Via their C-terminal Src homology 3 SH3 domains, PACSIN proteins bind to proline-rich domains of dynamin, synapsin and synaptojanin, three proteins also involved in vesicle endocytosis,
Trang 1Arndt Halbach1, Matthias Mo¨rgelin2, Maria Baumgarten2, Mark Milbrandt1, Mats Paulsson1
and Markus Plomann1
1 Center for Biochemistry and Center for Molecular Medicine (CMMC), Medical Faculty, University of Cologne, Germany
2 Department of Clinical Sciences, Section for Clinical and Experimental Infectious Medicine, University of Lund, Sweden
In eukaryotic cells, complex regulatory mechanisms
involving numerous proteins must operate to ensure
the temporal and spatial specificity of intracellular
membrane-trafficking pathways We and others have
identified three members of the protein kinase C and
casein kinase 2 substrate in neurons (PACSIN) protein
family, also named syndapin and focal adhesion
pro-tein 52 (FAP52), which participate in rearrangements
of actin networks during endocytosis [1–5] In contrast
to the neuron-specific PACSIN 1, other members of the PACSIN protein family show a broader tissue dis-tribution [2,4,6] Via their C-terminal Src homology 3 (SH3) domains, PACSIN proteins bind to proline-rich domains of dynamin, synapsin and synaptojanin, three proteins also involved in vesicle endocytosis, as well as to neural Wiskott–Aldrich syndrome protein
Keywords
F-BAR domain; membrane; oligomerization;
PACSIN 1; syndapin 1
Correspondence
M Plomann, Center for Biochemistry,
Medical Faculty, University of Cologne,
Joseph-Stelzmann-Str 52, D-50931
Cologne, Germany
Fax: +49 221 478 6977
Tel: +49 221 478 6944
E-mail: markus.plomann@uni-koeln.de
(Received 13 October 2006, revised
23 November 2006, accepted 4 December
2006)
doi:10.1111/j.1742-4658.2006.05622.x
The ability of protein kinase C and casein kinase 2 substrate in neurons (PACSIN)⁄ syndapin proteins to self-polymerize is crucial for the simulta-neous interactions with more than one Src homology 3 domain-binding partner or with lipid membranes The assembly of this network has pro-found effects on the neural Wiskott–Aldrich syndrome protein-mediated attachment of the actin polymerization machinery to vesicle membranes as well as on the movement of the corresponding vesicles Also, the sensing of vesicle membranes and⁄ or the induction of membrane curvature are more easily facilitated in the presence of larger PACSIN complexes The N-ter-minal Fes-CIP homology and Bin-Amphiphysin-Rvs (F-BAR) domains of several PACSIN-related proteins have been shown to mediate self-inter-actions, whereas studies using deletion mutants derived from closely related proteins led to the view that oligomerization depends on the formation of
a trimeric complex via a coiled-coil region present in these molecules To address whether the model of trimeric complex formation is applicable to PACSIN 1, the protein was recombinantly expressed and tested in four different assays for homologous interactions The results showed that PACSIN 1 forms tetramers of about 240 kDa, with the self-interaction having a KD of 6.4· 10)8m Ultrastructural analysis of these oligomers after negative staining showed that laterally arranged PACSIN molecules bind to each other via a large globular domain and form a barrel-like structure Together, these results demonstrate that the N-terminal F-BAR domain of PACSIN 1 forms the contact site for a tetrameric structure, which is able to simultanously interact with multiple Src homology 3 bind-ing partners
Abbreviations
ADAM, a disintegrin and metalloprotease; BAR, Bin-Amphiphysin-Rvs; BS3, bis[sulfosuccinimidyl]suberate; FAP52, focal adhesion protein 52; F-BAR, Fes-CIP4 homology and Bin-Amphiphysin-Rvs; FCH, Fes-CIP4 homology; GST, glutathione S-transferase; NMDA, N-methyl- D -aspartate; NOSTRIN, eNOS-trafficking inducer; N-WASP, neural Wiskott–Aldrich syndrome protein; PACSIN, protein kinase C and casein kinase 2 substrate in neurons; PACSIN 1-CS, PACSIN 1 carrying a C-terminal Strep II tag; PCH, pombe CDC15 homology; PSTPIP, proline, serine, threonine phosphatase-interacting protein; SH3, Src homology 3; Sulfo-EGS, ethylene glycol bis[sulfosuccinimidylsuccinate].
Trang 2(N-WASP), a stimulator of actin-related protein
2⁄ 3-induced actin polymerization [4,6] Accordingly,
the PACSIN proteins have been implicated not only in
vesicle endocytosis at the plasma membrane but in a
variety of membrane traffic events, most of which
occur at membranes of intracellular sorting
compart-ments [7,8]
PACSIN proteins also interact with specific
trans-membrane proteins, such as ADAM (a disintegrin and
metalloprotease) metalloproteinases [9,10], the CD95
ligand [11] and, in the case of PACSIN 1, the
phos-phodiesterase 6c [12] and the N-methyl-d-aspartate
(NMDA) receptor chain NR3A [13] These
interac-tions, most of which also involve the SH3 domains,
indicate a role of PACSIN proteins in the regulation
of the surface expression of some transmembrane
molecules by endocytosis For NR3A, we were able
to demonstrate an activity-dependent mechanism by
which PACSIN 1 regulates NMDA receptor
expres-sion at synapses during development [13]
PACSIN proteins represent a subgroup within a
lar-ger protein family, named pombe CDC15 homology
(PCH), displaying a similar arrangement of domains,
including at least one C-terminal SH3 domain and a
conserved N-terminal region (Fig 1A) The latter was
originally defined as the CDC15-N-terminal
(CDC15-NT) domain, spanning about 250 amino acids in
PAC-SIN proteins [4] Later, others distinguished between a
region covering the N-terminal circa 100 amino acids,
named the Fes-CIP4 homology (FCH) domain, and
the adjacent a-helical stretch, which is believed to form
a coiled-coil structure [14] So far, no function has
been reported for the FCH domain, whereas the
a-helical region has been shown to be responsible for oligomerization of PCH proteins [15–17] Recently, the whole N-terminal region, corresponding to the CDC15-NT domain and renamed the FCH and Bin-Amphiphysin-Rvs (BAR) (F-BAR) domain, has been characterized in more detail [18] Like classical BAR domains, this related domain is able to bind to lipid bilayers Moreover, the PCH proteins tested, including PACSIN⁄ syndapin 1, bind to liposomes containing phosphatidylserine and phosphoinositides, and are alone sufficient to deform them into tubules [18] In agreement with this, PACSIN 1 was recently identified
as the key interaction partner of dynamin 1 in synaptic vesicle endocytosis [19] In this process, PACSIN 1 is thought to bind to the synaptic plasma membrane, and induce a curvature in the membrane and neck for-mation prior to vesicle fission through the action of dynamin 1
Another proposed role for PACSIN proteins is in attachment of the actin polymerization machinery to vesicles after endocytosis They are believed to act as linkers between the endocytic protein dynamin and N-WASP, thereby directing the actin propulsion machinery to the site of vesicle fission [4,6] As several important interaction partners, including dynamin and N-WASP, interact with PACSIN’s single SH3 domain [4], only PACSIN oligomers would be able to act as linkers Previously, we showed that, in vitro, all three PACSIN proteins are able to to bind to each other and might exist as homo-oligomers and⁄ or hetero-oligomers [4] Recently, the interconnecting function of PACSIN oligomers was shown to be essential for PACSIN-mediated cytoskeletal rearrangements and
A
Fig 1 Domain structure and purification of recombinant PACSIN 1 (A) Domain struc-ture of PACSIN 1 with an N-terminal F-BAR domain and a C-terminal SH3 domain The F-BAR domain was formerly known as the CDC15-NT domain, and includes a con-served region, also named the FCH domain, and an a-helical polypeptide stretch postula-ted to act as a coiled-coil domain Addition-ally, PACSIN 1 contains two asparagine-proline-phenylalanine (NPF) motifs.
(B) Fractions eluted from a StrepTactin Sepharose column were purified by an additional ultracentrifugation step and tested for purity by SDS ⁄ PAGE under reducing conditions C, Coomassie Brilliant Blue; S, silver nitrate The arrow marks the recombinant PACSIN 1-CS (C) MALDI-TOF
MS analysis of the same purified PACSIN 1 sample.
Trang 3endocytosis [20] We report here that purified
PAC-SIN 1 exists as a 230–240 kDa tetrameric complex in
solution Although it shares many structural and
func-tional properties with other members of the PCH
pro-tein family, PACSIN 1 is distinct in that it forms
barrel-like homotetramers in vitro, held together by the
N-terminal F-BAR domain
Results
Purification and characterization of PACSIN 1
PACSIN 1, carrying a C-terminal Strep II tag
(PAC-SIN 1-CS) and expressed in HEK293 cells, was
puri-fied by a single affinity chromatography step The
homogeneity of the preparation was demonstrated by
reducing SDS⁄ PAGE followed either by staining with
Coomassie Brilliant Blue and silver nitrate (Fig 1B)
or by immunoblotting (data not shown)
MALDI-TOF MS analysis confirmed the purity (Fig 1C)
Secondary structure and thermal stability
of PACSIN 1
In order to analyze the conformation and thermal
sta-bility of PACSIN 1, the purified protein was studied
by CD spectroscopy Aliquots of PACSIN 1-CS
(100 lgÆmL)1) in 5 mm Tris⁄ HCl (pH 7.4) were
incu-bated at 20C, 37 C, or 60 C, and CD spectra were
recorded At the two lower temperatures, PACSIN 1
showed similar CD spectra with characteristic minima
in the 208–223 nm region, indicative of a high a-helical
content (Fig 2A) When PACSIN 1 CD spectra were
recorded at 60C, changes were observed, reflecting a
loss of a-helical structure (Fig 2A) This heat
denatur-ation was irreversible, as a subsequent decrease in
tem-perature was unable to restore the original structure
(results not shown) Furthermore, calculation of the
relative proportions of different secondary structure
elements by three different algorithms revealed that, at
37C, PACSIN 1 contains about one-third a-helix, a
portion of which is lost upon heating, accompanied by
an increase in b-structure (supplementary Table S1) A
melting point of 44C was determined from the
mid-point of the transition at 221 nm (Fig 2B)
Oligomeric structure of PACSIN 1 complexes
To determine the oligomerization state of PACSIN 1,
purified PACSIN 1-CS was analyzed by gel filtration
In a KCl-containing buffer at cytosolic ion strength,
PACSIN 1 eluted as a single peak between the marker
proteins aldolase (158 kDa) and ferritin (440 kDa)
(Fig 3A) The presence of PACSIN 1 in individual fractions was confirmed by immunoblotting (results not shown) For the calculation of the molecular mass
of the PACSIN protein complex, the Kavvalues of the marker proteins used as a standard were plotted against log Mr With this method, the molecular mass
of the native PACSIN complex was determined as being 234 kDa, corresponding to an oligomerization state of about 4.5 (Fig 3B, supplementary Table S2A) Furthermore, performing the same experiment with a bis(sulfosuccinimidyl)suberate (BS3)-crosslinked sample led to comparable results (Mr¼ 240 kDa; oligomeriza-tion state 4.6), showing that crosslinking with this rea-gent captures PACSIN complexes in a native state (Fig 3C, supplementary Table S2B)
We next used protein crosslinking to study intermo-lecular interactions between PACSIN 1 subunits Three homobifunctional imidoester reagents, disuccinimidyl suberate (DSS), BS3 and ethylene glycol
bis(sulfosuc-Fig 2 CD spectra of PACSIN 1 (A) PACSIN 1-CS protein (100 lgÆ
mL)1in 5 m M Tris ⁄ HCl, pH 7.4) was measured at 20 C (solid line),
37 C (dotted line) and 60 C (dashed line) Changes in the content
of a-helix and b-structure were observed between 37 C and 60 C (B) For the determination of the melting temperature, recordings were performed at 221 nm with a linear temperature gradient from
20 C to 80 C The midpoint of the conformational transition was
at 44 C.
Trang 4cinimidylsuccinate) (Sulfo-EGS) were tested for their ability to covalently link recombinantly expressed PACSIN 1-CS molecules All reagents crosslinked PACSIN 1 to dimers (Fig 4A, lanes 2 and 3 for BS3, and lane 4 for Sulfo-EGS) or, at higher crosslinker concentrations, to tetramers (Fig 4A, lanes 4–7 for
BS3, and lanes 5–7 for Sulfo-EGS) Sulfo-EGS differs from DSS and BS3 by having a slightly longer spacer arm (16.1 A˚ versus 11.4 A˚), and was less efficient in crosslinking PACSIN 1 to tetramers Adducts larger than tetramers were only occasionally observed at higher protein concentrations (50 lg versus 20 lg), corroborating the tetrameric structure indicated by the gel filtration experiments
MS analysis of crosslinked fractions confirmed the presence of dimers, but ionization was insufficient for the detection of higher oligomers (Fig 4B,C) The mod-erate increase in mass of PACSIN 1 monomers resulted from bound crosslinker molecules The actual presence
of tetrameric complexes in a BS3-crosslinked sample was confirmed by size exclusion chromatography, which resulted in a single symmetrical peak comparable to the previously analyzed native sample (Fig 3C)
As oligomerization is a prerequisite for the proposed function of PACSIN as a linking protein, surface plas-mon resonance was used to further support the pres-ence of a self-interaction between PACSIN 1 subunits and to determine the strength of this binding (Fig 5) High-affinity binding between PACSIN 1 monomers could indeed be detected (Fig 5), and a ka of 1.44·
105m)1Æs)1, a kDof 4.3· 10)3and a KDof 6.4· 10)8m were calculated (supplementary Table S3)
Purified full-length PACSIN 1-CS (Fig 1) was also submitted to electron microscopy after negative stain-ing with uranyl formate (Fig 6) The protein particles were heterogeneous in size, and closer examination revealed that both monomers and, predominantly, higher aggregates were present in the sample (Fig 6A) Most of the monomers formed elongated curved struc-tures, but fully extended 7–8 nm monomeric particles could occasionally be seen (Fig 6B) Dimers showed a lateral alignment of PACSIN molecules joined at one end Tetramers displayed a barrel-like structure, often with a more heavily stained hole in the middle and most mass at the periphery (Fig 6C,D) The average diameter of the tetramers was 8 nm Occasionally, par-ticles were oriented to give a top view (Fig 6D, top panel) We never observed a waist-like structure, which would have indicated that a rod-like coiled-coil a-helix might assemble the higher-order structure Electron microscopy of negatively stained recombinant F-BAR domains of PACSIN 1 again showed monomeric (Fig 6E, upper row) and dimeric (Fig 6E, lower row)
Fig 3 Analysis of PACSIN 1 oligomers by gel filtration (A) Elution
profile of PACSIN 1-CS from size exclusion chromatography using a
Superdex 200 column (solid line) The elution profile of a mixture of
thyroglobin (669 kDa), ferritin (440 kDa), aldolase (158 kDa),
ovalbu-min (43 kDa) and ribonuclease A (13.7 kDa) is shown as a dotted
line Numbers at the peaks represent the molecular masses of the
corresponding marker proteins The Y-axes show the relative
fluor-escence intensities at 280 nm, with the left axis corresponding to
the marker proteins, and the right axis to PACSIN 1-CS AU ¼
arbi-trary units (B) Plot of Kavvalues of marker proteins against log Mr.
Numbers at the open squares represent the molecular masses.
The relative mass of the native PACSIN complex was determined
as being about 234 kDa (closed square), corresponding to an
olig-omerization state of 4.5 (C) Analysis of BS 3 -crosslinked PACSIN 1
gave comparable results, with a molecular mass of 240 kDa and an
oligomerization state of 4.6 (closed square) Numbers at the open
squares represent the molecular masses of the corresponding
mar-ker proteins.
Trang 5particles, demonstrating that this domain is sufficient
for oligomerization
Discussion
The present characterization of the neurospecific
repre-sentative of the PACSIN proteins, PACSIN 1 [1,3],
supports the hypothesis that PACSINs act as linking
molecules in vesicular trafficking It was previously
shown that PACSIN proteins bind to both dynamin
and N-WASP, and that impairment of these
inter-actions leads to changes in actin dynamics, block of
endocytosis, and mislocalization of involved proteins
[4,6,17,20–22] As both binding partners are recognized
by the single C-terminal SH3 domain of PACSINs,
multiple simultaneous interactions are only possible if PACSINs form oligomers Members of the PCH fam-ily contain at least one a-helical polypeptide stretch, which is assumed to form a coiled-coil and thereby enable oligomerization Several studies on individual members of this protein family have shown their abil-ity to homo-oligomerize to dimers [proline, serine, threonine phosphatase-interacting protein (PSTPIP) and PSTPIP 2 [15]], or trimers [FAP52 [16] and (endothelial nitric oxide synthase) eNOS-trafficking inducer (NOSTRIN) [17]] We previously observed that all PACSIN isoforms are able to interact with each other in two-hybrid assays [4], and recently another study confirmed the ability of PACSIN proteins to self-associate [20] To determine the
A
B
C
Fig 4 Crosslinking of PACSIN 1 (A)
PACSIN 1 was incubated at 50 lgÆmL)1
with 0.5 l M –10 m M BS 3 (left panel) or at
20 lgÆmL)1with 0–5 m M Sulfo-EGS
cross-linker (right panel) and analyzed by reducing
SDS ⁄ PAGE on 5–15% gels The crosslinked
products are labeled (B, C) The products of
PACSIN 1 crosslinked with 25 l M (*, B) and
2.5 m M Sulfo-EGS (#, C) were analyzed by
MALDI-TOF MS.
Trang 6stoichiometry of PACSIN 1 oligomers, we expressed
recombinant PACSIN 1 in eukaryotic cells and, by use
of CD spectroscopy, confirmed that the protein was
correctly folded The content of a-helix in the
full-length protein was found to be 33.7–37.9%, depending
on the software used, which is significantly higher than
the 25.8% calculated for the closely related PACSIN 2
ortholog FAP52 [16] Heat treatment leads to an
irre-versible loss of a high proportion of these helices and,
interestingly, to an increase of b-structure
(supplement-ary Table S1)
When subjected to size exclusion chromatography,
PACSIN 1 eluted as a complex of about 234 kDa,
indicating that oligomers are formed in solution This
mass slightly exceeds that of a tetramer, which may
result from the shape of the PACSIN 1 complexes
when compared to the standard proteins A similar
oligomerization has been shown for a recombinantly
produced GST–NOSTRIN fragment [17], but here gel
filtration indicated a trimerization In a recent
publi-cation, it was suggested that PACSIN 1
predomin-antly forms dimers in vivo [20] This was concluded
from crosslinking studies in brain and cell extracts,
but less well-resolved higher molecular weight
com-plexes were also observed [20] To address the
appar-ent discrepancy with our gel filtration results, we used
increasing concentrations of three different
crossl-inkers with varying spacer arm lengths, and clearly
detected the preferential formation of PACSIN 1
dimers and tetramers In contrast to the other studies,
we avoided the use of cell lysates in which potential
exogenous interaction partners might be present and
Fig 5 Surface plasmon resonance binding curves obtained for
the PACSIN 1 self-interaction Antibodies to GST were coupled
(15 000 RU) to a CM5 sensor chip and saturated with GST–
PACSIN 1 or GST as a control PACSIN 1-CS was injected at
differ-ent concdiffer-entrations The binding curves shown have been corrected
by subtracting the values obtained with GST alone.
A
B
C
D
E
Fig 6 Electron microscopy of negatively stained recombinant PACSIN 1-CS and the PACSIN 1 F-BAR domain The overview (A) shows aggregates as well as monomeric PACSIN 1 molecules; the lower panels show selected monomeric (B), dimeric (C) and tetra-meric (D) particles The bottom panel (E) shows PACSIN 1 F-BAR monomers (upper row) and dimers (lower row) The picture at the lower right includes a monomer located next to the dimer The bars correspond to 10 nm.
Trang 7in which complexes may include proteins other than
PACSIN The analysis of PACSIN 1 self-association
by real-time surface plasmon resonance gave a KD of
6.4· 10)8m, which is comparable to the KD of
4.7· 10)9m calculated for FAP52 [16] Although
clo-sely related to PACSIN 2, FAP52 appears to
partici-pate in different processes In chicken embryo heart
fibroblasts, it localizes to focal adhesion contacts [5],
and this was never observed for any of the three
PACSIN isoforms
To complement our biochemical analysis, we also
employed electron microscopy of negatively stained
PACSIN 1 complexes; this confirmed the formation
of dimers and tetramers observed in solution The
PACSIN tetramers form a barrel-like structure in the
absence of any lipids or other proteins Taken together,
our results suggest that tetramers are the highest
oligo-mers formed by PACSIN 1, and that dioligo-mers may be
intermediates in the assembly The proportions of
dimers and tetramers detected may depend on the
ana-lytical method used None of the particles seen with
electron microscopy showed a waist-like structure,
which would have been indicative of assembly via a
coiled-coil a-helix Instead, subunit contacts appear to
be mediated by domains of globular shape These may
be the PACSIN 1 F-BAR domains, particularly as
elec-tron microscopy of isolated PACSIN 1 F-BAR
domains also shows oligomerization (Fig 6E) The
importance of this region for self-assembly has been
confirmed for PACSIN 1 [20], and also been reported
for other F-BAR domain-containing proteins [16,17]
The exact role of PACSINs is controversial,
especi-ally with regard to when and where PACSIN
mole-cules contribute to vesicle formation and removal
PACSIN proteins have been proposed to play a role in
the regulation of transferrin endocytosis However,
these findings were based on overexpression of either
isolated SH3 domains [23] or full-length proteins [4],
and may reflect an impairment of proper dynamin
localization, as overexpression of other
dynamin-bind-ing SH3 domains has comparable effects [22–24] A
recent study demonstrated that F-BAR domains are
able to bind to phospholipids, in particular to
mem-branes containing phosphatidylserine, and that they
are able to cause membranes to form tubules in vitro
[18] This suggests an involvement of PACSIN proteins
early in vesicle formation at donor membranes, which
has recently been confirmed for PACSIN 1 at nerve
terminals [19] Here, the phosphorylation-dependent
interaction of PACSIN 1 with dynamin 1 is essential
for synaptic vesicle endocytosis The authors propose a
new model in which PACSIN 1 induces membrane
curvature and⁄ or formation of a neck at endocytic
sites before dynamin 1 facilitates vesicle fission inde-pendently of the actin cytoskeleton PACSIN 1 F-BAR domain oligomers might be required for this function, as the related BAR domains need to dimerize
to be active [25] Also, PACSIN 1 tetramers localized around a vesicle neck could provide multiple docking sites for dynamin molecules
However, biochemical analysis revealed that PAC-SIN 1 is present in microsomal and cytosolic fractions from brain ([26]; unpublished results) and can only occasionally be detected at the plasma membrane [3,4,27] Immunofluorescence microscopy clearly shows that most endogenous PACSIN 1 molecules are distri-buted throughout the neuron, including synapses, pro-cesses and cell bodies [3,4] Also, proteomic studies of the composition of clathrin-coated vesicles [28], postsy-naptic densities [29] and brain plasma membranes [30] failed to identify PACSIN 1 Recently, we demonstra-ted a postsynaptic role for PACSIN 1 in regulating NR3A endocytosis [13], which may represent an exam-ple of PACSIN 1 acting as a linker molecule on moving
in neurons It has previously been shown that dynamin remains attached to vesicle membranes after scission, and serves as an anchoring site for actin tails [31,32] The proline-rich region of dynamin is essential for the formation of actin comet tails, indicating that interac-tion partners that bind via their SH3 domains to this region, such as PACSIN proteins, may also be neces-sary The role of PACSIN proteins in connecting the GTPase dynamin with N-WASP through oligomeriza-tion has recently been confirmed [20] However, tetramerization of PACSIN proteins provides a more efficient mode of interconnection
The results presented here show that PACSIN forms tetramers via its F-BAR domain Such tetramers may participate in synaptic vesicle endocytosis by deform-ing the corresponddeform-ing membrane, and⁄ or in the assem-bly of the vesicle docking site for actin-mediated propulsion [19,20] The increasing number of trans-membrane molecules identified as PACSIN-binding partners may represent the cargo molecules transpor-ted by these vesicles, and may provide the specificity of the PACSIN–vesicle association
Experimental procedures
Expression and purification of PACSIN 1
A full-length murine PACSIN 1 cDNA clone in pBlue-script [1] was used as template for PCR using AmpliTaq DNA Polymerase (Perkin Elmer, Wellesley, MA, USA) and specific primers (sense, 5¢-AAG CTT GCC ACC ATG TCT GGC TCC TAC GAT GAG GCC-3¢; antisense,
Trang 85¢-GCG GCC GCT ATA GCC TCA ACG TAG TTG G-3¢).
The amplified DNA fragment was cloned into the pCR2.1
vector (Invitrogen, Karlsruhe, Germany), and after sequence
confirmation was digested with HindIII and NotI and cloned
into the HindIII–NotI-digested expression vector
pCEP-puBM40-cStrep [33] This produced a fusion protein in which
a Strep II tag was placed in frame with the PACSIN 1 coding
region The plasmid was transfected into 293-EBNA cells by
electroporation, and the cells were subsequently selected for
puromycin resistance Cell pellets were lysed in NaCl⁄ Pi
(pH 7.5) containing 0.25 mm sucrose and 1 mm
phenyl-methanesulfonyl fluoride by sonification, and centrifuged at
20 000 g for 15 min at 4C (Beckman ultracentrifuge L7-55,
SW41 Ti rotor), and finally at 180 000 g for 2 h at 4C
Supernatants containing Strep II-tagged PACSIN 1 were
loaded on a StrepTactin Sepharose column (IBA, Gottingen,
Germany) at a flow rate of 0.5 mLÆmin)1 After being washed
with 10 column volumes of 100 mm Tris⁄ HCl (pH 8.0)
con-taining 1 mm EDTA and 1 mm phenylmethanesulfonyl
fluor-ide, the proteins were eluted with the same buffer containing
2.5 mm desthiobiotin The protein samples were resolved by
SDS⁄ PAGE, and analyzed either by Coomassie or silver
staining of the gel, or transferred to a poly(vinylidene
difluo-ride) membrane and detected with antibodies against
PAC-SIN 1 [1]
Glutathione S-transferase (GST) fusion proteins of
PACSIN 1 were produced by cloning cDNAs
correspond-ing to either the complete codcorrespond-ing region of PACSIN 1 or
the F-BAR domain (amino acids 1–285) into the pGEX-6P
vector (Amersham Pharmacia Biotech, Freiburg, Germany)
and then expressing in Escherichia coli (BL21) The fusion
proteins were purified by affinity chromatography on
gluta-thione–Sepharose 4B, and GST was removed by cleavage
with Precission protease (Amersham Pharmacia Biotech)
for some applications
Gel filtration analysis
For the size determination of purified PACSIN complexes,
freshly purified recombinant PACSIN 1-CS was dialyzed
against 10 mm Pipes⁄ KOH (pH 7.4) containing 100 mm
KCl, 3 mm NaCl and 3.5 mm MgCl2 The sample (50 lgÆ
mL)1) was applied to a Pharmacia SMART Superdex 200
column and analyzed at a flow rate of 10 lLÆmin)1 For
size calculation, the standard proteins ribonuclease A
(13.7 kDa), ovalbumin (43 kDa), aldolase (158 kDa),
fer-ritin (440 kDa) and thyroglobin (669 kDa) were treated
equally and analyzed The eluted fractions were monitored
at 280 nm by UV photometry
CD measurements
CD spectra were recorded in a Jasco (Gross-Umstadt,
Germany) J-715 spectropolarimeter PACSIN 1-CS was
dialyzed against 5 mm Tris⁄ HCl (pH 7.5), at a concentra-tion of 100 lgÆmL)1
Crosslinking assays Crosslinking assays were carried out using the three lysine side-chain-reactive crosslinkers (Pierce, Rockford, IL, USA), BS3, DSS and Sulfo-EGS The PACSIN 1-CS was added at a concentration of 20 or 50 lgÆmL)1 The reaction was carried out in a final volume of 40 lL in NaCl⁄ Pi
(pH 7.4) for 1 h at 4C and was stopped by the addition
of 10 lL of 1 m Tris⁄ HCl (pH 8.0)
MALDI-TOF MS For MALDI-TOF MS analysis, the samples were dissolved
in 5 lL of 0.1% aqueous trifluoroacetic acid MS was carried out in linear mode on a Bruker Reflex IV equipped with a video system, a nitrogen UV laser (Omax¼ 337 nm) and a HiMass detector (Bruker, Bremen, Germany) One microliter of the sample solution was placed on the target, and 1 lL of a freshly prepared saturated solution of sinapi-nic acid in acetonitrile⁄ H2O (2 : 1) with 0.1% trifluoroace-tic acid was added The spot was then recrystallized by addition of another 1 lL of acetonitrile⁄ H2O (2 : 1), which resulted in a fine crystalline matrix For recording of spec-tra, an acceleration voltage of 20 kV was used, and the detector voltage was adjusted to 1.9 kV About 500 single laser shots were summed into an accumulated spectrum Calibration was carried out using the single and double protonated ion signal of BSA for external calibration
Surface plasmon resonance binding assays Assays were performed using a Biacore 2000 (BIAcore AB) Coupling of antibodies to GST (BIAcore, Freiburg, Ger-many) to the CM5 chip was performed in 10 mm sodium acetate (pH 5.0), at a flow rate of 5 lLÆmin)1 A 6 min pulse of 0.05 mm N-hydroxysuccinimide⁄ 0.2 m N-ethyl-N¢-dimethylaminopropyl carbodiimide was used to activate the surface The antibodies to GST (30 lgÆmL)1) were injected for 7 min in 10 mm sodium acetate (pH 5.0), until the desired amount was coupled (15 000 RU), and excess reactive groups were deactivated by a 7 min pulse of 1 m ethanolamine hydrochloride (pH 8.5) The antibodies were saturated with GST–PACSIN 1 (100 lgÆmL)1), or, as a control, GST alone until saturation Measurements were carried out in NaCl⁄ Pi(pH 7.4) containing 2.5 mm desthio-biotin at a flow rate of 30 lLÆmin)1 The injection of 90 lL
of the PACSIN 1-CS solution (0.1–2 lm) and the 180 s association was followed by a 180 s dissociation Each ana-lysis was carried out a minimum of four times with two parallel samples After subtraction of the data obtained for GST, they were analyzed with biaevaluation software 3.0,
Trang 9according to the Langmuir model for 1 to 1 binding All
binding curves could be fitted with an accuracy of
v2< 0.5
Electron microscopy
Purified PACSIN 1-CS (10 lgÆmL)1) or a purified
recom-binant PACSIN 1 fragment containing the F-BAR domain
was adsorbed onto a 400-mesh carbon-coated copper grid,
which was rendered hydrophilic by glow discharge at low
pressure in air The grid was immediately washed with
two drops of water, and stained with 0.75% uranyl
for-mate for 15 s Specimens were observed in a Jeol JEM
1230 transmission electron microscope (Jeol, Tokyo,
Japan) operated at 60 kV accelerating voltage The images
were recorded with a Gatan Multiscan 791 CCD camera
(Gatan, Munich, Germany) Evaluation of the data from
electron micrographs was done as described previously
[34]
Acknowledgements
We would like to thank the Bioanalytical Laboratory
of the Center for Molecular Medicine Cologne for
the MS analysis This work was supported by
Deut-sche Forschungsgemeinschaft grant PL233⁄ 1-2 (to
M Plomann) and by a grant from the Ko¨ln Fortune
program of the Medical Faculty of the University of
Cologne
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Supplementary material
The following supplementary material is available online:
Table S1 PACSIN 1 secondary structure at different temperatures
Table S2 Calculation of Kavvalues for native (A) and crosslinked (B) PACSIN 1 complexes
Table S3 Surface plasmon resonance analysis of PACSIN 1 self-interaction
This material is available as part of the online article from http://www.blackwell-synergy.com
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