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OF YESHIVA UNIVERSITY
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3. Purification and molecular characterization
synaps@-€hriched clathrin assembly polypeptide.
of NPl85, a neuronal-specific and
Shengwen Li1, Michael Lisanti2
and Saul Puszkin3
From the Deparlment of Cell Biology, Harvard
Medical School, Harvard University, Boston,
Massachusetts 021152, Deparlment of Molecular
Pharmacology, Albert Einstein College of Medicine,
Bronx, New Yorkz, and Department of Pathology,
College of Physicians & Surgeons, Columbia
University Medical Center, New York, New York
1 00323.
Correspondence should be addressed to Dr Saul
Puszkin, Scientific Director of Health Sciences
Consultant Group Laboratories, HSCG, Lasker
Biomedical Research Building, 3960 Broadway,
Suite 305, Columbia University, New York, New York
10032
Tel: 212-342-7272; Fax: 212-342-7274;
E- m ai I : "sp220@col u m bi a.edu "
BIOQUIMICA Y PATOLOGIA CLINICA - Vol 62 - N' 1 - 1998
Resumen:
La NP185 es un polip6ptido especifico de las c6lu-
las neuronales y de una masa molecular de '185 kilo-
Daltons. Esta proteina la descubrimos originalmente
cuando prepar6bamos anticuerpos monoclonales
usando como antigeno el complejo proteico de cla-
trina extraido de veslculas cubiertas en cerebro de
bovino.
Dos anticuerpos monoclonales, que Ilamamos 8GB y
6G7, nos permitieron visualizar y caracterizar ante-
riormente la proteina NP185. Esas caracterlsticas
fueron estudiadas e identificadas bioqu[micamente y
en desarrollo embrionario. Encontramos tambi6n
que la proteina NP185 se expresaba en las c6lulas
tumorales de tejido renal PC12, cuando estas c6lu-
las son estimuladas con el Factor de Crecimiento de
Nervios (NGR. En esos estudios usamos cerebro de
embriones de pollo y encontramos que el momento
en que comienza la expresi6n gen6tica de NP185
coincide con la formaci6n funcional de las termina-
ciones sindpticas.
En el trabajo que describimos a continuaci6n usa-
mos m6todos basados en principios gen6ticos y he-
mos avanzado nuevamente en nuestro entendimien-
to sobre las propiedades de la proteina NP185. He-
mos elucidado las siguientes caracterlsticas:
A) Con un anAlisis de micro-secuencia de amino6ci-
dos obtuvimos cinco fragmentos de mol6culas pep-
tidas que pertenecen a distintas regiones del poli-
peptido NP185 de cerebro bovino. Estas regiones
tambi6n corresponden exactamente a las secuen-
cias que fueron reconocidas en la proteina de laucha
F1-20 y en la proteina de rata AP180 (llamada re-
cientemente AP3).
B) Usando m6todos establecidos anteriormente he-
mos demostrado que Ia proteina NP185/AP3 purifi-
cada por m6todos de gen6tica recombinante facilita
la reconstrucci6n de las jaulas de clatrina.
C) Usando borramiento de mutaciones gendticas
hemos delineado los sitios antig6nicos de dos anti-
cuerpos monoclonales diferentes que reaccionan
con la proteina NP185 de bovino. Estos sitios anti-
g6nicos corresponden a una regi6n de amino6cidos
de la proteina recombinante NP1B5/AP3 que tiene
60 residuos de aminodcidos.
D) La proteina recombinante NP185/AP3 puede ser
Li, Michael Lisanti and Saul Puszkin

4. 6
Purification and molecular characterization of NP185, a neuronal-specific and synapse-enriched ...
fosforilada con la enzima purificada de kinasa de ca-
seina tipo ll.
Finalmente, E) La proteina recombinante NP185/AP3
se acopla directamente a tubulina. En esa forma es-
timula la reorganizaci6n y formaci6n de jaulas de cla-
trina. Por lo tanto asumimos que la proteina
NP185/AP3 se encuentra involucrada en el transpor-
te regulado de veslculas cubierlas con clatrina en el
citoplasma de las c6lulas neuronales de cerebro.
BasAndonos en estos resultados proponemos que la
kinasa de caseina tipo ll, es una enzima que acom-
pafra a las veslculas cubiertas en c6lglas nerviosas
deltejido de cerebro. Las veslculas cubiertas de cla-
trina participan as[ en el transporte de porciones de
membrana sin6ptica y facilitan la secresi6n de sus-
tancias qulmicas neuronales de transmision de im-
pulsos nerviosos en cerebro.
Abstract
NP185, a neuronal-specific protein of 185 kDa, was
first discovered when we prepared monoclonal anti-
bodies (mAbs) against bovine brain clathrin coated
vesicles. Two mAbs, 8GB and 6G7, permitted us to
characterize this protein both biochemically and in
development (NP185 is expressed in a NGF-depen-
dent manner in PC12 cells). The expression of
NP185 coincides with synaptogenesis. ln this work,
we have further characterized this protein as follows:
i) Microsequence analysis of immuno-purified natlve
NP185 from bovine brain yielded five peptides that
corresponded exactly to the known sequences of
murine F1-20 and rat AP'l80 (renamed AP3); ii) Using
an established assay, we show that purified recom-
binant NP1B5/AP3 can facilitate clathrin cages
assembly; iii) Using deletion mutagenesis, we
mapped the epitopes of two distinct mAbs directed
against bovine NP185 to a 60 amino acid residue
region of the murine recombinant NP185/AP3; iv)
Recombinant NP185/AP3 can be phosphorylated by
purified casein kinase ll in vitro; and v) Recombinant
NP185/AP3 directly binds to purified brain tubulin.
Since NP185/AP3 binds to tubulin and stimulates the
clathrin assembly, it may be involved in the regula-
tion of the transport of clathrin-coated vesicles.
Casein kinase ll, an enzyme known to be present in
clathrin-coated vesicles, may play a role in the regu-
lation of NP185/AP3 for the promotion of clathrin
assembly.
Key words: NP185/AP3, phosphorylation, casein
kinase ll, tubulin binding, epitope mapping, microse-
quencing.
Running title: Characterization of neuron-specific
protein NP1B5/Ap3
List of Abbreviations: APlBO: assembly pblypep-
tide 180 kDa; CCV: clathrin coated vesicle; CKll:
casein kinase ll; NGF: nerve growth factor; NP185:
neuronal-specific protein 185 kDa; PBS: phosphate-
buffered saline; PC12: rat pheochromocytoma cell
line; RT-PCR: reverse transcriptase polymerase
chain reaction; SDS-PAGE: sodium dodecyl sulfate-
polyacrylamide gel electrophoresis.
lntroduction
Clathrin coated vesicles (CCVs) participate in various
intracellular transporl processes, such as receptor
mediated endocytosis and the retrieval of membrane
after exocytosis. Biochemical and morphological
evidence shows that clathrin-coated vesicles play a
major role in the nervous system-specifically in the
recycling of synaptic vesicle membranes after they
fuse and discharge their contents within the synaptic
cleft (Maycox et al., 1992). Neuronal-specific com-
ponents of clathrln coated vesicles have been iden-
tified. These include neuronal specific isoforms of
the clathrin light chains (LCa and LCb) (Jackson et
al., 1987; Kirchhausen et al., 1987b) and neuronal
specific isoforms of members of the assembly
polypeptides, AP-2 complex (Robinson, '1990;
Robinson, 1989) that facilitates the assembly and
attachment of clathrin to plasma membranes.
NP'l85 (Neuronal Protein of 185 kDa) is a major com-
ponent of clathrin coated vesicles (Kohtz and
Puszkin, 1988; Puszkin et al., 1992). This 185 kDa
protein was first identified when we elicited mAbs
against clathrin coated vesicles purified from bovine
brain (Kohtz and Puszkin, 19BB). Two mAbs (BGB,
6G7), which specifically recognized NP185, have
allowed us to study the distribution and localization
of NP 185 (Kohtz and Puszkin, '1988; Kohtz and
Puszkin, 1989; Perry et al., 1991; Perry et al., 1992;
Su et al., 1991), Unlike clathrin-the major coat pro-
tein of coated vesicles, - the expression of NP185 is
highly restricted. lt is only expressed in brain tissue
and selectively in neurons (Kohtz and Puszkin, 19BB;
Kohtz and Puszkin, 1989; Perry et al., 1991 ; Perry et
al., 1992; Su et al., 1991). ln PC'|2 cells, a modelfor
neuronal cell - systems, NP185 is dramatically
induced by stimulation with nerve growth factor
(NGF) during differention (Kohtz and Puszkin, 1988).
These developmental studies showed that expres-
sion of NP'l85 coincided with synaptogenesis (Perry
et al., 1991; Puszkin et al., 1992). This molecule is
enriched in mature synaptic terminals, associated
with clathrin light chains and casein kinase ll (Kohtz
and Puszkin, 1989; Su et al., 1991) .
However, little is known about the function of NP185
in brain cellular activities. Clues to the function of
Shengwen Li, Michael Lisanti and Saul Puszkin

5. 7
BIOQUIMICA Y PATOLOGIA CLINICA - Vol 62 - N' 1 - 1998
Microsequence Analysis.
The pure native bovine NP185 protein in gels was
excised and subjected to microsequencing at The
Protein Chemistry Group, Biotechnology Resource
Laboratory W.M. Keck Foundation (by Dr. Kathy
Stone, Yale University). lnternal peptides were
obtained by trypsin digestion of the purified native
bovine NP185. The enzymatic cleavage fragments
were separated on a narrow-pore (2.1-mm i.d.)
reverse-phase high-pedormance liquid chromato-
graphy (HPLC) using a dual-syringe Brownlee
micropump (Aebersold et al., 1987). The peptides
were sequenced by automated gas-phase sequena-
tor. The amino acid sequences of those peptides
were analyzed using the programs FASTA and
BLAST through the GenBank database.
lmmunoblotting
Mouse brain homogenates were prepared as
described previously (Sousa et al., 1992). Native
NP185 obtained from the high salt extract of bovine
brain CCVs (Kohtz and Puszkin, 1988) as well as
affinity purified protein were used for immunoblotting
analysis. Gel electrophoresis was adapted for the
brain preparations following the procedure of
Laemmli (1970). Sample preparations were loaded
onto 5-15% gradient SDS-PAGE gels. Proteins were
then transferred to nitrocellulose f ilters for
immunoblotting analysis with mouse anti-NP185
mAbs (Kohtz and Puszkin, 1988) or anti-Fl-20 mAbs
(kindly provided by Dr. E. M. Lafer) (Sousa et al.,
1990). Goat anti-mouse secondary antibody IgG
conjugated with alkaline phosphatase. After wash-
ings, the signals were detected by BCIP/NBT in
developing buffer.
Reverse Transcriptase-PCR cloning of the murine
F1-20 cDNA.
Mouse brain poly (A+) RNA (1 pg) was used for
reverse transcription with the first-strand cDNA syn-
thesis kit (Pharmacia) by following the manufactur-
er's instructions. The first-strand oDNA sample (15
;rl) was heated to 90'C for 5 minutes to denature the
RNA-oDNA duplex and to inactivate the reverse
transcriptase, then chilled on ice. The entire first-
strand cDNA sample was then amplified in PCR
using primers flanking the open reading frame of F1-
20 cDNA, forward: 5'-CTCACTCGAGGGCCGGT-
GAAGATGTC-3' ANd TEVETSE: 5,-CTCAACTCGA-
GMTCTTATCTGAAGTTTCC-3.' (Zhou et al., 1992;
Zhou et al., 1993).
The purified PCR product of the expected size was
end-repaired and ligated with Smal cut pGEX-3X to
generate the recombinant plasmid that expresses
GST-NP185/AP3 fusion protein. The recombinant
plasmids with correct orientation and exact reading
NP185 came from in vitro reassembly experiments
using purified clathrin triskelia (Su et al.' 1991)' ln
these experiments, NP185 acted as an assembly
promoter agent that facilitated the assembly of
clathrin into cages under conditions where clathrin
was unable to assemble on its own. NP185 behaved
like other assembly polypeptides-multiple-subunit
complexes termed AP-1 and AP-2 (AP, assembly
polypeptide)- that were expressed in most if not all
cell types (Keen, 1990; Robinson, 1992).
Ungewickell and colleagues have identified a 180
kDa protein that they termed AP180 (Ahle and
Ungewickell, 1986) , while Keen and colleagues dis-
covered pp155 (Keen and Black, 1986). In many
respects, NP185, AP180 and ppl55 share similar
properties (Ahle and Ungewickell, 1986; Keen and
Black, 1986; Kohtz and Puszkin, 1988), and they
were therefore renamed as AP3 (Murphy et al',
1991). However, this is based solely on immunologi-
cal cross-reactivity. lt was very difficult to purify a
sufficient amount of the native NP185 for microse-
quencing analysis since the native NP185 is
extremely sensitive to proteolysis' Thus, there was
no direct evidence to confirm the identity from the
amino acid sequence of purified native NP185'
Here, we provide direct molecular evidence for the
identity of NP185 with AP180/F1-20 (designated as
AP3). lmmuno-purification and microsequencing of
native bovine NP185 yielded five peptide sequences
that were identical to the known sequence of rat AP-
180 and murine F1-20 that is a protein identified by
immunoscreening of cDNA expression library. Using
the murine F1-20/AP3 cDNA we specifically mapped
the epitopes of two mAbs directed against NP185 to
a 60 amino acid residue region of NP1B5/AP3' Also,
we demonstrate that bacterial-expressed recombi-
nant murine NP185/AP3 functionally promoted the
assembly of clathrin triskelia into cages. Fudher
experiments showed that recombinant NP1B5/AP3
directly interacts with brain tubulin and is phospho-
rylated by casein kinase ll (CKll) in vitro.
Experimental Procedures
lmmuno-affinity purification of NPI 85.
Parlial purified native NP185 was isolated from
bovine brain clathrin coated vesicles (CCVs) (Schook
and Puszkin, 1985) by high salt extraction (Kohtz and
Puszkin, 1988). Extracted proteins were separated
on Sepharose CL6B and subsequently on Mono Q
ion-exchange chromatography. Fractions containing
native NP185 were passed through antibody affinity
column (See Figure 1A). Pure native bovine NP185
was eluted from this column, and separated by SDS-
PAGE. The protein was visualized by staining with
Coomassie blue.
Shengwen Li, Michael Lisanti and Saul Puszkin

6. purification and molecular characterization ot ttpiaE; **Gpecific and synapse-enriched "'
frame were determined by restriction enzyme map- and examined in a Joer 100-B electron microscope
ping and doubre_stranaeo oun sequencing. tne at an accereration vortage of B0 kv (su et ar' 1991)'
6si-Npras/AP3 fusion protein was obtained when
expressed in a suitabL Escherichia coti strain (8L21, Epitope-mapping of mAbs 8G8 and 6G7'
lacking Lon and ompT proteinases, Novagln, lnc')' For-expression of NP1B5/AP3 as a recombinant
GST-fusion protein, the cDNA for murine F1-20lAP3
purification of GST-N1g5/Ap3 fusion protein. was subcroned in frame into the murtipre croning site
purification of GST-Np18s/Ap3 protein-*". p"r- of the vector pGEX3X or pGEX-1LT' Deletion
formedusingglutathioneSepharose48columnmutantswerethencreatedbypartialdigestionwith
(Pharmacia) accoroing to man,tacturer s instruction Pst l or by PCR using primers that incorporated con-
with foilowing modificaiion (Li et ar., 1gg5; Li et ar., venient restriction sites' Fusion proteins were then
1996a;Marston,1986).Briefly,theinducedcellsexpressedinE'colistrainBL2lafterlPTGinduction'
were harvested by row speed centrifugation. p"tt"t purified and used as the substrate for immunobrot-
was washed by the oltrer'ot 25 mM rris-ibr, pH 7's, iilo 1v1n
mAbs BGB and 6G7 directed against native
10 mM EDTA; 85 mM NaCl and 50 mM glucose' NP1B5'
Proteinase inhibitors were immediately added to the
cells and the cells wlre frozen in liquid nitrogen. After ln Vitro phosphorylation
thawing, the sample was treated by Polytron and ! Yjlf
pnotphorytation was performed as described
sonicated and centrifuged. The supernatant was previousty (L! et
-al.,.
1,996b)' Recombinantly
diluted in pBS (rsomr'r"NaCl, 16 mM Na2HPO4,4
"*pr".r"i
purified caseinJ(inase ll (uBl', lnc') was
mM NaH2Po4, pH 7'3), plus 1% Triton X-100, and incubated with GSTalone, GST-FL-NP1B5/AP3 (Full-
apptied to the grutaih'rone-agarose affiniiy Lotmn leLsJn
NP185/AP3 fused to GST) bound to glu-
that specificatty ninJs io csi-po,tion of fusion pro- tathione sepharose 91T.. After 2 hour incubation
tein. After washing for eight times, the fusion protein with rotation at 4oc' GST or GST-NP185/AP3 bound
was eruted with reduced grutathione-coniaining to beads were washed extensivery with a buffer con-
buffer. All manipulations were performed at 4"C (i; taining SDS and deoxycholate (Li et al'' 1996b;
cord room) or on ice, and a* sorutions were ice-cord pendergast et ar., 1gg'1 ; Pendergast et al', 1991)' The
and contained the folowing mixture oi-piot."r" Np1B5-tK1 comprex or GST alone bound to the
inhibitors: phenytmethylsulfonyl ttuorioe it mM), beads was then incubaied with kinase reaction
aprotinin (2 mg/ml), pepstatin (1 mg/mli, anO reu- buff-er-containing 20 mM Hepes' 5 mM MgCl2' 1 mM
peptin (1 mg/ml).
'rr PsPorq'"' ' "'Y' '
Mncl2, y-32p-ATe for 5-10 minutes at room temper-
ature. The reaction was stopped by adding the SDS
crathrin assembry assay. sampre buffer and boiring at 100 oc for 5 min. After
An estabrished assay was used to evaruate the effect .DS-'AGE, dried ger was subjected to autoradiog-
ofnativebovineNPl85andrecombinantraphytovisualizephosphorylatedproteins.
NP185/AP3 on clathrin assembly into cages (Su et
al., 1gg1). Clathrin triskelions were pLrified as lnteraction of brain tubulin and recombinant
described previously following the procedure of NP185/AP3
winkler and stanley (winkler and stanley, 1983). The We h.ave adopted an established assay to determine
effect of Np1g5 on the polymerization oiclathrin was the interaction of brain tubulin and recombinant
determined by layering purified clathrins incubated NP1B5/AP3 (Li et al" 1995)' Brain tubulin was puri-
with either purified n"tlr" bovine Np18s or the puri- fied by DEAE chromatography (gift from Dr. David R'
fied GST-Np1B5/Ap3 fusion protein, on top ol a'1o%o colman, columbia University). Approximately 5 pg of
sucrose solution. The samples were centrifuged at either GST or purified GST-NP185/AP3 fusion pro-
100,0009 to permit polymerized structures to sedi- teins bound to glutathione-agarose beads were
ment at the bottom of the sucrose layer. Aliquots extensively washed with phosphate-buffered saline
from the top solution and the ,.rr.p"nd"d pellets (PBS) (six times) and incubated with 2 pg of brain
were assayed by either immunoblotting or f[tSR to iubulin in 1 ml of PBS containing proteinase
detect clathrins and Np1g5. The pelletl were resus- inhibitors by rotating overnight in the cold room
pended for electron microscopy to visualize the type (4"C)' After binding, the beads were extensively
of structure formed. washed (for six t1n":) with PBS containing pro-
Approximately 10 pg of the pellet containing NP185- teinase innioitors' Finally' bound proteins were elut-
induced clathrin cage polymers were susp-ended in ed with 100 ml of elution buffer containing PBS' 10
0.1 M Tris buffer pfiZ.O and used tor nelative stain- mM reduced glutathione and proteinase inhibitors'
ing. Samples were placed on Formvar-tiated grids, The eluate was mixed 1:1 with 2 X SDS-sample
negatively staineJ with 1 %o uranyl acetate, air dried buffer and separated on a 10% sDS-polyacrylamide
Li. Michael Lisanti and g:qleg94!

7. r9
BIOQUIMICA Y PATOLOGIA CLINICA - Vol 62 - N' 1 - 1998
from 15 freshly obtained bovine brains; See
Experimental Procedures). The five internal peptide
sequences obtained are listed in Table l. These five
peptides did not show homology to any known
genes in the GenBank in 1992 except that they cor-
responded exactly to the known sequence of murine
F1-20 phosphoprotein and rat AP1B0, which were
deposited into the GenBank but not yet published.
These results clearly demonstrate that at the prima-
ry amino acid sequence level, native bovine NP185
and AP3 (murine F1-20/ral AP1B0) are apparently
identical which may be due to extremely similar
genes. ln support of this assumption, immuno-puri-
fied native bovine NP185 reacted with a monoclonal
antibody probe raised against murine F1-20 (Figure
gel electrophoresis (SDS-PAGE) and analyzed by
Western blotting with anti-tubulin (1:1000 dilution)
monoclonal antibody. Horse radish peroxidase-con-
jugated secondary antibodies (1:5000 dilution;
Amersham Corp.) were used to visualize bound pri-
mary antibodies by enhanced chemiluminescence
(ECL).
Results
lmmuno-purif ication and microsequencing analy-
sis of native bovine NP185: ldentity with murine
F1-2O phosphoprotein and rat AP180.
Native bovine NP185 is a large molecule (185 kDa)
and extremely sensitive to proteinase degradation. lt
is masked by dominant 180 kDa of clathrin heavy
chain and very acidic so that it was escaped from
cietection with any conventional techniques such as
SDS-PAGE and Coomassie blue staining, Native
NP185 tightly associates with other molecules such
as assembly polypeptides of 100 kDa and 50 kDa.
Thus, it was difficult to purify native NP185 as a sin-
gle species and in sutficient amounts for protein
microsequencing. Therefore, uncovering of the mol-
ecular structure of the protein awaited. purification
from recommbinant 185 expressed molecules.
Here, we have developed a procedure to successful-
ly purify native bovine NP185 to be homogeneity
through differential centrifugation, different buffer
extraction, gel f iltration chromatography, ion
exchange chromatography, immuno-affinity chro-
matography, and HPLC (outlined in Figure 1A).
Through the immuno-affinity chromatography with
anti-NP185 mAb, native bovine NP185 was eluted in
two peaks: a minor peak (Peak 1) and a major peak
(Peak 2) (Figure 1B). These two peaks may represent
two NP185 isoforms (Kohtz and Puszkin, 1988)' The
fraction of the peak 1 represents an isoform of
NP185 that has a low binding affinity for it's antibody
and it was eluted first from the column. The fraction
of the peak 2 represented an isoform with high affin-
ity with the anti-NP185 mAb. After immuno-atfinity
chromatography, the purified protein appeared as a
single band by SDS-PAGE analysis and Coomassie
Blue staining (Figure 1C, lane 3). The fraction from
the major peak (Peak 2) was used for further analy-
sis. Purified native NP185 did noi cross-react with
antibodies directed against other coated vesicle
components such as clathrin or the assembly
polypeptides (not shown).
We then performed microsequencing analysis of
immuno-purified native bovine NP185. As the N-ter-
minus was blocked (Li and Puszkin, 1991, unpub-
lished observations), NP185 was digested with
trypsin and internal peptides were isolated by HPLC
(15.5 mg protein: - 100 picomoles, approximately
so$
$$e
a.S
60' ,.''
..'$9 d.f
'
Figure 2. Recognition of purified native bovine NP185
by anti-NP't85 and anti-F1-20 mAbs.
Purified native bovine NP185 (isolated as described in
Figure 1) was resolved by SDS-PAGE and electroblotted
onto nitrocellulose membranes. Strips were cut and
probed with anti-NP185 mAbs (8G8 and 6G7)and anti-F1-
20 mAb. The arrow marks the position of purified full-
length native bovine NP-185' Note that some minor degra-
dation products are observed, as native NP185 is
extremely sensitive to proteolysis.
Murine F1-20 is a neuronal phosphoprotein of
unknown function identified by immunoscreening of
oDNA expression library (Zhou et al., 1992) ; rat
AP180 is a 180 kDa clathrin assembly polypeptide
(Ahle and Ungewickell, 1986). Rat AP1B0 and murine
F1-2O are now believed to represent the rat and the
murine forms of the same gene product (Morris et
Shengwen Li, Michael Lisanti and Saul Puszkin

8. CCVs were decoated with 0.5
M NaBr
I
V
Centrifuged 35,000 rpm [Ii45),
M Tris pH 7.8 and 0.5
75 min, 4 oC
Discard pellets (membrane
fractions)
Supernatant was concentrated with ammonium
sulfate and dialyzed against column loading buffer.
I
Sepharose CLOe cotJmn chromotography for sepa-
ration of clathrin, NP185, assembly polypeptides.
I
MonoQ column cnroJatography for separation of
clathrin, NP185, assembly polypeptides.
I
Y
lmmuno-affinity column chromatography coupled
with anti-NP185 mAb to purify Np185 to
homogeneity.
I
V
NP1 85
I
Y
Trypsin digestion
I
lnternal peptides sfparateA by HpLC
I
Y
Protein microsequeccing
to
Purification and molecular characterization of NP185, a neuronal-specific and syn"ps"**i"f*a ...
Figure 1. lmmuno-affinity purification of native
NP185 from extracts of bovine brain coated vesicles.
Fraction Nu-bers
B) lmmuno-affinity chromatography with anti-
NP185 mAb indicates that there are two iso-
forms of native NP185 from bovine brain. peak
1: minor NP185 peak, in which the NpIBS iso-
form has low affinity to the anti-Np1g5 mAb
therefore eluted first from the column; peak 2:
major NP185 peak, which was used for further
analysis and microsequencing, in which the
NP185 isoform has high affinity to the Np.185
mAb therefore eluted second from the column.
C) Purified native bovine Np1BS. Left,
Coomassie Blue stainingi Right, lmmunoblot
analysis with anti-NP185 mAb 8GB. Lane 1, puri-
fied clathrin coated vesicles; lane 2, soluble
extract of clathrin coated vesicles; lane 3, puri-
fied native bovine NP185. Note abbreviations:
HC, clathrin heavy chain; LC, clathrin light
chains. Note that the purified native bovine
NP185 does not react with an anti-clathrin heavy
chain antibody and anti-assembly polypeptide
antibody, indicating the purity of the native
bovine NP185 (not shown).
A) Purification scheme. Native bovine Np1g5 was suc-
cessfully
.purified by differential centrifugation, sizing
columns (gel filtration columns), ion exchange column
S:.? $.9glumn), and immuno-affinity chromatogra-
phy. Anti-NP185 mAb (8G8)was couptjO to CNBr_acti_
vated Sepharose 48 for the immuno-affinity chro_
matography.

9. BIOOUIMICA Y PATOLOGIA CLINICA - Vol 62 - N' 1 - 1998
Clathrin triskelia can self-assemble into regular coat
structures-clathrin cages in vitro under certain con-
ditions (e.9., pH 6.5) even in the absence of mem-
branes, lt is, however, strictly dependent on protein
cofactors, namely assembly polypeptides under cer-
tain conditions (e.9., pH 7.5). Using an established in
vitro assembly assay, we have shown that native
bovine NP185 can induce clathrin to assemble under
conditions that do not favor clathrin assembly (Su et
al., 1991). ln this sense, native NP185 behaves simi-
larly to other assembly polypeptide complexes,
namely AP-1 and AP-2 (Keen, 1990).
To investigate whether recombinant NP185/AP3 can
function as a facilitator of clathrin assembly, we puri-
fied the full-length recombinant fusion protein GST-
NP185/AP3 by affinity chromatography on glu-
tathione agarose and gel-elution (Figure 44). Like
native bovine NPl85, recombinant NP185/AP3 pro-
moted the assembly of purified clathrin triskelia into
clathrin cages. As observed with other assembly
polypeptides, the addition of recombinant
NP1B5/AP3 induced the formation of clathrin cages
of a relatively uniform diameter, 98Yo of the cages
with 60-80 nm in diameter (Figure 4 B). As a control,
GST failed to induce the formation of clathrin cages
(Figure 4 C). Additional controls, recombinant NP185
alone or clathrin alone) did not show any clathrin
cage structures under the same conditions (not
shown). Furlher SDS-PAGE and immunoblotting
analysis show that the reassembled cages contained
intact recombinant NP185/AP3 and clathrins (not
shown).
Epitope mapping of anti-bovine-NP185 mAbs
(8G8 and 6G7).
We mapped the epitopes of mAbs directed against
bovine NP185 by deletion mutagenesis of the murine
NP1B5/AP3 oDNA (Figure 5). Deletion mutants were
generated by complete and pafiial digestion with
Pstl; smaller fragments of NP'185/AP3 were generat-
ed by PCR amplification using selected primers as
described in Experimental Procedures. The epitopes
for both mAbs directed against bovine NP185 (8G8
and 6G7) mapped to the same region of the predict-
ed NP185/AP3 amino acid sequence (411-471) as
shown by immunoblot analysis of the deletion
mutants (Figure 5; Table ll). The position of this epi-
tope relative to other known antibody epitopes is
summarized in Figure 6.
ln vitro phosphorylation of NP185/AP3 by purified
casein kinase ll.
AP3 was previously identified as phosphoprotein in
vivo by several laboratories. Puszkin and colleagues
Shengwen Li, Michael Lisanti and Saul Puszkin
al., 1993; Zhou et al., 1993). Keen and colleagues
have used the term AP3 for the isoforms of bovine
pp155, rat AP1B0, bovine NP185 (Murphy et al.,
1991) and murine F1-20 (Morris et al., 1993; Zhou et
al, 1993).
Recombinant expression and assembly promot-
ing activity of purified recombinant NP185/AP3.
We isolated the cDNA for murine F1-20 by reverse
transcriptase-polymerase chain reaction (RT-PCR)
from mouse brain mRNA. The sequence of PCR
product was confirmed by restriction mapping and
DNA sequencing. The entire coding region was sub-
cloned into the vector pGEX-3X for recombinant
expression as a GST-fusion protein. lmmunoblot
analysis revealed that the recombinant NP1B5/AP3
fusion protein is specifically recognized by an anti-
NP185 mAb (8GB) and an anti-F1-20 mAb (Figure 3).
These results independently suppotl our results from
microsequencing analysis of immuno-purified native
bovine NP185.
Coomassie Blue mAb 8G8 mAb F1-20
|PTG+++
Figure 3. Recombinant expression of full-length F1-
20lNP185(AP3) as a GST fusion protein.
Total proteins from the E. coli cells containing the recom-
binant plasmid encoding recombinant F1-2OlNP185(AP3)
were resolved by SDS-PAGE. A plus "+" indicates that
IPTG was used to induce expression of the F1-20 fusion
protein. Left, Coomassie Blue staining; Middle,
lmmunoblot analysis with anti-NP185 mAb 8G8; Right,
lmmunoblot analysis with anti-F'l-20 mAb (F'1-20). Note
that recombinant F1-20lNPl85(AP3) is specifically recog-
nized by both anti-NP185 (8G8) and anti-F1-20 monoclon-
al antibodies. Full-length GST-F1 -20lNP1 85(AP3) migrated
with an apparent molecular weight ol - 210 kDa and is
indicated by arrow.

10. 12
Purification and molecular characterization of NP185, a neuronal-specific and synapse-enriched ...
A
-!!l-
!s
r
i
O
-r!
Figure 4. Purified recombinant NP185/AP3 fusion protein promotes the assembly of clathrin triskelions into
cages.
The GST-FL-NP185/AP3 fusion protein was first purified by affinity chromatography on glutathione agarose and then
resolved by SDS-PAGE. ln order to obtain the full-length protein product, the band corresponding to the full-length GST-
F1-20/NPl85(AP3)fusion protein (as indicated by immunoblot using anti-NP185 mAb) was excised from the gel and
eluted according to the manufacturer's instructions (Amicon). After gel-elution, the full-length fusion protein was used to
assay for clathrin assembly activity.
A) Lane'1 , Total proteins from cells containing the recombinant plasmid induced by IPTG was resolved by SDS-PAGE
and stained by Commassie blue; lane 2, GST-F1 -20l NP185 (AP3) fusion protein purified by affinity chromatography on
glutathione agarose and gel-elution.
B) Purified GST-F1 -20l NP185 (AP3) fusion protein promotes the assembly of clathrin triskelions into cages. Clathrin was
incubated with the purified full-length fusion proteins in Tris buffer at pH 7.5, and the protein mixture was layered on top
of a 1O%o sucrose solution and centrifuged at high speed in an airfuge for 2 hours. The pellet formed under the sucrose
layer was resuspended and an aliquot deposited on a Fromvar-coated grid. Scanning electron microscopy shows that
the formation of clathrln cages are induced by the bacterially expressed GST-F1 -20l NP185/AP3 fusion protein. X
120,000. Note that clathrin triskelions can not assemble into cages at pH 7.5 without the presence of assembly polypep-
tides. The white bar indicates 100 nm.
C) Purified GST alone did not stimulate clathrin triskelions to form cages under the same condition as the recombinant
NP185/AP3. The white bar indicates 100 nm.
N.M C.]Hd
1 -P185 FuIl-l€Er] c t2
-
E@
L lts
21t 17,
a7, s
&lm
316 !?2
g! t12
gl ato
ar! ai,
Figure 5. Epitope mapping of anti-NP185 mAbs 8G8 and 6G7.
A) Schematic diagram summarizing the immuno-reactivity of
mAbs 8G8 and 6G7 with a panel of murine NP185/AP3 fusion pro-
teins. The nucleotide positions included within a given insert are
as indicated at the endpoints. Convenient restriction sites used in
these constructions are also as indicated. Note that both anti-
NP185 mAbs 8G8 and 6G7 recognize an epitope within murine
NP185/AP3 amino acid residues 411-471 (Corresponding to
nucleotide positions 1233-1416).
B) The epitopes for both mAbs
8G8 and 6G7 map to murine
NP185/AP3 residues 411-471.
Lane '1
, total proteins from E.coli
BL21 cells expressing GST
fusion protein encoding amino
acid residues 411-471 of murine
NP185/AP3 (Corresponding to
nucleotide positions 1233-1416) i lane 2, total proteins of cells expressing GST alone;
lane 3, total proteins of cells expressing full-length GST-F1-20/ NPl85 (AP3). Left
panel, immunoblot analysis with anti-NP185 mAb 8G8; right panel, immunoblot analy-
sis with another anti-NP185 mAb 6G7.
mAb 8G8 mAb 6G7
t?3 123
*!1
i:Y
6-
6
I
-l
k
Li, Michael Lisanti and Saul Puszkin

11. laa AP3: NP185/F1-20IAP180 eo2aa
ru^
4t 1 47
t=epilopes,or 6Abs 8G8, 6G7 (Enli'NPl85)
4OO 766
cpiloP6,or F1'20 mAb (anlrFl'20)
s62 , ,741
epilopo lor AP180 mAb (anli'APl80)
;
BIOOUlt,llCA Y PATOLOGIA CLINICA - Vol 62 - N" 1 - 1998
GST
GST-NPIES + r
I:'P-ATP + "
CKll - +
t3
Figure 6. Schematic diagram summarizing the epi-
toles of anti-NP185' anti-F1-20, and anti-AP180 mon-
oclonal antibodies.
found that native bovine NP185 associates with
casein kinase ll activity (CKll) (Kohtz and Puszkin,
1989; Su et al., 1991). Ungewickell and colleagues
have demonstrated that AP1B0 is phosphorylated in
serine residues in clathrin-coated vesicles from
bovine brain (Ahle and Ungewickell, 1986; Morris et
al., 1990). Similarly, with metabolic labeling, Keen
and colleagues show that pp155 is phosphorylated
in serine residues (Keen and Black, 1986; Murphy et
al., 1994). Lafer's group suggested that F1-20 is a
phosphoprotein using phosphatase treatment and
phosphatase inhibitors (Zhou et al., 1992)' Taken
together, NP185/AP3 is phosphorylated presumably
by serine kinases. lndeed, cDNA sequence analysis
revealed that there are seven consensus motifs for
casein kinase ll in murine F1-20 (Zhou et al', 1992)'
However, there is no evidence to support that AP3
can be directly phosphorylated by casein kinase ll'
ln this work, we studied the serine phosphorylation
of NP1B5/AP3 by casein kinase ll in vitro' We recon-
stituted that serine phosphorylation of NP185/AP3
by purified recombinant casein kinase ll in vitro'
Recombinant full-length NP'185/AP3, expressed as a
GST-fusion protein, was incubated with purified
recombinant casein kinase ll in the presence of [g-
32P]ATP. Under these conditions, only NP185 under-
went casein kinase ll-mediated phosphorylation'
GST alone failed to be phosphorylated by CKll,
despite the fact that it contains serine residues'
Omission of either casein kinase ll or [y-32P]ATP
prevented phosphorylation, indicating that this
phosphorylation is ATP- and CKll-dependent (Figure
7). Recombinant NP'185/AP3 appeared not to be
phosphorylated by either purified recombinant Src
tyrosine kinase or purified recombinant Fyn-tyrosine
kinases (not shown).
Figure 7. ln vitro phosphorylation of recombinant full-
length murine NP185/AP3 by casein kinase ll'
puritieo full-length NP185/AP3 (GST-FL-NP1B5)
expressed as a GST fusion protein or GST alone was sub-
jected to in vitro phosphorylation with purified recombi-
nant casein kinase ll and 1-32P-ATP in kinase reaction
buffer. After SDS-PAGE, phosphorylated proteins were
visualized by autoradiography. Note that each reaction
contained equivalent amounts of GST and purified full-
length recombinant NP185/AP3, although only the recom-
binint NP185iAP3 undergoes phosphorylation.
Phosphorylation of purified full-length recombinant
NP185iAP'3 was specifically dependent on addition of
both casein kinase ll and 1-32P-ATP. Omission of either
casein kinase ll and y32P-ATP prevented phosphoryla-
tion.
lnteraction of NP185 with brain tubulin
Previous findings suggested ihat native bovine
NP185 is associated with brain tubulin(Kohtz and
Puszkin, 1989; Su et al., 1991)' Here, we used an
established assay to determine the potential direct
interaction between brain tubulin and recombinant
NP1B5/AP3 (Li et al., 1995). We expressed full-length
NP185/AP3 (GST-FL-NPl85 as indicated in Figure 8)
as a GST fusion protein. After purification by affinity
chromatography on glutathione-agarose, GST-
NP185/AP3 fusion protein bound on the beads were
incubated with purified brain tubulin. After extensive
washings, GST fusion proteins bound to glutathione-
agarose beads were specifically eluted with reduced
glutathione-containing buffer' As this treatment
releases GST fusion proteins, it also indirectly releas-
es proteins bound to GST fusion proteins; non-
specifically bound proteins should remain attached
to the beads under these conditions. To evaluate
brain tubulin binding, these eluates were then sub-
jected to SDS-PAGE analysis and immunoblotting
with anti-tubulin monoclonal antibody' As compared
with a critical control GST alone, brain tubulin bound
specifically to full-length NP1B5/AP3, but not to GST
Li, Michael Lisanti and Saul Puszkin

12. .st* +e€
, zrJ' .( -ltc,t cr#' c9' cr+
alone (Figure 8). This is the first indication that
NP185/AP3 can directly bind to brain tubulin.
Figure 8. lnteraction of brain tubulin and recombinant
NP185/AP3.
GST-FL-NP185/AP3 (Bound to glutathione-agarose
beads) was incubated with brain tubulin purified by DEAE
column chromatography (gift from Dr. David R. Colman).
GST-FL-NP'185/AP3 represents full-length recombinant
NP1B5/AP3 (residues '1
-902) expressed as a GST fusion
protein. After binding, extensive washing (six times), and
eluted with reduced glutathione, bound tubulin was visu-
alized by Western blotting with a tubulin-specific mono-
clonal antibody. As a critical control, binding v/as com-
pared with GST alone. ln addition, note that antibody
binding was specifically dependent on the addition of
tubulin, demonstrating that the antibody does not cross-
react with either recombinant NP185/AP3 or GST.
Equivalent amounts of GST and GST-FL-NP1B5/Ap3
(about 1 ug per binding experiment) were used in binding
experiments. For each binding experiment, 0.2 ug of
native brain tubulin was used which was purified by DEAE
chromatography.
Discussion
Among the principal results of this study was the
purification and molecular characterization of
NP185, a synapse-specific protein. We learned that
the microsequence analysis of immuno-purified
native bovine NP185 yielded five peptides that cor-
respond exactly to the known sequences of murine
F1-20 and rat AP'180, which was recenily designated
as AP3; also using an established assay, we showed
that purified recombinant Np'195/Ap3 can act as a
facilitator of clathrin assembly. With deletion muta-
genesis, we mapped the epitopes of two distinct
mAbs directed against Np1g5 to a 60 amino acid
residue region of murine Np1g5/Ap3; we detected
ll.at NP185/AP3 is phosphorytated by casein kinase
rr rn vrtro and that Np185/Ap3 direcily binds to brain
tubulin.
t4
Purification and molecular characterization of NP185, a neuronal-specific and synapse-enriched ...
Bovine NP185 has been discovered and character-
ized biochemically in our lab (Kohtz and puszkin,
1988; Kohtz and Puszkin, 1989;Su et al., 1991) , and
immuno-histochemically in mouse and chicken
(Perry et al., 1991; Perry et al., 1992; Puszkin et at.,
1992) ; while murine F1-20 has been cloned by
immunoscreening of oDNA expression library with-
out any evidence showing a relationship with other
proteins (Zhou et al., 1992). lndeed, a survey of pre-
viously published repods indicates that NP1B5,
pp155, AP1B0 and F1-20 have a number of striking
similarities: i) expression only in neuronal cells (Kohtz
and Puszkin, 1988; Sousa et al., 1992) ; ii) synapse-
specific (Perry et al., '199'l; Sousa et al., 1992) ; iii) atl
are phosphoproteins (Keen and Black, 1986; Morris
et al., 1989; Morris et al., 1990; Zhou et al., 1992) ; iv)
all are acidic and with anomalous migration on SDS-
PAGE (Ahle and Ungewickell, 1986; Keen and Black,
1986; Kohtz and Puszkin, 19BB; Zhou et al., 1992) ;
v) all are developmentally regulated in their expres-
sion (Perry et al., 1991; Sousa et al., 1992); and vi) all
are different isoforms (Kohtz and Puszkin, 1988; this
repofi; Zhou et al., 1992, 1993; Morris, et al., 1993).
However, to date there was no direct evidence for
the molecular identity of purified native NP185 with
the other forms. Thus, our current results now direct-
ly demonstrate that native bovine NP185 shares
identity with rat AP1B0, murine F1-20, bovine pp155,
They may represent the different species-specific
isoforms of this protein, namely AP3.
One of the conclusions of our work is that
NP185/AP3 functions as an adaptin-like molecule
since recombinantly-expressed NP1 85/AP3 induces
in vitro clathrin assembly. Previously, we showed that
parlially purified preparations of native bovine NP185
induce rapid and effective in vitro assembly of clathrin
molecules into closed cages (Su et al., '1991)
; identi-
cal results were obtained by others with purified
native AP1 80 (Ahle and Ungewickell, 1986). However,
preparations of native NPl 85 or native APi 80 used in
those assays may contain assembly polypeptides
AP2 contaminants, which, as observed, bind to
NP185/AP3, and could be responsible for inducing
coat assembly (Kohtz and Puszkin, 1988). To resolve
this ambiguity, we obtained recombinanfl y-expressed
and purified NP1B5/AP3 from bacterial cell lysates.
Consequently, we showed that the preparation of
recombinant NP1Bs/AP3 is sufficient to induce
clathrin cage formation, acting as a facilitator of
clathrin-assembly in vitro. Thus, NP185/Ap3 is a
bona fide assembly polypeptide as defined by this in
vitro assay system used to characterize the assembly
activity of other more well-established assembly
polypeptide complexes-namely AP-1 and Ap-2.
This confirmed the findings made independenily by
Lafer and colleagues (Ye and Lafer, 1995).

13. 15BIOQUIMICA Y PATOLOGIA CLINICA - Vol 62 - N" 1 - 1998
Two of the monoclonal antibodies directed against brain acquires high significance. lt's transient deote-
nativebovinebrainNPl85,BGBand6GT,havebeen tion in neuroblastoma cells blocks neuritogen-esis,
invaluable for the study of native NP185 structure indicating CKll regulates neuritogenesis. tn rieuronat
and function. We have used these antibodies for bio- cells, rapid responses to signal transmission Lt"chemical characterization of native NP'185 (Kohtz place in the synapse. Specific subcellular tocaiiza-
and Puszkin, 1988; Kohtz and Puszkin, 1989; Su et tion of CKll might be needed to limit its activity to
al., 199'1) , examining developmental expression potentialtargets involved in regulated rapiO synaptic
(Perryetal., 1991) ,detectionof nativeNPl85inthe vesicle recycling or fast axonal transport to the
neuromuscular junction (Perry et al., 1992) , as well synapse. As such, anchoring of CKll to subcellular
as in immuno-affinity purification of native NP185 organelles may sequesterthe kinase to appropriate
(this report). Since both mAbs can recognize NP185 substrates and compartmentalize its activity in the
by immunoblotting after transfer from SDS-PAGE synapse. lndeed, the bacterially-exiressed
gels, it is reasonable to conclude that the epitopes NP'185/AP3 fusion protein binds to brain'tubulin.
are sequential, not conformational. Here, we have Thus, NP185/AP3 mediates the interaction of vesic-
mapped the epitopes of these mAbs to a discrete 60 ular organelles with cytoskeletal protein, e.g,, by
amino acid residue region of NP1B5/AP3 that con- binding to tubulin present at the opening bale of
tains two casein kinase ll motifs for potential phos- these organelles. These correlations suggest an
phorylationof NP185/AP3fl-able2).Therefore,these increased impodance of NP185 in regulaiing CKll
two anti-NP185 mAbs may be useful for functional activity for higher neuronal functions. ln support of
characterization of NP185/AP3 phosphorylation this interpretation, a recent report shows that casein
sites both in vivo and in vitro. ln addition, as sum- kinase ll does play an imporlant role in Ap-1 func-
marized in Fig. 6, the epitope of murine F1-20 mAb tions for downstream sorting event of theTrans-Golgi
was found in the middle of F1-20 sequence- vesicularmembrane network (Mauxion et al., 19g6).
between amino acid residue 400 and residue 760 ln conclusion, NP185 interacts with majorfunctional
(Zhou et al., 1992) ; while the epitope of rat AP180 molecules, structural molecules, and membrane in
mAb was defined to reside between amino acid brain. This role is assigned in nature to molecules
residue 562 and residue 744 (Morris et al., 1993). who can also be responsible for disease conditions.
These four mAbs could be used as complementary This exciting possibility awaits furlher study.
molecular probes to further study the structure and
function of AP3. These types of studies may be Acknowledgments
required when the involvement of these polypetides We thank the members of Dr. Saul Puszkin,s labora-
in diseases of the nervous system is elucidated. The tory for their excellent technical assistance and help-
significance of its brain specificity is an indication ful discussions. We thank Dr. Eileen M. Lafer
thatthosetypesof studiesareoverdue. (University of Pittsburgh) for providing anti-F1-20
Native NP185 has been shown to be associated with mAb and Dr. David Colman (Coiumbia University) for
casein kinase ll (CKll) (Kohtz and Puszkin, 19Bg; Su the purified bovine brain tubulin. This work *"r.rp_
etal', 1991). NP185 isthrefore involved in membrane ported in part by grants from the United States
transport events associated with neurite develop- National lnstitutes of Health (NlH) NSi 2467 and
ment like found in PC12 cells (Kohtz and Puszkin, NS26113. Dr. Saul Puszkin has been Jacob Javitz
19BB). Since casein kinase ll (CKll) is enriched in the awardee by NIH for excellence in Neuroscience. Dr.
brain and its involvement in neuritogenesis is well Michael Lisanti is an awardee of Research Grants by
established(Ulloaetal., 1993;Wongetal,, 1996),the NlH. Dr. Shengwen Li was supported in part by a
regulated interactions of these two molecules in Research Fellowship award by NlH.
Shengwen Li, Michael Lisanti and Saul puszkin

14. t6
Purification and molecular characterization of NP185, a neuronal-specific and synapse-enriched ...
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Shengwen Li, Michael Lisanti and Saul Puszkin