Islet Cell Autoantigen of 69 kDa Is an Arfaptin-related Protein Associated with the Golgi Complex of Insulinoma INS-1 Cells*
Folker Spitzenberger
¶,
Susan Pietropaolo ||,
Paul Verkade **,
Bianca Habermann 
,
Sandra Lacas-Gervais
,
Hassan Mziaut
,
Massimo Pietropaolo || and
Michele Solimena
** 
From the
Experimental Diabetology and the
Department of Internal Medicine III, "Carl
Gustav Carus" Medical School, University of Technology Dresden, 01307
Dresden, Germany, the ||Division of Immunogenetics,
Diabetes Institute, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15213, the **Max Planck Institute for
Molecular Cell Biology and Genetics, 01307 Dresden, Germany, and

Scionics GmbH, 01307 Dresden,
Germany
Received for publication, December 27, 2002
, and in revised form, March 24, 2003.
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ABSTRACT
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Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still
unknown function. Involvement of ICA69 in neurosecretion has been suggested by
the impairment of acetylcholine release at neuromuscular junctions upon
mutation of its homologue gene ric-19 in C. elegans. In this
study, we have further investigated the localization of ICA69 in neurons and
insulinoma INS-1 cells. ICA69 was enriched in the perinuclear region, whereas
it did not co-localize with markers of synaptic vesicles/synaptic-like
microvesicles. Confocal microscopy and subcellular fractionation in INS-1
cells showed co-localization of ICA69 with markers of the Golgi complex and,
to a minor extent, with immature insulin-containing secretory granules. The
association of ICA69 with these organelles was confirmed by immunoelectron
microscopy. Virtually no ICA69 immunogold labeling was observed on secretory
granules near the plasma membrane, suggesting that ICA69 dissociates from
secretory granule membranes during their maturation. In silico
sequence and structural analyses revealed that the N-terminal region of ICA69
is similar to the region of arfaptins that interacts with ARF1, a small GTPase
involved in vesicle budding at the Golgi complex and immature secretory
granules. ICA69 is therefore a novel arfaptin-related protein that is likely
to play a role in membrane trafficking at the Golgi complex and immature
secretory granules in neurosecretory cells.
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INTRODUCTION
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Originally identified by immunoscreening of an islet cDNA expression
library with prediabetic sera, islet cell autoantigen of 69 kDa
(ICA69)1 is an
evolutionary conserved gene with homologues in the nonmammalian model
organisms Drosophila melanogaster and Caenorhabditis elegans
(13).
Like most antigens of type 1 diabetes, ICA69 is enriched in pancreatic
-cells and neurons (4).
Use of alternative exons in the 5'-untranslated region of the ICA69 gene
can affect its tissue expression
(5). Although ICA69 deficiency
in mice does not induce any obvious phenotype, the knockout of its C.
elegans homologue compromises neurotransmission
(3,
6). This finding has led to the
hypothesis that ICA69, despite lacking membrane anchoring signals, is linked
to neuronal synaptic vesicles (SVs) and the related synaptic-like
microvesicles (SLMVs) of endocrine cells
(3).
We now show that ICA69 is mostly associated with the Golgi complex and, to
a less extent, with immature secretory granules (ISGs) of insulinoma cells.
ICA69 is similar to arfaptins, which act as effectors of the small GTPases ARF
and Rac (7,
8). Thus, ICA69 is likely to
play a role in vesicular transport regulated by small GTP-binding proteins at
the Golgi complex and ISGs in
-cells and secretory cells in general.
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EXPERIMENTAL PROCEDURES
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MaterialsThe following antibodies were used:
affinity-purified rabbit antibodies against residues 471483 of human
ICA69 (pICA69) (1) and against
ICA512 ectodomain (9); mouse
monoclonal antibodies against human ICA69 (mICA69); GAD65
(9);
'COP (Drs. J.
Fuellekrug and K. Simons, Max Planck Institute for Molecular Cell Biology and
Genetics, Dresden, Germany); the 18-kDa fragment of secretogranin II (Dr. S.
Tooze, Cancer UK, London, UK)
(10,
11); giantin (Dr. H.-P. Hauri,
Biozentrum, Basel, Switzerland)
(12); TGN38, GM130, syntaxin
6, and carboxypeptidase E/H (BD Transduction Laboratories, Heidelberg,
Germany); ARF1 (Affinity Bioreagents, Golden, CO); proinsulin (nonreactive
against insulin) (Research Diagnostics); synaptophysin (Synaptic Systems,
Goettingen, Germany); transferrin receptor (Zymed Laboratories, South San
Francisco, CA); insulin and
-tubulin (Sigma), goat anti-rabbit and
anti-mouse IgGs conjugated to Alexa 488 or Alexa 568 (Molecular Probes, Inc.,
Eugene, OR); and goat anti-rabbit and goat anti-mouse IgGs conjugated with
gold particles (Electron Microscopy Sciences, Fort Washington, PA). All other
reagents were from Sigma, unless otherwise specified.
ImmunofluorescenceMouse brains were fixed and immunolabeled
as described (13). Rat INS-1
cells, hamster HIT-15, and mouse MIN6 insulinoma cells were cultured and
processed as described
(1316).
For
-tubulin staining, cells were fixed with cold methanol (-20 °C)
for 5 min and labeled as described
(17). Antibodies were diluted
as follows: pICA69 (1:200) and mICA69 (1:20). All other antibodies were used
according to the providers' instructions. Confocal microscopy was performed
with an LSM 510 station (Carl Zeiss, Jena, Germany). For drug treatments,
cells were incubated for 1 h or 30 min in normal medium plus 20 µg/ml
nocodazole or 2 µg/ml BFA, respectively. For mild destabilization of the
microtubule cytoskeleton, cells were processed as described
(18).
Biochemical ProceduresRat brain and INS-1 cells were
homogenized and centrifuged as described
(19,
20). Postnuclear supernatants
(PNS) were spun at 150,000 x g for 1 h at 4 °C. The
resulting high speed supernatant was collected while the high speed pellet was
brought back to the original volume with homogenization buffer. Protein
concentrations were measured with the BCA reagent (Pierce). For Western
blotting, proteins were separated by 10% SDS-PAGE and blotted with the
following antibodies: pICA69 (1:1,000), mICA69 (1:200), and synaptophysin
(1:10,000). Immunoreactivity was detected by chemiluminescence (Amersham
Biosciences) using a LAS-1000 Bioimaging System (Fujifilm, Tokyo, Japan).
Immunoprecipitation was carried out essentially as described
(21). 600 µl of the
extracts were incubated overnight at 4 °C with 10 µl of mICA69, 25
µl of pICA69, or 10 µl of rabbit IgGs as a negative control.
In Vitro Transcription/TranslationIn vitro
transcribed/translated [35S]Met-labeled fragments of human ICA69
(N-terminal fragment: residues 1239; C-terminal fragment: residues
230483) were incubated with mICA69 for 16 h and then for 45 min with
protein G-Sepharose (Amersham Biosciences) at 4 °C. An IgG1
immunoglobulin was used as negative control. Immunoprecipitates were separated
by SDS-PAGE and visualized by exposing autoradiography films for 1215 h
at -80 °C.
[
-32P]GTP OverlayINS-1 cells were
processed for immunoprecipitation as described above.
[
-32P]GTP overlay assay was performed as previously
described (22). Radiolabeling
was detected with the Bio-Imager Analyzer BAS-1800II (Fujifilm).
Subcellular FractionationRat brain subcellular fractions
and purified SVs (23) were
kindly donated by Dr. W. Huttner (Max Planck Institute for Molecular Cell
Biology and Genetics, Dresden, Germany) and Dr. R. Jahn (Max Planck Institute
for Biophysical Chemistry, Goettingen, Germany). Fractions were immunoblotted
with pICA69 and synaptophysin antibodies. Fractionation of INS-1 cells on
continuous sucrose density gradients (0.41.8 M sucrose) was
performed as described
(2426).
Fractions were immunoblotted for ICA69 (pICA69), synaptophysin, TGN38 (1:200),
GM130 (1:200), syntaxin 6 (1:500), transferrin receptor (1:2,500),
carboxypeptidase E (CPE) (1:2,000), and the 18-kDa fragment of secretogranin
II (1:400). Semiquantitative measurement was performed with the Image Reader
2.2 software of the LAS-1000 imaging system (Fujifilm).
Immunoelectron MicroscopyImmunoelectron Microscopy on
gradient fractions was performed as described
(27) using antibodies pICA69
(1:50) and anti-TGN38 (1:25) followed by goat anti-rabbit and anti-mouse
antibodies conjugated with 10- and 6-nm gold particles, respectively.
Immunoelectron microscopy on ultrathin cryostat sections was performed as
described (28) with minor
variations. INS-1 cells were fixed with 4% paraformaldehyde and 0.05%
glutaraldehyde in phosphate-buffered saline. Sections were simultaneously
labeled with pICA69 (1:10) and anti-giantin (1:10) or anti-insulin (1:100)
antibodies, followed by 6- and 12-nm gold-conjugated anti-mouse and
anti-rabbit antibodies, respectively. As controls, primary antibodies were
omitted.
Data Base Searches and Structural PredictionsData base
searches were carried out with the programs BLASTP and PSI-BLAST
(29) using standard parameters
against the nonredundant NCBI protein data base (release of November 27,
2001). Multiple sequence alignments were produced with the program ClustalX
(30,
31) and manually refined.
Structure prediction was done using the programs 3D-PSSM
(32) and PredictProtein
(33).
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RESULTS
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Characterization of Two Antibodies Directed against Distinct Epitopes
of ICA69 The localization of ICA69 in neurons and insulinoma cells
was investigated by employing two distinct antibodies directed against ICA69.
The first antibody consists of affinity-purified rabbit IgGs (polyclonal
ICA69, or pICA69), which were raised against residues 471483 at the
C-terminal end of ICA69 (1).
The second antibody was a monoclonal IgG (mICA69), which was obtained by mouse
immunization with full-length recombinant ICA69 expressed in bacteria as a
fusion protein with glutathione S-transferase. To map the epitope
recognized by mICA69, the N-terminal (amino acids 1239) and C-terminal
(amino acids 230483) halves of ICA69 were independently expressed as
35S-labeled polypeptides by in vitro
transcription/translation and incubated either with mICA69 or control mouse
IgG for immunoprecipitation followed by SDS-PAGE and autoradiography
(Fig. 1A). This
analysis shows that mICA69 selectively immunoprecipitated the N-terminal ICA69
polypeptide, whereas control mouse IgG did not react with either ICA69
fragment. Whereas the N-terminal fragment of ICA69 has a molecular mass of 28
kDa, it migrated as a 35-kDa protein by SDS-PAGE. The aberrant electrophoretic
mobility of the N-terminal domain may account for the overall slower migration
of native ICA69, which has a molecular mass of 54.6 kDa but migrates as a
60-kDa protein under the present conditions for SDS-PAGE
(1)
(Fig. 1B). By Western
blotting on PNS from rat brain (Fig.
1B) and INS-1 cells
(Fig. 1C), both mICA69
and pICA69 antibodies recognized indeed a protein doublet of 60 kDa
(Rf = 0.34 and 0.37, respectively). Given its detection
with two antibodies directed against distinct ICA69 epitopes, this 60-kDa
protein doublet is likely to represent ICA69. The amino acid sequence of ICA69
contains a second methionine at position 26. Initiation of translation at this
alternative methionine may explain the presence of a protein doublet upon
in vitro translation (Fig.
1A) and in cell extracts
(Fig. 1, B and
C). Moreover, mICA69 and pICA69 antibodies
immunoprecipitated the same 60-kDa protein doublet from INS-1 cell extracts
(Fig. 1D) and produced
an identical particulate staining by immunocytochemistry
(Fig. 2A).
Partitioning of ICA69 between cytosolic and particulate fractions
(Fig. 1C) suggested
that a pool of the protein is membrane-associated, despite lacking membrane
targeting motifs.

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FIG. 1. Recognition of ICA69 by two antibodies against distinct ICA69
epitopes. A, [35S]Met-labeled in vitro
transcribed/translated (IVTT) N-terminal (amino acids 1239;
lanes 1 and 2) and C-terminal (amino acids 230483;
lanes 3 and 4) fragments of human ICA69 immunoprecipitated
(IP) with mICA69 (lanes 2 and 4) or nonspecific
IgG1 as control (lanes 1 and 3). Analysis of
immunoprecipitates by SDS-PAGE and autoradiography showed that mICA69
recognizes an epitope located within the N-terminal half of ICA69. B
and C, Western blotting with mICA69 (B, lane 2; C, lanes
13) and pICA69 (B, lane 3; C, lanes 46)
on rat brain PNS (B) and INS-1 cell fractions (C). ICA69
(60-kDa doublet) is present in the PNS, high speed supernatant (HSS),
and high speed pellet (HSP) of INS-1 cells. D, following
cross-immunoprecipitation and Western blotting (WB), pICA69 (lane
2) and mICA69 (lane 3) recognized the same 60-kDa protein
doublet. Nonspecific rabbit IgGs were used for immunoprecipitation as controls
(lane 4).
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FIG. 2. Confocal microscopy for ICA69 and markers of SLMVs, SVs, and SGs.
A, mICA69 (pseudogreen) and pICA69 (pseudored)
produced a nearly identical staining pattern in the perinuclear region of
INS-1 cells (orange-yellow pseudocolor). N, nucleus.
B, high power magnification of INS-1 cells double stained for ICA69
(pseudored) and synaptophysin (pseudogreen). While
synaptophysin was enriched at neurite-like cell extensions (long
arrow), ICA69 was concentrated in the perinuclear region
(arrowhead). C, ICA69 (pseudogreen) did not
co-localize with ICA512 (pseudored), a protein enriched in MSGs.
DF, distribution of ICA69 (pseudored) in mouse brain.
Double labelings with GAD65 (D and E) and synaptophysin
(F) as markers of SVs (both in pseudogreen). D,
ICA69 immunoreactivity was prominent in Purkinje cells (PC, arrow),
but not in the molecular layer (ML) or granular layer (GL),
where most synaptic terminals of the cerebellar cortex are found. E
and F, high power magnifications showing that ICA69 is associated
with perinuclear structures (arrowheads), whereas it does not
co-localize with GAD65 (E) or synaptophysin (F) in synaptic
terminals around the cell bodies (arrows) and axon hillocks
(asterisks) of Purkinje cells.
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ICA69 Is Not Enriched in Neuronal Synaptic Vesicles and
Synaptic-like-Microvesicles of INS-1 CellsPrevious studies
suggested an association of ICA69 with neuronal SVs and SLMVs of insulinoma
cells (3). Double
immunofluorescence on INS-1 cells, however, showed that ICA69 immunoreactivity
was concentrated in the perinuclear region
(Fig. 2A), whereas
virtually no signal was found at the tip of neurite-like extensions where
synaptophysin and SLMVs are concentrated
(Fig. 2B)
(34,
35). ICA69 distribution was
also different from that of ICA512, a protein enriched in mature secretory
granules (MSGs) near the plasma membrane
(Fig. 2C)
(9). Double immunofluorescence
on mouse cerebellar sections showed that ICA69 is found in the perinuclear
region of Purkinje cells (Fig. 2,
DF). This pattern did not resemble that of GAD65,
which is associated with SVs of GABA-ergic synapses around Purkinje cells and
throughout the cerebellar cortex (Fig. 2,
D and E)
(24,
36). There was also no
co-localization of ICA69 with synaptophysin
(Fig. 2F), a general
marker of SVs and synaptic terminals
(34). Similar observations
were made upon subcellular fractionation of rat brain synaptosomes. As
expected (37), synaptophysin
was concentrated in the LP2 fraction (crude SVs) and further enriched in the
sucrose-gradient vesicle fraction (purified SVs), whereas ICA69 was most
abundant in the LP1 (large membranes and cytoskeleton) and LS2 (synaptosol)
fractions (not shown). These data indicated that ICA69 is neither enriched on
neuronal SVs nor on SLMVs and MSGs of insulinoma cells.
Localization of ICA69 to the Golgi Complex and Immature Secretory
Granules by Confocal MicroscopyWe compared the perinuclear pattern
of ICA69 with that of proteins enriched in the Golgi compartment of INS-1
cells (Fig. 3,
AF). ICA69 was co-localized in part with TGN38
(Fig. 3A) and giantin
(Fig. 3B). Giantin is
a general marker of the Golgi complex
(12), whereas TGN38 is
enriched at the trans-Golgi network (TGN)
(38,
39). ICA69 also partially
overlapped with the peripherally associated Golgi proteins
'COP
(Fig. 3C)
(4043)
and GM130 (Fig. 3D)
(44). Finally, ICA69
overlapped in part with proinsulin, which is found in ISGs, but not in MSGs
(Fig. 3E)
(45,
46). It also co-distributed
with insulin in the Golgi region but not near the plasma membrane
(Fig. 3F). The
significant, albeit partial, colocalization of ICA69 with Golgi markers GM-130
and TGN38 was confirmed in hamster insulinoma HIT-cells and mouse insulinoma
MIN-6 cells (Fig. 4,
AI). These results suggested that ICA69 is broadly
associated with the Golgi complex and, to a much lesser extent, with ISGs.

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FIG. 3. Confocal microscopy for ICA69 (pseudored), markers of the Golgi
complex and SGs in rat insulinoma INS-1 cells (pseudogreen).
ICA69 overlaps significantly with TGN38 (A) and giantin (B).
Partial overlap was also found with 'COP (C), with which
the co-localization extended to structures outside the Golgi region, and with
GM130 (D). Double labelings for ICA69 and proinsulin showed some
co-distribution in the perinuclear region (E). F, ICA69
co-distributed with insulin in the perinuclear area (arrowhead) but
not near the plasma membrane, where MSGs accumulate (long arrow).
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FIG. 4. Confocal microscopy for ICA69 (pseudored, left panels) and
markers of the Golgi complex (pseudogreen, central panels) in hamster
HIT (AF) and mouse MIN6 (GI) insulinoma
cells. ICA69 partially overlaps significantly with TGN38
(AC) and GM-130 (DI). Merged images
are shown in the right panels.
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Localization of ICA69 at the Golgi Complex and Immature Secretory
Granules by Immunoelectron MicroscopyBy immunoelectron microscopy
on cryoultrathin sections of INS-1 cells, ICA69 was found on Golgi stacks
close to giantin (Fig.
5A) and at budding sites of vesicles from the Golgi
complex (Fig. 5B) and
SGs (Fig. 5C).
Overall, ICA69 was found on a minority of SGs
(Fig. 5E), as
identified by double gold immunolabeling with insulin (not shown).
ICA69-positive SGs were most abundant in the central region of the cell
(Fig. 5D), with a mean
distance from the plasma membrane that was about twice the mean distance of
ICA69-negative SGs and SGs in general (Fig.
5F). These data indicated that ICA69 is associated with
the Golgi complex, whereas a minor pool is also found on ISGs, which are
significantly more distant from the plasma membrane than the total of the
SGs.

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FIG. 5. Ultrastructural localization of ICA69 in INS-1 cells. In double
labelings, the staining with the pICA69 antibody (12-nm gold) was compared
with the staining for giantin (6-nm gold). ICA69 co-localizes with giantin on
Golgi cisternae (A). In addition, it is found on SGs (A),
which sometimes seem to be budding from the Golgi (B). C,
ICA69 is also present on vesicles which appear to emerge from SGs. D,
ICA69-negative SGs concentrate near the plasma membrane of INS-1 cells,
whereas ICA69-positive SGs are preferentially found toward the cell center.
E, ICA69 was detected on 27 (15.7%) of 172 SGs counted in 10 INS-1
cells. F, the mean distance of the ICA69-positive SGs from the plasma
membrane (840 nm, S.E. = 99 nm) was about twice the mean distance of the
ICA69-negative SGs (415 nm, S.E. = 37 nm; p = 0.000022) and of the
overall mean distance of all measured SGs from the plasma membrane (482 nm,
S.E. = 37 nm, p = 0.00057). The bar in D represents
150 nm for A and B, 100 nm for C, and 250 nm for
D.
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Enrichment of ICA69 on the Golgi Complex by Subcellular
FractionationFractionation of INS-1 cell extracts on sucrose
density gradients separated two main peaks of ICA69 at 0.9 and 1.1
M sucrose (Fig.
6A). These fractions included the peaks of the TGN
proteins TGN38 and syntaxin 6 (0.9 M sucrose) and the early Golgi
marker GM130 (1.1 M sucrose)
(Fig. 6B, left
panel). The soluble pools of ICA69 and GM130 were recovered in light
fractions (0.40.5 M sucrose), whereas low levels of ICA69
were detected in 1.21.4 M sucrose fractions. These last
fractions contained the first peak of the SG marker CPE
(Fig. 6B, middle
panel), indicating the presence of ISGs, which are lower in density than
the MSGs. A second pool of CPE was found in denser fractions (1.51.7
M sucrose) together with p18, a marker of MSGs
(11,
47). The peaks of ICA69 were
slightly shifted from those of synaptophysin and transferrin receptor, which
overlapped in 1.0 M sucrose fractions
(Fig. 6B, right
panel) (48). The gradient
fractions were also examined by immunoelectron microscopy. The 0.9
M sucrose fraction contained membranous, ribbon-like structures
that resemble Golgi stacks and were positive for ICA69 and TGN38
(Fig. 6C, left
panel), whereas the 1.35 M sucrose fraction contained SGs
positive for ICA69 (Fig.
6C, right panel). These data confirmed the
association of ICA69 with Golgi membranes and, to a lesser extent, with
ISGs.

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FIG. 6. Distribution of ICA69 by subcellular fractionation of INS-1 cells on
sucrose density gradients (0.41.8 M). A, equal
volumes of each fraction were immunoblotted with pICA69. B, profile
distribution of different organelle markers in the gradients as determined by
immunoblotting and chemiluminescence. Levels of ICA69 were compared with those
of TGN38, syntaxin 6, and GM130 as Golgi markers (B, left panel), CPE
and p18 as SG markers (B, middle panel), and synaptophysin and
transferrin receptor as markers of SLMVs and recycling endosomes, respectively
(B, right panel). ICA69 mostly co-distributed with late and early
Golgi markers (fractions 9 and 14, 0.9 and 1.1 M sucrose,
respectively) and to a lesser extent with the first peak of CPE (fractions
1618, 1.21.4 M sucrose), corresponding to ISGs.
C, fractions 9 (0.9 M sucrose) and 17 (1.35 M
sucrose) were examined by immunoelectron microscopy. Fraction 9 (left
panel) contained Golgi-like structures positive for ICA69 (12-nm gold,
arrowhead) and TGN38 (6-nm gold, arrow); fraction 17
(right panel) was enriched in SGs positive for ICA69. Sucrose
molarity was as follows: 0.43 ± 0.02 M (fraction 1); 0.74
± 0.02 M (fraction 5), 0.93 ± 0.03 M
(fraction 10), 1.16 ± 0.04 M (fraction 15), 1.46 ±
0.07 M (fraction 20), 1.84 ± 0.04 M (fraction
25).
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The Distribution of ICA69 Is Nocodazole- and
BFA-sensitiveWe analyzed whether drugs that affect the
organization of the Golgi complex alter the distribution of ICA69 in INS-1
cells. Nocodazole treatment caused a major loss of ICA69 immunoreactivity in
the perinuclear region (Fig.
7A). Some ICA69, however, could still be detected on
TGN38-labeled Golgi fragments scattered throughout the cells. Treatment of
cells with nocodazole followed by taxol causes a redistribution and mild
destabilization of the microtubule cytoskeleton similar to what occurs in the
prophase of mitosis (18,
49). In these conditions,
ICA69 had a bipolar distribution that overlapped with TGN38 at opposite sides
of the nucleus (Fig.
7B), near the centrioles
(Fig. 7C). BFA causes
the release of coatomers from Golgi membranes and the retrieval of resident
Golgi enzymes into the endoplasmic reticulum by inhibiting guanine nucleotide
exchange on ARF (50). It also
leads to the formation of a hybrid TGN-endosomal system near the
microtubule-organizing center (MTOC) by inhibiting the TGN-endosome recycling
pathway
(5154).
ICA69 immunoreactivity was significantly reduced upon BFA treatment (compare
Fig. 7, DF,
with Figs. 2,
3, and
7, B and C),
suggesting a redistribution of ICA69 in the cytosol, similar to what has been
shown for
'COP
(55). A pool of ICA69,
however, retained a juxtanuclear position within one or two brightly stained
clusters that were distinct from GM130-positive Golgi remnants
(Fig. 7E)
(44,
56) but that still contained
some TGN38 (Fig. 7D)
and especially syntaxin 6 (Fig.
7F). Syntaxin 6 participates in the TGN-endosomal
recycling pathway and is also found on ISGs
(11,
57). These observations
implied that the perinuclear concentration of ICA69 is microtubule-dependent
and emphasized the relationship of ICA69 with the Golgi complex.

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FIG. 7. ICA69 distribution upon pharmacological treatments of INS-1 cells.
A, after nocodazole treatment, the perinuclear staining for ICA69
(pseudored) disappeared. Residual ICA69 labeling was associated with
TGN38-positive Golgi fragments (pseudogreen) (A, arrows).
B, upon mild destabilization of the microtubule cytoskeleton by
nocodazole/taxol treatment, ICA69 immunoreactivity (pseudored)
overlapped with TGN38 (pseudogreen) in a bipolar manner at opposite
sides of the nucleus, near the centrioles, as visualized with
anti- -tubulin antibody (pseudogreen) (C). D,
BFA treatment reduced ICA69 immunoreactivity, whereas the integral membrane
protein TGN38 redistributed to the endoplasmic reticulum. E, the
residual ICA69 immunoreactivity was concentrated in one or two bright clusters
close to the MTOC (not shown), which did not overlap with
GM130-positive structures (pseudogreen) (E). A large
fraction of ICA69 co-localized instead with syntaxin 6 (pseudogreen)
(F).
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ICA69 Is Related to ArfaptinIterated BLAST searches with
the ICA69 sequence against current protein data bases indicated that the
N-terminal region of ICA69 is significantly similar to arfaptins
(Fig. 8A), with an
expect value of 2e-70 at convergence of PSI-BLAST searching. Back
PSI-BLAST searches with human arfaptin 1 identified the ICA69 family with an
expect value of 9e-73 at the third iteration. Arfaptin 1 and 2 are
cytosolic proteins that bind small GTPases of the ARF family
(8) and Rac1
(7). Arfaptin 1, in particular,
is recruited to Golgi membranes by ARF1-GTP and acts as negative regulator of
ARF1 function (8,
58,
59). Arfaptin is composed of
three
-helices that dimerize to form the binding interface for small
GTPases. Both dimer subunits contribute to a domain that is structurally
related to the Dbl homology domain of Tiam, a guanine nucleotide exchange
factor for Rac
(6062).
The secondary structure of ICA69 N-terminal half is predicted to be primarily
-helical (Fig.
8A) and coincides with that of human arfaptin 2, as
deduced from its tertiary structure
(63). Thus, it is likely that
ICA69 folds like arfaptins and that ICA69 dimerization generates a Dbl
homology-like domain that binds small GTPases. Through this interaction, ICA69
may participate in regulating membrane trafficking at the Golgi complex and
ISGs. These functional implications were sustained by the finding that both
mICA69 and pICA69 antibodies co-immunoprecipitated a yet unidentified small
GTP-binding protein from INS-1 cell extracts
(Fig. 8B). This small
GTP-binding protein comigrated with ARF1
(Fig. 7B) but did not
react with the anti-ARF1 antibody following immunoblotting of the material
immunoprecipitated by the ICA69 antibodies (not shown). Interestingly, several
of the residues involved in the binding of arfaptin 2 to small GTPases are not
conserved in ICA69 (Fig.
8A), suggesting that ICA69 may interact with small
GTPases other than ARF1 or Rac1.

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FIG. 8. ICA69 is related to arfaptins. A, sequence alignment of the
N-terminal region of human ICA69 (residues 1274) and ICA69 homologues
with members of the arfaptin family from different species (Hs, Homo
sapiens (arfaptin 1, NP_055262
[GenBank]
; arfaptin 2, NP_036534
[GenBank]
; Ica69, NP_071682
[GenBank]
);
Mm, Mus musculus (arfaptin 2, BAB26070
[GenBank]
; Ica69, XP_193008
[GenBank]
); Rn,
Rattus norvegicus (Ica69, NP_110471
[GenBank]
); Dm, Drosophila
melanogaster (arfaptin, NP_650058
[GenBank]
; Ica69, NP_649283
[GenBank]
); Ag, Anopheles
gambiae (arfaptin, EAA10493
[GenBank]
; Ica69, EAA11288
[GenBank]
); Ce, Caenorhabditis
elegans (Ica69, NP_491216
[GenBank]
)). Conserved residues are highlighted in
yellow. Residues highlighted in dark gray (arfaptin family)
or light gray (ICA69 family) are conserved within the arfaptin or
ICA69 family but differ between the two. The red circles indicate
conserved residues among arfaptins that participate in the binding between
arfaptin 2 and Rac1 (63) but
that significantly differ in ICA69. -Helices of arfaptin and ICA69 are
shown in green and blue, respectively. B,
[ -32P]GTP overlay assay on immunoprecipitates (IP)
from INS-1 cell extracts obtained with mICA69 (lane 1), pICA69
(lane 3), and mouse anti-ARF1 (lane 2) or rabbit pre-immune
IgGs (lane 4) as positive and negative controls, respectively. Both
anti-ICA69 antibodies immunoprecipitated an unidentified small GTP-binding
protein.
|
|
 |
DISCUSSION
|
---|
We have shown here that ICA69 is peripherally associated with the Golgi
complex and ISGs. This conclusion is consistent with results obtained by
confocal microscopy, immunoelectron microscopy, and subcellular fractionation.
Additional support for these findings has been obtained using pharmacological
treatments that affect the organization of the Golgi complex and post-Golgi
membrane compartments.
Previous studies suggested that the C. elegans homologue of ICA69,
termed ric-19, is associated with neuronal SVs
(3). A deletion mutant of
ric-19, in particular, was shown to confer resistance to the
acetylcholinesterase inhibitor aldicarb, thus implying a reduced secretion of
acetylcholine from SVs at neuromuscular junctions. Confocal microscopy on
brain sections, however, did not reveal a co-localization of ICA69 with SV
markers. In INS-1 cells ICA69 also did not co-distribute with markers of
SLMVs, the counterpart of neuronal SVs in peptide-secreting endocrine cells
(64). Furthermore, ICA69 was
not found on purified brain SVs nor co-distributed with SLMVs upon
fractionation of INS-1 cells. Thus, our data do not support the notion that
ICA69 is enriched on SVs/SLMVs. This conclusion, however, is not in conflict
with the observation that mutation of ric-19 hinders the secretion of
acetylcholine. Impaired secretion may result from alterations in membrane
trafficking upstream of SVs/SLMVs.
Our findings point to a tight connection of ICA69 with the trans-face of
the Golgi complex, from which SGs and precursors of SVs/SLMVs originate.
Immunoelectron microscopy showed the presence of ICA69 on SGs in the process
of budding from the TGN. Upon microtubule disruption (nocodazole treatment),
microtubule reorganization (nocodazole, taxol treatment), Golgi-endoplasmic
reticulum fusion and TGN-endosome fusion (BFA treatment), a pool of ICA69
remained associated with membranes positive for TGN38. Upon treatment with
BFA, ICA69 also co-localized extensively with syntaxin 6 near the MTOC.
Syntaxin 6 is a soluble N-ethylmaleimide-sensitive attachment protein
receptor protein of the TGN and early endosomes
(65) that was shown to
redistribute around the MTOC in BFA-treated cells
(66).
Besides being enriched at the TGN, a minor pool of ICA69 is found on ISGs,
whereas it is virtually absent from MSGs. Occasionally, ICA69 was detected on
what appeared to be small vesicles pinching off the membrane of SGs. Whereas
the presence of a coat was not readily apparent, such profiles could
conceivably represent budding clathrin-coated vesicles that remove proteins
not destined to MSGs (57,
67,
68). Protein removal from ISGs
is part of the maturation process that leads to the formation of MSGs
(69,
70). The departure of ICA69
from ISGs together with these vesicles could explain its absence on MSGs. In
this respect, ICA69 resembles again syntaxin 6, which is also associated with
ISGs but not with MSG of
-cells
(11,
57). Additional studies will
be necessary to determine whether ICA69 plays an active role in the maturation
process of ISGs.
The similarity of ICA69 with arfaptins strongly suggests that ICA69
participates in membrane trafficking. Arfaptin 1 binds to ARF proteins in
their GTP-bound conformation
(8). ARFs, in turn, regulate
membrane dynamics by promoting the recruitment of coat proteins on membranes
as well as by affecting the activity of phospholipid-modifying enzymes and the
organization of the actin cytoskeleton
(71,
73). In addition to ARFs,
arfaptin 2 can also bind Rac and has therefore been proposed as a potential
mediator of cross-talk between ARF and Rac
(7,
63,
74). Given the sequence
similarity of the N-terminal region of ICA69 with the arfaptin Dbl
homology-like domain, it is conceivable that ICA69 dimers might also bind
small GTPases. Additional studies will be required to identify its binding
partners. Thus, our findings raise the possibility that ICA69 acts in concert
with small GTPases in regulating membrane dynamics at the Golgi complex and
ISGs. The wide tissue expression of ICA69
(1,
4,
5), suggests that this
regulation may not be restricted to neuroendocrine cells.
Evidence that ICA69 is found on the TGN and ISGs of INS-1 cells provides
additional support to the notion that in type 1 diabetes autoantibodies are
preferentially directed toward proteins associated with the post-Golgi
secretory machinery of
-cells
(75). Interestingly, ICA69 has
also been recently identified as an autoantigen in Sjögren's syndrome
(6), an autoimmune disorder
that affects primarily the exocrine cells of the salivary gland.
Autoantibodies of patients with Sjogren's syndrome are typically directed
against proteins associated with the Golgi complex
(76). The association of ICA69
with Golgi membranes and insulin-containing secretory granules could therefore
account for the occurrence of ICA69 autoimmunity in both Sjogren's syndrome
and type 1 diabetes. Future studies will determine the contribution of ICA69
to membrane trafficking and the significance of autoimmunity against ICA69 in
diseases affecting secretory cells.
 |
FOOTNOTES
|
---|
* This work was supported by grants from the National Institutes of Health
(to M. S. and M. P.), the American Diabetes Association (to M. S. and M. P.),
and the Alexander von Humboldt Foundation (to M. S.). The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶ Recipient of a Meddrive grant of the Medical School at the University of
Technology Dresden. 

To whom correspondence should be addressed: Experimental Diabetology,
"Carl Gustav Carus" Medical School, University of Technology
Dresden, Fetscherstr. 74, 01307 Dresden, Germany. Tel.: 49-351-458-6611; Fax:
49-351-458-6330; E-mail:
michele.solimena{at}mailbox.tu-dresden.de.
1 The abbreviations used are: ICA69, islet cell autoantigen of 69 kDa; BFA,
brefeldin A; CPE, carboxypeptidase E; GAD65, glutamic acid decarboxylase of 65
kDa; ISG, immature secretory granule; MTOC, microtubule organizing center;
PNS, postnuclear supernatant; MSGs, mature secretory granules; SG, secretory
granule; SVs, synaptic vesicles; SLMVs, synaptic-like microvesicles; TGN,
trans-Golgi network. 
 |
ACKNOWLEDGMENTS
|
---|
We thank P. De Camilli for critical discussion and reading of the
manuscript; A. De Matteis for discussion and sharing preliminary data on the
relationship of ICA69 with arfaptins; W. Huttner and R. Jahn for purified SV
preparations; M. Wilsch-Bräuniger for advice; J. Fuellekrug, K. Simons,
S. Tooze, G. Warren, H.-P. Hauri, and J. Saraste for the gift of antibodies;
and P. Meda and R. Sherwin for insulinoma cell lines. We thank J. Graessler
and H.-E. Schroeder for encouragement, I. Unfried for skillful technical
support, and K. Pfriem for excellent assistance.
 |
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