellular compartment to which proteins are
ultimately targeted. This may be of particular relevance to TGN38 given
the demonstration of a role for Ser-331 in return of the protein from
the cell surface to the TGN (5). The greater affinity of interaction
shown here between TGN38 and µ2 compared with µ1 might account for
the observed traffic of TGN38 to the cell surface and its subsequent
internalization as opposed to µ1-mediated transport direct from the
TGN to lysosomal compartments. Similar differences in binding affinity
of TGN38 to adaptor complexes at the level of the sorting endosome
could mediate transport to the TGN as opposed to entering a recycling
pathway such as that seen for the transferrin receptor.
In summary, we have shown using three complementary approaches that the
complete cytosolic tail of TGN38 interacts with the µ2 subunit of the
AP2 adaptor complex. The interaction has been shown to occur in free
solution, intracellularly (using the two hybrid system) and also by
recruitment of µ2N to immobilized TCN38 tails (IAsys
measurements). The equilibrium dissociation constant
(Kd) for the interaction is 58 nM. The
interaction has been shown to be dependant not only on the tyrosine
residue of the internalization motif of TGN38 but also on the serine
residue upstream of this YXX
motif.
We thank Dr. M. S. Robinson (University of Cambridge) for providing cDNAs of µ1 and µ2, Drs. S. Hollenberg, P. Bartel, and S. Fields for the two hybrid vectors and yeast strain, Dr. M. Dickens (University of Bristol) for help and advice on two hybrid system, Drs. S. Mayes and P. Buckle of Affinity Sensors for loaning and providing excellent technical assistance for the IAsys system, and Drs E. P. Roquemore and B. J. Reaves for the TGN38 mutant constructs.