Receptor induced polymerization of coatomer
Constanze Reinhard*‡, Marcus Weidler†‡, Cordula Harter*, Martina Bremser*, Kai Sohn*, J.
Bernd Helms*, Paul Rösch†, and Felix Wieland*§)
*
Biochemie-Zentrum Heidelberg (BZH), Ruprecht-Karls-Universität Heidelberg
†
Institut für Struktur und Chemie der Biopolymere, Universität Bayreuth
‡
contributed equally to this publication
§
) Corresponding author:
Prof. Dr. Felix Wieland
Biochemie-Zentrum Heidelberg (BZH)
Ruprecht-Karls-Universität Heidelberg
Im Neuenheimer Feld 328
D-69120 Heidelberg
Tel.:
+49-6221-544150
Fax.: +49-6221-544366
E-mail:
felix.wieland@urz.uni-heidelberg.de
1
ABSTRACT
Coatomer, the coat protein complex of COPI vesicles, is involved in the budding of these
vesicles, but the underlying mechanism is unknown. Towards a better understanding of this
process, the interaction between coatomer and the cytoplasmic domain of a major
transmembrane protein of COPI vesicles, p23, was studied. Peptides analogous to the
cytoplasmic domain of p23 form -helices and adopt a tetrameric state as determined by
nuclear magnetic resonance spectroscopy. Interaction of coatomer with this tetramer results in
a conformational change and polymerization of the complex in vitro. This changed
conformation is also observed in vivo, i.e. on the surface of authentic, isolated COPI vesicles.
Based on these results we propose a mechanism by which the induced conformational change
of coatomer results in its polymerization, and thus drives formation of the bud on the Golgi
membrane during biogenesis of a COPI vesicle.
2
COPI-coated vesicles mediate protein transport in the early secretory pathway (1-3). The
COPI coat consists of a small G protein, ADP-ribosylation factor 1 (ARF (4, 5) and
coatomer, a hetero-oligomeric protein complex of seven subunits (- COPs, for coat
proteins) (6-8). COPI bud formation is initiated by membrane recruitment of ARF1, which in
its GTP-bound form (9, 10), together with members of the p24 family, provides membrane
binding sites for coatomer (11, 12). Subsequent coat assembly leads to membrane deformation
and the morphological appearance of a bud (13). To gain insight into the mechanism
underlying this process, we investigated the interaction with coatomer of p23 (14), a member
of the p24 family. p23, a type I transmembrane protein, is highly enriched in COPI vesicles
and is present in a ratio to coatomer of approximately 4:1 (14). The cytoplasmic domain of
p23 (YLRRFFKAKKLIE) is structurally similar to a classical dilysine motif (KKXX) for
retrieval to the endoplasmic reticulum (ER) (15-18), and binds coatomer with the same
efficiency as the KKXX motif (14). p23 binding, however, depends on its phenylalanine
residues as well as its lysine residues.
The essential components needed for the biogenesis of additional coated vesicles are defined
as well. Clathrin coated (19) and COPII-coated vesicles (20) have been generated in vitro
from defined constituents. Most recently, a conformational change was shown upon binding
of GTP to Dynamin (21), and this structural change was correlated to the process of pinching
off a coated vesicular intermediate. However, two questions remain: (i) how are coat proteins
normally bound and (ii) how does this association lead to a mechanical deformation of a
membrane.
We describe here a conformational change of the COPI-coat protein complex, coatomer,
induced by binding to the cytoplasmic domain of a major transmembrane protein of COPI
vesicles, p23. This conformational change in vitro leads to polymerization of the coat
complex. This changed conformation of coatomer is also observed in vivo, i.e. on the surface
3
of an authentic isolated COPI vesicle. NMR-studies show that the small cytoplasmic tail
domains of p23 adopt a tetrameric state, and offer a model as to how the amino acid residues
essential for binding coatomer are positioned within the tetramer on the surface of the
membrane. We propose that the induced conformational change drives polymerization and
subsequent deformation of the Golgi membrane during biogenesis of a COPI vesicle.
4
MATERIALS AND METHODS
Coatomer was isolated as described in (22). Purified COPI vesicles were prepared as
described in (23) (vesicles were stored in 25 mM HEPES-KOH pH 7.4, 250 mM KCl, 2.5
mM Mg-Acetate, 40% sucrose at -80°C). For Western blots shown in this study, an anti-COP antibody was used which was generated against the recombinant protein (24).
Synthetic Peptides. The sequences of the synthetic peptides are shown in Fig. 1A. Dimers
were formed by disulfide bridges linking the peptides via N-terminally introduced cysteine
residues. For this purpose, newly synthesized monomeric peptides were oxidized in 20 %
dimethyl sulfoxide in water for 48 h. Subsequently the dimers were isolated by high pressure
liquid chromatography.
Precipitation of Coatomer. Pure soluble coatomer (0.09 µM in buffer 1 (25 mM HEPESKOH pH 7.4 and 100 mM KCl)) was incubated with increasing concentrations of peptides
(monomeric peptides: 100-1000 µM, dimeric peptides: 10-100 µM) for 1 h at room
temperature (RT). Precipitates were pelleted by centrifugation at 40.000 g for 15 min at 4°C.
Pellets and supernatants were analyzed by SDS-PAGE and immunoblotting using anti-COP
antibodies. Quantification of precipitated and soluble coatomer was performed by scanning in
an linear range the -COP signal detected by enhanced chemiluminescence (ECL, Amersham).
Limited Proteolysis of Coatomer and COPI Vesicles. For experiments with isolated
coatomer, pure, soluble protein complex (0.09 µM in buffer 1) was incubated either with 50
µM p23wt-d, p23AS-d or Wbp1p-d under the conditions as described above. Thereafter,
5
thermolysin was added to a final concentration of 0.008 µM and digestion performed for the
times indicated. Proteolysis was stopped with EDTA (final concentration 40 mM). Proteolytic
fragments were analyzed on 7.5-15% gradient SDS-gels and immunoblotting.
For experiments with COPI vesicles, 25 µl of a suspension of purified COPI vesicles
containing  0.5 µg coatomer (final concentration  0.009 µM) were diluted to a final
concentration of 10% sucrose and incubated with 50 µM p23AS-d in a total volume of 100 µl
for 30 min at RT. Thereafter, thermolysin was added to a final concentration of 0.008 µM,
digestion was performed for 0-2 hours and proteolysis was stopped with EDTA (final
concentration 40 mM). Protein was precipitated with chloroform/methanol, and separated on
7.5-15% gradient SDS-gels under reducing conditions. COPs were analyzed by
immunoblotting. Coatomer (0,5 µg, 0.009 µM) was incubated under identical conditions
either with 50 µM p23wt-d or p23AS-d in total volume of 100 µl (25 mM HEPES-KOH pH
7.4, 250 mM KCl, 2.5 mM Mg-Acetate, 10% sucrose), and digested and analyzed as described
for COPI vesicles.
Determination of the Stoichiometry between Precipitated Coatomer and p23 Peptide.
p23wt-d and p23AS-d (10 nmol in 10 µl of water) were labelled with 125Iiodine (1 mCi)
using IODO-GEN iodination reagent (10 µg, Pierce) according to manufactures’ instructions.
Iodide was removed by chromatography of soluble peptides on SEP-PAK C18 cartridges
(Millipore) and washing with 10 ml of water. Peptides were eluted from the columns with
75% acetonitrile, 0.1% trifluoracetic acid in water. Upon addition of 0.5% (v/v) pyridin the
solvents were evaporated in a Speed Vac. The peptides were dissolved in water and stored at 4
°C. Specific radioactivity for p23wt-d was 3-5104 cpm/pmol and for p23AS-d 4-6104
cpm/pmol. The iodinated peptides were used within 4 weeks. 500 pmol p23wt-d containing
8105-1106 cpm in a total volume of 20 µl of buffer 1 (25 µM p23wt-d, specific activity
6
between 1,6 and 2103 cpm/pmol) was incubated with increasing concentrations of coatomer
(3.6-7.2 pmol) for 30 min at RT. Precipitates were pelleted by centrifugation at 40.000 g for
15 min, 4°C. Pellets were washed three times with buffer 1 containing 500 pmol non
radioactive p23wt-d and centrifuged at 40.000 g for 15 min at 4°C. Thereafter, the amount of
precipitated peptide was analyzed by counting radioactivity of the pellets (signals detected
were in the range of 1-3104 cpm above background). Precipitation of coatomer was
determined by analyzing pellets and supernatants on 7.5% SDS-gels and immunoblotting. The
same experiments were performed with 125Ip23AS-d.
Nuclear Magnetic Resonance Spectroscopy of p23wt-m and p23wt-d. The solution
structures of p23wt-m and p23wt-d were determined by 2D 1H-NMR spectroscopy (4.0 mM
peptide (p23wt-d) or 7.0 mM peptide (p23wt-m), pH 3.6 in 9:1 H2O:D2O, 8:2 H2O:d2-TFE
(v/v), 7:3 H2O:d2-TFE, or 6:4 H2O:d2-TFE, 100 mM potassium phosphate buffer, 50 mM
NaCl, 280 K). Complete sequence-specific assignments of backbone and side-chain protons
were obtained by total correlation spectroscopy (TOCSY) with 80 ms mixing time (25, 26),
correlation spectroscopy (COSY) (27), and NOE spectroscopy (NOESY) (mixing times 150
ms and 300 ms) (28). All spectra were acquired on a Bruker DRX600 spectrometer using
standard pulse sequences (29). Saturation of the water signal was accomplished by
presaturation during the relaxation delay. 4096  512 data points were collected with a
spectral width of 6024 Hz in both dimensions. All two-dimensional spectra were multiplied
by a squared sinebell function phase shifted /2, /4 or /8. Base line and phase correction to
6th order was used. In addition to the standard Bruker spectrometer control software, the
NDEE 2.0 software package (Software Symbiose, Inc., Bayreuth, Germany) was used for data
processing.
7
Restrained And Unrestrained Molecular Dynamics calculations.
Simulated annealing and refinement calculations (34) were performed on DEC Alpha
workstations with X-PLOR V3.840 (35) using standard protocols. The 3JN coupling constants
were determined by cross-peak measurements in a COSY spectrum. The NOE intensities were
classified as strong, medium, and weak corresponding to proton-proton distances of 1.8 - 2.7
Å, 1.8 - 4.0 Å, and 1.8 - 5.5 Å, respectively. Stereospecific assignments for the methyl,
methylene, and aromatic protons have not been performed, and appropriate corrections were
added for constraints including pseudoatoms (30-32). Torsion angles were derived from 3JN
coupling constants (33). An interval of 30° around the experimental value was allowed.
Frequency degenerated cross-peaks were incorporated into the structure calculations as
‘ambiguous’ in order to extract as much structural information as possible from the NOESY
spectrum (35). Subsequently, the proton-proton distances in the calculated structures were
determined using the program ‘BackCalc_db 2.0’ (Software Symbiose, Inc., Bayreuth,
Germany) and compared with the combinations of distances possible for each frequency
degenerated NOESY cross-peak. If only one set of the possible distance combinations was
fulfilled in more than 50% of the calculated structures, the distance information was used in
further structure calculations. This procedure was repeated several times.
For the unrestrained molecular dynamics simulation (MD) a water/TFE box consisting of
3392 water and 768 TFE molecules with a spatial extension of 6.2 x 6.2 x 6.2 nm was
generated (36, 37). The structure with the lowest energy was chosen as the starting structure
for the MD calculations that were performed according to published procedures (36-40).
Size Exclusion Chromatography of Monomeric and Dimeric p23wt. Size exclusion
chromatography was performed on a SMART System (Pharmacia Biotech) using the
8
Superdex Peptide PC 3.2/30. The column was equilibrated with buffer 1 at a flow rate of 40
µl/min at 8°C. Peptides were dissolved in buffer 1 at a concentration of 1 mg/ml (Fig. 7A, B)
or 0.1 mg/ml (Fig. 7D) and injected as indicated in the figure. Chromatography of the peptides
was performed at a flow rate of 20 µl/min at 8°C and fractions of 40 µl were collected.
9
RESULTS
p23-tail Peptide Induced Polymerization of Coatomer. To study the interaction of p23 with
isolated coatomer, we incubated the complex with a synthetic peptide analogous to the
cytoplasmic domain of p23 (p23wt-m, Fig. 1A), and after centrifugation observed aggregation
of coatomer. At a peptide concentration of about 750 µM, half of the soluble coatomer is
precipitated (Fig. 1B). Based on the  4:1 stoichiometry between p23 and coatomer in COPI
vesicles, we reasoned that the active form of p23 might be an oligomer. Indeed, a dimeric
form of p23wt-m, p23wt-d (Fig. 1A), precipitates 50 % of coatomer at about one hundredth
the concentration of p23wt-m (Fig. 1C). Unrelated proteins like bovine serum albumin and
phosphorylase are not precipitated under identical conditions. Furthermore, a peptide, p23AS
(Fig. 1A), that lacks the amino acid residues known to be essential for binding coatomer (14),
does not precipitate coatomer in considerable amounts, neither in its monomeric nor its
dimeric form (Fig. 1B, C). A peptide analogous to the cytoplasmic domain of Wbp1p (41), an
ER-resident transmembrane protein which is not part of the transport machinery but rather
cargo for retrieval (17), binds coatomer with the same efficiency as the p23-tail peptide.
However, both the monomeric and the dimeric form of this peptide (Fig. 1A), did not cause
significant precipitation of the complex (Fig. 1B, C).
Limited Proteolysis of Coatomer. The aggregation of coatomer observed with the p23wt
peptides is likely to involve a conformational change of the complex. To probe such a
conformational change by limited proteolysis, coatomer was incubated with thermolysin in the
presence of the various peptides used in the precipitation. Since -COP has been identified as
a binding partner of both, ER-retrieval motif (24) and the cytoplasmic domain of p23 (42),
10
proteolysis of this subunit was monitored. -COP is partially cleaved to yield a prominent
fragment of about 59 kD (Fig. 2, A-C, 1 h). In the presence of p23 wt-d this 59 kD fragment is
proteolytically degraded into various fragments (Fig. 2A, 2 and 3 h). In contrast, in the
presence of p23AS-d, the 59 kD fragment is clearly more stable (Fig. 2B, 2 and 3 h). Even
after 3 h in the presence of the mutated peptide only one additional fragment of about 52 kD is
observed. Partial digestion in the presence of Wbp1p-d resulted in a pattern very similar to
that with p23AS-d (Fig. 2C). The increased susceptibility to protease of -COP together with
polymerization of coatomer specifically induced by p23wt-d demonstrate a conformational
change of coatomer upon binding to the peptide.
The increased susceptibility to protease of coatomer observed in its aggregated state in vitro
led us to investigate whether this conformational change also occurs under conditions that
reflect an in vivo situation, i.e. on the surface of an isolated COPI vesicle. To this end, purified
COPI vesicles (23) were submitted to limited proteolysis with thermolysin. The result is
shown in Fig. 3. Strikingly, although coatomer is densely packed on the surface of the vesicle,
the -COP 59 kD- domain is highly susceptible to the protease (Fig. 3A), similar to the
precipitated state of coatomer (Fig. 3B), and different to soluble coatomer, where the -COP
domain again is much more stable (Fig. 3C) (Note that the conditions of proteolysis for
technical reasons were different in the experiments shown in Figs. 2 and 3). These results
strongly indicate a physiological role of the observed conformational change in the biogenesis
of a COPI vesicle.
11
Stoichiometry of p23wt-d and Coatomer. A stoichiometry of p23 to coatomer of about 4 to
1 has been determined in isolated COPI vesicles (14). If the observed conformational change
of coatomer with subsequent precipitation reflected a physiological mechanism, then a similar
stoichiometry would be expected in the precipitate between the p23 tail peptide and coatomer.
To determine the stoichiometry in the precipitates, various amounts of coatomer were
incubated with a precipitating amount of
125
I-labelled dimerized p23wt peptide, and after
centrifugation, the amount of peptide in the precipitates was determined by counting their 125Iradioactivity. The result is shown in Fig. 4. A linear increase of radioactivity is observed with
increasing amounts of coatomer, and the stoichiometry calculated from the specific
radioactivity of the dimerized peptide and coatomer (100% of the input coatomer was
precipitated at each concentration) can be determined from the resulting slope. From an
average of four experiments, 2.2 moles of dimerized p23wt peptide bind to 1 mole of
coatomer under precipitating conditions. This is in good agreement with approximately 4
molecules of p23 bound to 1 coatomer in a COP I vesicle. As a control, 125I-labelled p23AS-d
peptide did not lead to amounts of radioactivity precipitated above background (Fig. 4).
Structure of the Cytoplasmic Domain of p23. The strikingly higher efficiency of p23wt-d to
aggregate coatomer might imply that the dimeric peptide adopts a distinct and defined
structure in solution. Therefore, a complete structural analysis of p23wt-m and p23wt-d by
NMR techniques was performed in aqueous buffer solution with addition of various amounts
of trifluoroethanol (TFE) (43). According to sequential short-range dNN(i,i+1) NOEs, mediumrange d(i,i+3), d(i,i+3), and d(i,i+4) NOEs (Fig. 5A, B), both, p23wt-m and p23wt-d
form -helices from L2 to I12, fringing at Y1 and E13. The four residues involved in
coatomer binding, F5, F6, and K9, K10 (14), are located at the same side of the helix (Fig. 6B,
C). Remarkably, this helix clearly shows amphipathic character, its hydrophobic face
12
composed of residues Y1, F5, A8, and I12. Amphipathicity is increased by a salt bridge
between K9 and E13, stable in molecular dynamics simulations. The root-mean-square
deviation (RMSD) for the backbone heavy atoms of the 10 structures with lowest internal
energies of p23wt-m is 0.17 Å (Fig. 6A) (table 1). The structure of p23wt-d, calculated under
the assumption of a monomeric state of the dimerized peptide, could not satisfactorily explain
15 long-range NOEs between I12 and Y1 on one hand and I12 and F5 on the other hand. This
implies oligomerization of p23wt-d at least to a p23wt-d dimer, analogous to a tetrameric
form of the p23 cytoplasmic domain. No indication for an oligomerization state of the
dimerized peptide higher than a dimer, corresponding to four -helices, could be observed.
In order to analyze this dimerization of p23wt-d biochemically, size exclusion
chromatography was performed at pH 7.4 without the addition of TFE, and under the
conditions used for NMR spectroscopy. p23wt-m and p23wt-d reduced by dithiothreitol elute
in a single peak (Fig. 7A). p23wt-d, however, gives rise to two peaks (Fig. 7B): Peak 2 elutes
with the same retention time as p23wt-m (Fig. 7A), indicating very similar Stokes’ radii of the
two species, whereas peak 1, judged from its retention time, represents a p23wt-d dimer.
Accordingly, rechromatography of the fraction corresponding to peak 1 in Fig. 7B results in
an elution profile similar to the p23wt-d sample applied in Fig. 7B. However, a different ratio
of peak sizes is observed with peak 2 enlarged at the expense of peak 1 (Fig. 7C), indicating
an equilibrium of the two states of the peptide. This equilibrium depends on the concentration
of the peptide (Fig. 7D), revealing its bimolecular nature. Thus, peak 1 is caused by the
dimeric, and peak 2 by the monomeric state of p23wt-d. Size exclusion chromatography
clearly confirms a dimeric state of p23wt-d even in the absence of TFE (Experiments carried
out under the conditions used for NMR spectroscopy yielded identical results).
13
Thus, the following criteria must be met in order to delineate the solution structure of p23wtd: (i) according to gel filtration the dimerized peptide forms a dimer, i.e. a quaternary structure
containing four monomers. (ii) amino acids with identical sequence positions were
magnetically equivalent, suggesting identical conformations for the monomers and a highly
symmetrical quaternary structure. (iii) long range NOEs observed between residues I12 and
between Y1 and I12 and F5, restrict the distance between these residues to less than 5 Å
suggesting an antiparallel arrangement of two dimerized peptides (Fig. 6C).
The overall structure of the p23wt-d dimer is bipartite (Fig. 6C). The -helices are well
defined, and a hydrophobic core is formed through interactions of the side chains of residues
F5, A8, L11, and I12 of the two dimers. A halfcircle is formed by residues K9 and K10 of two
antiparallel helices (Fig. 6C) separating the corresponding diphenylalanine motifs of the two
antiparallel -helices (Fig. 6C). This quaternary structure "FKF motif" is present twice in the
tetrameric arrangement. Thus, p23wt-d dimer comprises a tetramer of the cytoplasmic domain
of p23. Likewise, the stoichiometry found for both p23 and coatomer in isolated COPI
vesicles and in coatomer precipitated by p23 wt-d was approximately 4:1. Thus, we propose
that this tetramer is the species active in coatomer binding in vivo (Fig. 8).
14
DISCUSSION
We have shown here that interaction of coatomer with the cytoplasmic domain of p23 leads to
a conformational change in its -subunit and concomitant precipitation of coatomer. This
conformational change is specific, in that a Wbp1p peptide which also binds coatomer via it's
ER-retrieval motif (24) failed to induce this conformational change. The very same
conformation of -COP is found in coatomer under conditions that reflect an in vivo situation,
namely on the surface of authentic, Golgi-derived COPI vesicles. The structure of p23 that
triggers the conformational change is a tetramer of four equivalent -helical domains, as
judged from nuclear magnetic resonance spectroscopy and molecular dynamics simulations. A
tetrameric form of this domain is confirmed by size exclusion chromatography and
determination of its stoichiometry in the precipitated coatomer complex. The structure arising
from tetramerization of the p23-tail domain represents a symmetrical molecule of two
equivalent half's, two "FKF"-motifs (Fig. 6 C). We figure that one such motif is directed
towards the surface of the Golgi membrane and may well be stabilized by interactions with
membrane lipid headgroups, and the other motif protrudes from the membrane and serves as a
"plug" for coatomer.
In vitro precipitation of coatomer was recently observed in the presence of some bivalent
aminoglycoside antibiotics (44), and interpreted in terms of aggregation of coatomer by
crosslinking. In light of the data described here these aminoglycosides may well fit into the
binding site for the cytoplasmic domain of p23 that resides in the -subunit of the complex
(42) and induce its conformational change.
15
It is of note that a Wbp1p-peptide with a characteristic ER-retrieval motif does bind coatomer
but, in contrast to the cytoplasmic domain of p23, does not trigger a conformational change of
the complex. Thus, the two classes of domains have distinct and different functions:
interaction of the Wbp1p-type of peptide might serve the sorting of cargo to be retrieved to the
ER into retrograde COPI vesicles. p23, however, may represent part of the machinery of a
COPI vesicle. Not only does it bind to coatomer during the budding reaction, but it also
triggers a conformational change that leads to polymerization of the complex (Fig. 8).
The stoichiometry described here in the precipitate between p23 peptide and coatomer and the
solution structure of the dimeric peptide strongly favour a tertramerized state of p23 when it
binds to coatomer in vivo. Similarly, other members of the p24 family known to bind
coatomer and bearing a diphenylalanine and dilysine motif might interact in a tetrameric state
with coatomer. Thus receptor induced polymerization of coatomer might represent a general
mechanism for COPI vesicle formation. However, at present it is not known in which
stoichiometries the various p24 family members interact with each other (45) or whether they
are able also to form homooligomers. In any case, various oligomers would allow different
classes of COPI vesicle to exist, specified by the different p24 members they contain.
According to this model, oligomerization of p23 and its relatives must be regulated in vivo.
Such regulation might occur via interaction of p24 members with cargo, if p24 members serve
as cargo receptors (46-48). Another candidate to regulate oligomerization of p24 members is
ARF 1, the recruitment of which to Golgi membranes precedes binding of coatomer.
However, such a role of ARF 1 in oligomerization of p24 members waits to be elucidated.
As coatomer is used in many cycles, an important question concerns a reversibility of the
conformational change of complex. Since ARF 1 is a component of COPI vesicle and known
16
to be involved in the uncoating process by hydrolysis of its bound GTP (49), this energy
providing step might well help to reverse the conformational change of coatomer and thus
dissociate the coat.
In general, receptor induced polymerization of coat proteins during their recruitment would
strongly increase the efficiency of the coating process, and with the resulting geometry ruled
by the nature of the conformationally changed proteins, may well be the driving force to shape
a membrane.
17
FIGURE LEGENDS
Fig. 1. Precipitation of coatomer with synthetic peptides corresponding to the COOH-termini
of p23wt, p23AS and Wbp1p. (A) Peptides used in this study. p23wt represents the
cytoplasmic domain of p23 which binds coatomer depending on its dilysine and
diphenylalanine motifs, whereas p23AS lacks these residues and therefore does not bind
coatomer. Wbp1p represents the cytoplasmic domain of a subunit of the yeast Noligosaccharyl transferase complex bearing a characteristic KKXX ER-retrieval motif known
to bind coatomer. (B, C) Precipitation of coatomer (0.09 µM) with monomeric peptides (B)
and dimeric peptides (C). Only p23wt (filled squares) precipitates the complex, whereas
mutated p23 peptide (p23AS, filled circles) and Wbp1p (filled triangles) have no effect. The
error bars indicate standard errors (n=3).
Fig. 2. Limited proteolysis of coatomer. Proteolysis of coatomer (0.09 µM) with thermolysin
(0.008 µM) in the presence of dimerized peptides (50 µM), p23wt-d (A), p23AS-d (B) and
Wbp1p-d (C). Western blot analysis of proteolytic fragments of the -COP subunit reveals a
better susceptibility of this subunit to the protease in the presence of p23wt-d (A, lanes 2, 3) as
compared to p23AS-d (B, lanes 2, 3) and Wbp1p-d (C, lanes 2, 3).
Fig. 3. Limited proteolysis of COPI vesicles. (A) Suspension of purified COPI vesicles
containing  0.009 µM coatomer treated with thermolysin (0.009 µM) in the presence of
p23AS-d (50 µM) as a substrate for the protease. Western blot analysis of proteolytic
fragments of -COP reveals a prominent fragment of about 59 kD (lane 2) which is highly
susceptible to the protease (lane 3). (B) Coatomer (0.009 µM) incubated with p23wt-d (50
18
µM) and treated with thermolysin under the same conditions as used for the COPI vesicles
yields a result comparable to that of COPI vesicles (lanes 2, 3). In contrast, partial digestion in
the presence of p23AS-d (50 µM) yields a different result: the 59 kD fragment is more stable
(C, lanes 2, 3).
Fig. 4. Stoichiometry between coatomer and p23wt-d. 125I-labelled p23wt-d (filled squares) or
p23AS-d (filled circle) were incubated with increasing concentrations of coatomer . The
amounts of peptide in the precipitates were determined by counting the
125
I radioactivity. The
error bars indicate standard errors (n=4).
Fig. 5. NOEs indicative of secondary structures. The width of the lines represents the relative
strength of the NOEs; a gray line indicates that the NOE could not be identified because of
spectral overlap. (A) p23wt-m, (B) p23wt-d.
Fig. 6. (A) Superimposition of the 10 structures with lowest internal energies of p23wt-m
obtained from simulated-annealing molecular dynamics using NOE-derived an dihedral
restraints. The heavy atoms of all structures are shown. (B) Ribbons depiction of p23wt-m
showing the secondary structure, diphenylalanine (green) and the dilysine (blue) motif. Top:
view perpendicular to the helix axis; bottom: parallel view. A similar representation from the
structure of p23wt-d dimer, representing the tetramer of the cytoplasmic domain of p23, is
shown in (C).
Fig. 7. Dimer formation of p23wt-d confirmed by size exclusion chromatography. (A)
Monomeric p23wt (p23wt-m: 1 mg/ml, inject 20 µl). (B) Dimeric p23wt (p23wt-d: 1 mg/ml,
inject 20 µl). Peak 1 represents the dimerized form of p23wt-d, peak 2 the monomeric form of
19
p23wt-d. (C) Rechromatography of peak 1 of (B) (40 µl). The appearance of both peak 1 and
2 demonstrates an equilibrium between monomeric and dimeric states of p23wt-d. (D)
Dimeric p23wt (p23wt-d: 0.1 mg/ml, inject 40 µl). The concentration dependence of the
equilibrium of peak 1 and 2 indicates a bimolecular event. Thus, peak 1 is caused by the
dimeric and peak 2 by the monomeric state of the peptide.
Fig. 8. Hypothetical model for bud formation. According to this model, interaction of
coatomer with a tetramer of p23 induces a conformational change of the complex. This leads
to its polymerization on the surface of a membrane resulting in the formation
Table I. Energy contribution to the structure and deviation from standard geometry. Etotal, total
energy; EvdW, van der Waals energy; ENOE, effective NOE energy term resulting from a
square-well potential function; ELJ, Lennard-Jones part of the electrostatic energy. The
Lennard-Jones potential was not used during any refinement stage. All calculations were
carried out using the standard X-PLOR force field and energy terms. The values are mean
values over 10 refined structures. #Because of line-broadening the 3JN could not be
determined reliably for p23wt-d dimer.
20
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Acknowledgements
We thank Dr. Suzanne Pfeffer, Dr. Kai Simons, and Dr. Graham Warren, for critically reading
the manuscript, and Dr. Heinrich Sticht for his X-PLOR protocols. This work was supported
by grants of the Deutsche Forschungsgemeinschaft (SFB 352, to C. H., J.B. H., F. W.), the
Human Frontier Science Program (to F. W.) and the Fonds der Chemischen Industrie (to F.
W., P. R.).
24