Báo cáo khoa học: The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway doc

The fate of newly synthesized V-ATPase accessory subunit Ac45
in the secretory pathway
Vincent Th. G. Schoonderwoert, Eric J. R. Jansen and Gerard J. M. Martens
Department of Animal Physiology, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, the Netherlands
The vacuolar H
+
-ATPase (V-ATPase) is a multimeric
enzyme complex that acidifies organelles of the vacuolar
system in eukaryotic cells. Proteins t hat interact with t he
V-ATPase may play a n i mportant role in controlling the
intracellular localization and activity of the proton pump.
The neuroendocrine-enriched V-ATPase a ccessory subunit
Ac45 may represent such a protein as it has been shown to
interact with the membrane sector of the V-ATPase in only a
subset of organelles. Here, we examined the fate of newly
synthesized Ac45 in the secretory pathway of a neuroendo-
crine cell. A major portion of intact  46 -kDa Ac45 was
found to be N-linked glycosylated to  62 kDa and a minor
fraction t o  64 kDa. Trimm ing of the N-linked glycans
gave rise to glycosylated Ac45-forms of  61 and  63 kDa
that are cleaved to a C-terminal f ragment of 42–44 kDa (t he
deglycosylated form is  23 kDa), and a previously not
detected  22-kDa N-terminal cleavage fragment (the
deglycosylated form is  20 kDa). Degradation of the
N-terminal fragment is rapid, does not occur in lysosomes
and is inhibited by b refeldin A. Both the N - and C-terminal
fragment pass the medial Golgi, as t hey become partially
endoglycosidase H resistant. The Ac45 cleavage event is a
relatively slow process (half-life of i ntact Ac45 is 4 –6 h) and
takes place in the e arly secreto ry pathway, as it is not affected
by brefeldin A and monensin. Tunicamycin inhibited

N-linked g lycosylation of Ac45 and interfered w ith t he
cleavage process, suggesting t hat Ac45 need s proper folding
for the cleavage to occur. Together, our results i ndicate that
Ac45 folding and cleavage occur slowly and early in the
secretory pathway, and that the cleavage event may be linked
to V-ATPase activation.
Keywords: acidification; regulated secretory pathway; post-
translational modification; vacuolar proton ATPase;
Xenopus.
Acidification of organelles in eukaryotic cells is required for
a variety of cellular processes, such as the release of ligands
from receptors during endocytosis and the hydrolysis of
macromolecules in lysosomes [1–3]. In the secretory path-
way, the lumen gradually acidifies from endoplasmic
reticulum (ER) to Golgi to secretory granules (reviewed in
[4]). The pH of the lumen of the ER, Golgi, and trans-Golgi
Network ( TGN) is  7.3,  6.4, and  6.0, re spectively, and
is similar in regulated and nonregulated secretory cells
[5–10]. The significance of the pH in the ER remains to be
established, although it seems likely that ER processes such
as protein glycosylation and f olding depend on it. The low
pH in the Golgi has been shown to be important for the
regulation of protein–protein interactions [11,12] and the
activity of the N-glycan processing enzyme sialyltransferase
[13]. In the TGN, an acidic pH is necessary for the proper
processing of proproteins [14] and for the condensation of
regulated secretory proteins, which is important for their
targeting to immature secretory granules [15–17]. Immature
secretory granules mature and become progressively more
acidic (pH o f  5.5 [18–20]). Granular acidification further

concentrates regulated proteins [21], while nonregulated
proteins are sorted away into clathrin-coated vesicles that
pinch off from the maturing granule [22–24]. Furthermore,
the acidic granular pH is necessary for the processing
enzymes to efficiently cleave the prohormones [25].
Acidification of intracellular compartments is established
and maintained by the vacuolar H
+
-ATPase (V-ATPase).
This multimeric enzyme complex consists of at least 13
different subunits that have been classified into a membrane
integral sector (V
0
) and a peripheral sector (V
1
) [26,27]. The
V-ATPase V
1
sector contains the c atalytic site which
hydrolyses ATP to translocate protons across the mem-
brane by the proton-pore f orming V
0
sector. In the ER, the
assembly of the V -ATPase starts with the V
0
-sector and m ay
be completed in t his c ompartment by the build-up of the V
1
onto the V
0

[28,29]. Given the pH in the ER, the V-ATPase
should be considered as being essentially inactive in this part
of the secretory pathway. An active V-ATPase is required
further downstream in the secretory pathway but it is not
known in which compartment the V-ATPase becomes
active and which mechanism is involved in the targeting of
the V-ATPase to the various secretory pathway compart-
ments. V-ATPase interacting proteins, such as the accessory
subunit Ac45, may play an important role in this targeting
process, as Ac45 has been shown to interact with the
Correspondence to G. J. M. Martens, Department of Animal Physi-
ology, Nijmegen Center for Molecular Life Sciences, University of
Nijmegen, Geert Grooteplein Zuid 28, 193RT, 6525 GA Nijmegen,
the Netherlands. F ax: + 31 24 3615317, Te l.: + 31 24 3610564,
E-mail:
Abbreviations: Baf, bafilomycin A 1; BFA , brefe ldin A ; E ndoH,
endoglycosidase H; ER, endoplasmic reticulum; NDGA, nordi-
hydroguaiaretic acid; NIL, neurointermediate lobe; PC2, prohormone
convertase 2; POMC, proopiomelanocortin; TGN, trans-Golgi net-
work; V-ATPase, vacuolar H
+
-ATPase.
Note: a web page is available at
/>(Received 26 October 2001, revised 6 February 2002, accepted 8
February 2002)
Eur. J. Biochem. 269, 1844–1853 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02831.x
membrane sector of the V-ATPase in only a s ubset of
organelles [30]. A c45 was initially isolated from bovi ne
chromaffin granules and identified as a type I t ransmem-
brane protein of 45 kDa [30]. However, N-terminal

sequencing of the isolated 45-kDa protein and the cloning
of full-length Ac45 cDNA re vealed that the isolated protein
represents a c leaved fragment o f a larg er protein [ 30,31]. In a
differential screening strategy aimed at identifying genes
that are involved in the biosynthesis and release of peptide
hormones, we isolated a cDNA (X1311) encoding Ac45 of
the amphibian Xenopus laevis [32]. Th e melanotrope cells of
the Xenopus intermediate pituitary were used f or this
screening approach because the activity of these neuroen-
docrine cells can be physiologically stimulated by placing
the animal on a black background. The cellular activation
results in the production and release of large amounts of the
proopiomelanocortin (POMC)-derived melanophore-stimu-
lating hormone, which causes pigment dispersion in dermal
melanophores, thereby darkening the skin [33]. Approxi-
mately 10 times more Ac45 transcripts have been found in
the melanotrope cells of animals adapted to a b lack
background compared to those of white-adapted animals
[32], suggesting that Ac45 has an important role in the
regulated secretory pathway o f neuroendocrine cells.
Here, we examined in detail the fate of the Ac45 protein
in the melanotrope cells of Xenopus intermediate pituitary.
We found that in these cells, the folding and proteolytic
cleavage of intact Ac45 is slow, and occurs in the early
secretory pathway where activation of V-ATPases is
required.
MATERIALS AND METHODS
Animals
South-African clawed toads, Xenopus laevis,werebredand
reared in the aquarium facility o f the Department of Animal

Physiology of the University of Nijmegen. Animals were
adapted to a black background by keeping them in black
buckets under constant illumination fo r at least three w eeks
at 22 °C. All experiments were carried out under the
guidelines of the D utch law concerning animal welfare.
Biochemicals and antibodies
Rabbit polyclonal antisera 1311C and 1311N, directed
against a synthetic peptide comprising the 12 C-terminal
amino-acid residues of Xenopus Ac45 and against a
recombinant fragment of Xenopus Ac45 (comprising ami-
no-acid residues Gly68 to Pro388 with a hexahistidine tail at
its N-terminus; numbering according to [34]), respectively,
have been described previously [34] (Fig. 1). Rabbit poly-
clonal antiserum 1311NC was raised against a recombinant
polypeptide corresponding to amino-acid residues P ro208–
Ser381 (numbering according t o [34]) of Xenopus Ac4 5
expressed in E. coli as a fusion protein with a hexahistidine
tag at its C-terminus (Cogon, Hilden, Germany) (Fig. 1).
Brefeldin A (BFA), monensin, nordihydroguaiaretic acid
(NDGA), chloroquine, and tunicamycin were purchased
from Sigma (St Louis, MO, USA). Leupeptin was from
Roche Diagnostics (Mannheim, Germany) and bafilomycin
A1 (Baf) from Wako Pure Chemical Industries (Osaka,
Japan).
Metabolic labeling of
Xenopus
neurointermediate lobes
and immunoprecipitation analysis
Neurointermediate lobes (NILs) from black-adapted Xen-
opus laevis were dissected and preincubated in methionine-

and cysteine-free c ulture medium [ 6.7 mL L15 medium
(Gibco-BRL, Gaithersburg, MD, USA), 3 mL milli-Q
water, 10 lgÆmL
)1
kanamycin, 1% antibiotic-antimycotic
solution (Gibco-BRL), 8 mg CaCl
2
, 3 mg bovine serum
albumin and 2 mg glucose] for 30 min at 22 °C. P ulse
labeling of newly synthesized proteins was performed by
incubating the lobes in methionine/cysteine-free culture
medium containing 5 mCiÆmL
)1
[
35
S]Met/Cys (Promix,
Amersham, Buckinghamshire, UK) for 1 h at 22 °C.
Subsequent chase incubations were in culture medium
supplemented with 5 m
ML
-methionine, 2.5 m
ML
-cysteine
and 10% fetal bovine serum. BFA (2.5 lgÆmL
)1
)was
present during the pre-, pulse and chase incubations, unless
stated otherwise. NDGA (30 l
M
) was present only during

the chase incubation. In some experiments, lobes were first
incubated overnight in the absence or presence of
10 lgÆmL
)1
tunicamycin in culture medium containing
10% fetal bovine s erum (Gibco-BRL). For immunoprecipi-
tation analysis, lobes w ere homogenized on ic e in lysis b uffer
(50 m
M
Hepes pH 7.2, 140 m
M
NaCl, 10 m
M
EDTA,
1% Tween-20, 0.1% Triton X-100, 0.1% deoxycholate)
containing 1 m
M
phenylmethanesulfonyl fluoride and
0.1 m gÆmL
)1
soybean trypsin inhibitor. Homogenates were
cleared by centrifugation (10 000 g,7minat4°C), and
used for protein deglycosylation (see below), or directly
supplemented with 0.1 volume of 10% SDS and diluted
10-fold in lysis buffer before addition of anti-Ac45 antise-
rum (1 : 500 d ilution). Immune complexes w ere precipitated
with protein-A–Sepharose (Pharmacia Biotech, Uppsala,
Sweden) and subjected to SDS/PAGE [35]. Gels were
processed for fluorography and radiolabeled proteins were
detected by autoradiography.

Immunoblotting
NILs dissected from black-adapted Xenop us laevis were
incubated overnight at 22 °C in culture medium with 10%
fetal bovine serum in the absence or presence of drugs, or
directly homogenized in lysis buffer containing 1 m
M
phenylmethanesulfonyl fluoride and 0.1 mgÆmL
)1
soybean
trypsin inhibitor. Lysates were cleared by centrifugation
(10 000 g,7min,4 °C) and u sed for protein deglycosylation
(see below) or immediately denatured in sample buffer at
Fig. 1. Antigenic epitopes used t o produce Ac45 re gion-specific antisera.
Recombinant proteins comprising residues Gly68 to Pro388 and
Pro208-Ser381, and a synthetic peptide corresponding to the 1 2
C-terminal amino-acid residues of Xenopus Ac45 were used to produc e
rabbit polyclonal antisera 1311N, 1311C, and 1311NC, respectively.
Ó FEBS 2002 The fate of Ac45 in the secretory pathway (Eur. J. Biochem. 269) 1845
95 °C for 5 min Proteins were separated by SDS/PAGE
and electrotransferred to nitrocellulose (Schleicher &
Schuell, Dassel, Germany). Membranes were blocked and
washed with blocking buffer (100 m
M
NaCl; 100 m
M
Na
2
PO
4
; 1% Tween-20) containing 5% low-fat dry milk.

Blocking buffer with 2% low-fat dry milk was used for
further washing steps and incubations with primary and
secondary antibodies. The secondary antibody used was an
peroxidase-conjugated anti-(rabbit IgG) Ig (Sigma, St
Louis, MO, USA) at a dillution of 1 : 3000. Peroxidase
activity was detected using the Lumilight system (Roche
Diagnostics, Mannheim, Germany).
Deglycosylation of proteins
Proteins were treated with endoglycosidase H ( EndoH)
(Roche Diagnos tics, Mannheim, Germany) to r emove high-
mannose N-glycans from glycoproteins. Lysates were boiled
for 10 min in 50 m
M
Na-citrate buffer (pH 5.5) containing
0.1% SDS, gradually cooled to RT, and incubated
overnight in the absence or presence of 40 m UÆmL
)1
EndoH at 37 °C. Proteins were deglycosylated by
N-glycosidase F (Roch e Diagnostics, Mannheim, Germany)
to remove both high-mannose and complex oligosacchar-
ides. For this purpose, protein lysates were boiled for
10 min in 10 m
M
Hepes (pH 7.4) containing 0.1% SDS,
cooled down t o RT, supplemented with 0.5% Nonidet P-40,
100 l
M
phenylmethanesulfonyl fluoride and 100 lgÆmL
)1
soybean trypsin inhibitor, and incubated o vernight at 37 °C

with or without 5 U N-glycosidase F p er mL.
RESULTS
Intact newly synthesized Ac45 is N-linked glycosylated
To study the biosynthesis of Ac45, we raised, in addition to
the previously produced antisera 1311N and 1311C (Fig. 1;
[34]), a third anti-Ac45 antiserum (1311NC; against a
recombinant protein comprising Xenopus Ac45 residues
208–388; Fig. 1). Following a 1-h pulse labeling of neuro-
intermediate lobes (NILs) from black-adapted Xenopus,the
1311N and 1311C antisera detected a newly synthesized
protein of  62 kDa and a less abundant protein of
 64 kDa (Fig. 2, lanes 1 and 10). Both proteins represent
intact forms o f Ac45 a nd vary only in the degree of N-linked
glycosylation, as deglycosylation of these radiolabeled
proteins by N-glycosidase F led to an  46-kDa protein
(Fig. 2 , lanes 4 and 13). The mobility of the two intact forms
increased slightly during subsequent chase incubations of
4 h and 8 h, giving rise to products of  61 and  63 kDa
(Fig. 2 , c ompare lane 1 with 2 and 10 with 11). This minor
shift in mobility is likely due to a change in the N-linked
sugars (possibly oligosaccharide trimming), as deglycosyla-
tion of these proteins again yielded a product of  46 kDa
Fig. 2. Deglycosylation allows detection of the Ac45 processing products
by region-specific polyclonal antisera. NILs from black-adapted
Xenopus were pulsed for 1 h with [
35
S]Met/Cys and then chased f or
the indicated time periods. Total lobe extracts were directly subjected
to immunoprecipitation with antisera 1311N, 1311C or 1311NC, or
deglycosylated by N-glycosidase F or EndoH prior to immunopre-

cipitation. Precipitated proteins were resolved by SDS/PAGE and
visualized by fluorography. Migration positions of intact and
processed form s o f Ac 45 are indicated. Lane 18 with increase d co ntrast
is depicted in lane 18¢. Note that some of the immunoprecipitates
contain 37- kDa glycosylated or 3 5-kDa deglycosylated POMC t hat
bound nonspecific ally (aster isk).
1846 V. Th. G. Schoonderwoert et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 2, lanes 5, 6, 14 and 15). The amount of 61- and 63-
kDa Ac45 decreased during the 8-h chase (half-life 4–6 h) as
a r esult o f a cleavage e vent ( see b elow). Both the 61- and 63-
kDa forms are not immunoprecipitated by the 1311NC
antiserum (Fig. 2, lanes 19–21). Presumably, the N-linked
glycans prevent det ection of these forms as t heir removal by
N-glycosidase F results in the immunoprecipitation of the
deglycosylated 46-kDa intact form by this antiserum
(Fig. 2; lanes 22–24). These results show that newly
synthesized Ac45 is N-linked glycosylated to a major
product of  62 kDa and a minor product of  64 kDa
that are subsequently processed to  61- and  63-kDa
products.
Newly synthesized Ac45 is cleaved
In the melanotrope cells of the Xenopus NIL, intact
N-linked glycosylated Ac45 is intracellularly cleaved to a
C-terminal fragment of  40 kDa. Although the  40-kDa
product could be detected by Western blotting with the
C-terminally directed anti-Ac45 serum 1311C, this antise-
rum did not immunoprecipitate the newly synthesized form
of this fragment [32]. However, after optimization of the
immunoprecipitation conditions, we detected the newly
synthesized C-terminal product with antiserum 1311C as a

diffuse band of 42–44 kDa (Fig. 2, lanes 11 and 12). With
antisera 1311N and 1311NC, we could not precipitate this
product (Fig. 2, lanes 2 and 3, and 20 an 21), possibly
because of the presence of numerous N-linked glycans in
this region of the protein (Fig. 1). Indeed, after removal of
the N-linked glycans by N-glycosidase F, all three antisera
(1311N, 1311NC and 1311C) immunoprecipitated this
fragment in its deglycosylated forms, namely as proteins of
 23 and  24 kDa (Fig. 2, lanes 5, 14 and 23, and 6, 15
and 24, respectively). During the chase i ncubation, the
mobility of t he deglycosylated C-terminal fragment shifted
from  23 kDa to  24 kDa (Fig. 2, lane 14 and 15),
probably as the result of a post-translational m odification.
The amount of the deglycosylated C-terminal Ac45
cleavage fragment, with a size of  23 kDa after 4 h of
chase and  24 kDa after 8 h of chase, increased during
the chase incubation (Fig. 2, lane 14 and 15), as was
expected because of the progressive cleavage of intact 61/
63-kDa Ac45. Thus, from these data, we conclude that
newly synthesized  61/63-kDaAc45iscleavedto
C-terminal products of 42–44 kDa (with deglycosylated
forms of  23 and  24 kDa).
Identification of the N-terminal Ac45 cleavage product
In contrast to what holds for the C-terminal cleavage
fragment of Ac45 [30,34], the N-terminal cleavage fragment
has not been identified y et. However, after optimization o f
the immunoprecipitation conditions, from newly synthe-
sized Xenopus NIL proteins we precipitated with antiserum
1311N a low-abundant product of  22 kDa (Fig. 2, lanes
1–3). Because of the following we conclude that this  22-

kDa product is the glycosylated form of the N-terminal
Ac45 cleavage fragment. First, the size of this product is in
line with the predicted size of the N-terminal fragment that
remains following cleavage of intact 61/63-kDa Ac45 to the
42–44-kDa C-terminal product. Furthermore, both the
 22-kDa fragment and its deglycosylated  20-kDa form
(see below) are not immunoprecipitated with the two
antisera raised against more C-terminally located regions of
Ac45 (1311C and 1311NC, Figs 1 and 2, lanes 10–15 and
19–24). Finally, t he N-terminal Ac45 fragment contains o ne
potential N-linked glycosylation site (Asn128; numbering
according to [34]), and this s ite appears to b e used, as
N-glycosidase F t reatment of the NIL lysate prior to immu-
noprecipitation causes a shift in the mo bility of the 22-kDa
product to  20 kDa (Fig. 2, lanes 4–6). The amount of the
N-terminal fragment would be expected to increase during
the chase period because of t he progressive cleavage of
intact Ac45. However, during the chase incubation a
decrease in the level of the N-terminal fragment was found,
suggesting that this cleavage p roduct may be subjected to an
intracellular degradation process. This circumstance may
also explain why the N-terminal Ac45 fragment has not
been detectable by Western b lotting [34].
Transport of newly synthesized Ac45 to the Golgi
In the medial Golgi, N-linked oligosaccharides can be
modified to two broad classes, namely complex oligosac-
charides and high-mannose oligosaccharides. Both types
of oligosaccharides can be removed completely from
proteins by treating them with N-glycosidase F. In contrast,
endoglycosidase H (EndoH) removes only high-mannose

oligosaccharides. The acquisition of resistance of an
N-glycosylated protein to EndoH, which requires t he action
of glycosylation enzymes localized in the medial Golgi, can
thus be used to determine whether a glycosylated protein
has entered the medial compartment [36]. We determined
whether the intact or the cleavage products of Ac45 acquire
resistance to digestion with EndoH. Extracts of pulse-
chased NILs were subjected to EndoH before immunopre-
cipitation with antisera 1311N, 1311C or 1311NC. All
three anti-Ac45 antisera immunoprecipitated from the
EndoH-treated NIL lysate a newly synthesized product of
 46 kDa. This product corresponds with the intact newly
synthesized deglycosylated Ac45 protein that was immuno-
precipitated from NIL lysates that were treated with
N-glycosidase F, in dicating that intact Ac45 is sensitive to
EndoH. This finding implies that i ntact Ac45 is cleaved in a
compartment before the medial Golgi. Antisera 1311N and
1311C immunoprecipitated from the EndoH-treated and
the N-glycosidase F-treated lysates similar amounts of the
 46-kDa product (Fig. 2, lanes 7–9 and 16–18). In
contrast, the 1311NC antiserum precipitated a considerably
lower amount of t his product from the EndoH-treated than
from the N-glycosidase F-treated lysates (Fig. 2, compare
lanes 22–24 with 25–27). Probably, the presence of the
N-acetylglucosamine residues remaining after EndoH
digestion [37], but removed by N-glycosidase F [38], lowers
the affinity of the Ac45 product for the 1311NC antiserum.
This possibility may also explain why this antiserum was
not able to detect significant amounts of the  23-kDa
C-terminal cleavage product in EndoH-treated lysates

(Fig. 2 , lanes 17 and 18).
In addition to the  23-kDa product, the 1311C anti-
serum detected also a low-abundant product of  26-kDa in
the EndoH-treated lysate (Fig. 2, lanes 17 and 18/18¢ ). This
product was not detected in the N-glycosidase F-treated
lysate (Fig. 2, lane 15), indicating that it represents a
C-terminal Ac45 cleavage form of which most, but not all,
Ó FEBS 2002 The fate of Ac45 in the secretory pathway (Eur. J. Biochem. 269) 1847
N-glycans are sensitive t o EndoH. The amount of the
 23-kDa product in the EndoH-treated lysates remained
constant during the chase, whereas the analysis of the
N-glycosidase F-treated samples clearly indicated an
increase in the total amount of this fragment (Fig. 2,
compare lanes 14 and 15 with 17 and 18). These findings
suggest that at first, all the N-linked sugars on the
C-terminal cleavage product are sensitive to EndoH
(EndoH treatment gives an  23-kDa product), and that
during the chase some of t he N-glycans on the C-terminal
cleavage product become resistant to EndoH (resulting in
an  26-kDa product). The N-linked sugar on the
N-terminal cleavage product also acquired resistance t o
EndoH, as w e found a f aint band of  22 kDa i n the
EndoH-treated extracts that is absent in the total lysates
of these samples (Fig. 2, lanes 8 and 9, and data not
shown) .
Western blot analysis was employed to study the steady
state levels of E ndoH-sensitive and EndoH-resistant forms
of Ac45. In line with the results of biosynthetic studies,
EndoH treatment of the NIL lysate prior to Western blot
analysis with the 1311C antibody again resulted i n t he

detection of an  23-kDa and an  26-kDa product
(Fig.3,lane2).Theintensityofthe 23-kDa band is
higher than that of the  26-kDa band, indicating that in
the steady state situation the  23-kDa product is the
major form in the EndoH-treated lysate. As expected,
deglycosylation by N-glycosidase F resulted in the detec-
tion of the  23-kDa C-terminal cleavage product (Fig. 3,
lane 3). As this product is more abundant in the EndoH-
treated NIL lysate than the  26-kDa product, we
conclude that at steady state, most of the g lycosylated
42–44-kDa C-terminal cleavage products contain N-linked
glycans that are sensitive to EndoH.
Together, these results demonstrate t hat the cleavage of
intact 61/63-kDa Ac45 occurs before the medial G olgi,
and that in this compartment the N-glycan on the
N-terminal and some of the N-glycans on the C-terminal
cleavage product are converted to complex oligosaccha-
rides.
BFA inhibits the degradation of the N-terminal Ac45
fragment
As the N-terminal fragment was not detected by immu-
noblotting, we hypothesized that it may be degraded
intracellularly. We examined this possibility by affecting
Ac45 transport through the secretory pathway via drugs
that interfere with intracellular prote in transport, namely
the fungal metabolite brefeldin A ( BFA) and the s odium
ionophore monensin. BFA causes fusion of Golgi mem-
branes with the ER and the retention of newly synthesized
proteins in a l umenal milieu c haracteristic o f the early
compartments of the secretory pathway [39]. In addition,

BFA blocks the exit of proteins from the TGN [40] as we
have recently shown for several regulated secretory proteins
in Xenopus melanotrope cells [41]. Monensin interferes with
protein transport between Golgi compartments [42]. Fur-
thermore, to examine if t he N-terminal cleavage product i s
degraded in lysosomes, a number of compounds that
interfere with lysosomal function were used. Leupeptin is a
thiol protease i nhibitor that i nhibits d egradation of proteins
in lysosomes [43]. T he weak base chloroquine and the
V-ATPase-specific inhibitor bafilomycin A1 (Baf) are
known to inhibit lysosomal and endosomal enzymes by
disturbing the intralumenal p H [1]. Baf may also affect the
transport of intact Ac45 or its cleavage products in a post-
TGN compartment, e.g. in Xenopus melanotrope cells [41].
To examine the effects of the above-mentioned drugs,
Xenopus NILs were incubated overnight in the absence or
presence of a drug, and the lobes w ere lysed and subjected to
Western blotting with the 1311N or 1311C antiserum. The
N-terminal cleavage fragment o f Ac45 did not accumulate
when NILs were incubated in the presence of monensin or
the lysosomal inhibitors leupeptin, chloroquine and Baf.
However, in NILs incubated in the presence of BFA, an
 22-kDa product had clearly accumulated (Fig. 4A). This
product could b e deglycosylated with N-glycosidase F to
 20 kDa and was not detected with the 1311C antibody
(data not shown), indicating that this product represents
the N-terminal fragment of Ac45. The drugs used did
not significantly change the amount of the 42–44-kDa
Fig. 4. Effect of inhibitors of intracellular transport and lysosomal
function on the degradation of the N-terminal 22-kDa Ac45 cleavage

fragment. NILs dissected from black-adapted Xenopus were incubated
overnight in medium w ith no d rugs, bref eldin A ( BFA, 2.5 lgÆmL
)1
),
chloroquine (Chl, 100 l
M
), bafilomycin A1 (Baf, 1 l
M
), leupeptin
(Leu, 100 lgÆmL
)1
), or mo nensin (Mon, 100 n
M
). Proteins we re
extracted from these NILs, separated by SD S/PAGE, transferred to
nitrocellulose and probed with the anti-Ac45 serum 1311N to detect
the 22-kDa N-terminal fragment (A) or 1311C to detect the 42 to
44-kDa C-terminal fragment (B).
Fig. 3. Steady-state levels of EndoH-sensitive and -resistant forms of the
C-terminal Ac45 cleavage fragment. To tal NIL extracts from black-
adapted Xenopus were incubated overn ight with no enzyme (lane 1),
EndoH (lane 2), or N-glycosidase F (lane 3). Reactions were stopped
by adding SDS sample buffer, and th e samples were subjected to SDS/
PAGE and immunoblotting, using 1311C.
1848 V. Th. G. Schoonderwoert et al. (Eur. J. Biochem. 269) Ó FEBS 2002
C-terminal Ac45 product (Fig. 4B), suggesting that cleavage
of intact Ac45 was not affected.
Next, w e sought to determine whether not only the steady
state levels but also th e amount of the newly synthesized N -
terminal cleavage fragment is affected by BFA. For this

purpose, we pulse-chased NILs in the absence or presence of
BFA, and performed immunoprecipitation analyses with
antibodies 1311N and 1311C. In line with t he Western
blotting results, the presence of BFA did not affect the
cleavage of intact Ac45. However, in the presence of BFA,
intact Ac45 does not migrate as a 61/63-kDa product, but
rather as a single product of  61 kDa (Fig. 5, lanes 2 and
3). Apparently, the redistribution of Golgi enzymes to the
ER induced by BFA [44] results in the premature trimming
of the N -linked sugars. BFA-treatm ent also led to the
accumulation of the  22-kDa N-terminal cleavage frag-
ment during the first 4 h of chase (Fig. 5, lane 2). However,
in the next 4-h chase period with BFA, the amount of the
cleaved N-terminal product did not increase, presumably
because this fragment was degraded (Fig. 5, lane 3). Thus,
BFA leads to an accumulation of the N-terminal f ragment,
but does not prevent the degradation process. These data
indicate that the BFA-indu ced transport block of proteins
out of the ER still allows cleavage of intact Ac45, and
support our notion that cleavage of Ac45 occurs i n t he early
secretory pathway. We also conclude that degradation of
the N-terminal  22-kDa cleavage fragment is inhibited by
BFA, and seems to occur after the N-linked sugar acquires
EndoH resistance and not in the endosomal-lysosomal
system.
BFA leads to the accumulation of the newly synthesized
C-terminal Ac45 cleavage fragment
At steady state, the C-terminal Ac45 cleavage fragment is
the predominant form of Ac45 present in the melanotrope
cells of the NIL (Fig. 3 , lane 2). However, in the biosyn-

thetic studies the amount of newly synthesized C-terminal
cleavage product was lower than one would expect on the
basis of the amount of intact glycosylated Ac45 that is
cleaved to the C-terminal product. Surprisingly, immuno-
precipitates from extracts of BFA-incubated NILs (Fig. 5)
show, in addition to the newly synthesized N-terminal
fragment, a high amount of newly synthesized C-terminal
Ac45 cleavage product, much higher than detected in NILs
that were incubated in the absence of BFA (Fig. 2, lane 1 –3,
10–12). Possibly, the region of the C-terminal cleavage
fragment to which the 1311C antibody was directed (the
cytoplasmic tail of Ac45) is more accessible to t he antibody
in the presen ce of BFA. A binding candidate may b e COPI,
as BFA is known to dissociate COPI from Golgi mem-
branes [45,46]. Alternatively, and more likely, BFA led to
the accumulation of the C-terminal cleavage fragment of
Ac45 in the ER-Golgi, thereby preventing the C-terminal
fragment from obtaining its normal conformation or from
associating with i ts normal p artner (e.g. t he V-ATPase
enzyme complex). In case of the possibility of epitope
unmasking, one would expect to find equal amounts of the
C-terminal cleavage product to be immunoprecipitable
from radiolabeled NILs when BFA is either present
constantly or added at a later stage of the chase period.
However, from NILs pulse-chased in the continuous
presence of BFA (Fig. 6, lane 1) or chased first in the
presence and then in t he absence of BFA (Fig. 6, lane 2 ), the
amount of immunoprecipitated C-terminal cleavage prod-
uct is much higher than from NILs chased fi rst in the
absence and then in the presence of BFA (Fig. 6, lane 3).

Therefore, we conclude that the more efficient detection of
the C-terminal cleavage product of Ac45 i n the presence of
BFA can not be attributed to an unmasking of the 1311C
epitope by, e.g. COPI-dissociation. To further support
this notion, we used the lipoxygenase inhibitor
nordihydroguaiaretic acid (NDGA), a drug acting similar
to BFA but preventing dissociation of COPI from Golgi
membranes [47]. As for BFA, the presence of NDGA
during the chase incubation a llowed the efficient detection
of the newly synthesized C-terminal product (Fig. 7, lane 1
and 2) and thus COPI dissociation is not involved.
Together, we conclude that inhibition of ER to Golgi
transport prevents the C-terminal Ac45 cleavage product
Fig. 5. BFA a llows immunoprecipitation of the N- and C-terminal Ac45
cleavage fragments. NILs from black-adapted Xenopus were pulsed for
1hwith[
35
S]Met/Cys and su bsequently chased for the ind icated t ime
periods in the presence of BFA. Ac45 products were immunoprecipi-
tated with both t he 1311N and 1311C antibody. Precipitated proteins
were resolved by SDS/PAGE and visualized by fluorography.
Migration positions of intact and processed forms of Ac45 are
indicated. Note that some o f the immunoprecipitates contain 37-kD a
POMC and  70 kDa p rohormone convertase PC2 that bound
nonspecifically (asterisk).
Ó FEBS 2002 The fate of Ac45 in the secretory pathway (Eur. J. Biochem. 269) 1849
from adopting its normal conformation or from interacting
with its binding partner.
Tunicamycin inhibits N-linked glycosylation and cleavage
of intact Ac45

As N-linked glycosylation of intact Ac45 precedes its
cleavage, we wondered whether inhibition of N-glycosyla-
tion by tunicamycin would affect the cleavage event. In the
absence of tunicamycin, newly synthesized Ac45 was
detected with the 1 311N antiserum a s the intact glycosylated
 62–64-kDa form, with the mobility s hifting to  61–
63 kDa during the subsequent chase period. The cleavage
process caused the amount of the intact glycosylated form
of Ac45 to decrease during the chase p eriod (Fig. 8, lane 1–
3). In the presence of tunicamycin, Ac45 is immunoprecip -
itated as a product of  46 kDa (Fig. 8, lane 4–6). The size
of this  46-kDa unglycosylated product is similar to the
size of intact Ac45 deglycosylated with N-glycosidase F
(Fig. 2 , lane 4–6), indicating that tunicamycin prevents
N-linked glycosylation of intact Ac45. Interestingly, the
processing of the  46-kDa unglycosylated intact form of
Ac45 was clearly affected (Fig. 8, lanes 4–6). Even after 8 h
of chase a high amount of the  46-kDa unglycosylated
intact form of Ac45 is still present. These findings demon-
strate that tunicamycin not only inhibits N-linked glycosy-
lation but also cleavage of Ac45, suggesting that N-linked
glycosylation of intact Ac45 is necessary to allow its
cleavage.
DISCUSSION
Acidification of organelles is important fo r numerous
intracellular processes. In the regulated secretory pathway,
acidification is mainly required for the sorting of proteins
and processing of prohormones [1]. The lumen of the
organelles o f t he regulated secretory pathway gradually
Fig. 6. BFA leads to the accumulation of the C-terminal cleavage

product of Ac45. NILs from black-adapted Xenopus were pulsed for
1hwith[
35
S]Met/Cys in the presence of BFA, and chased for two
subsequent periods of 4 h in the absence or p resence BFA. Ac45
products were immunoprecipitated with antibody 1311C, separated by
SDS/PAGE and visualized by fluorography. The migration positions
of intact and processed forms of Ac45 are indicated. No te that some of
the immunoprecipitates contain 37-kDa POMC and  70 kDa PC2
that bound nonspecifically (aste risks).
Fig. 7. NDGA allows immunoprecipitation of the C-terminal fragment
of Ac45. NILs from b lack-ad apted Xenopus were pulsed for 1 h with
[
35
S]Met/Cys and subseq uently chased for the indicated time period s in
the presence of NDGA or BFA. Ac45 products were immun oprecip-
itated with antibody 1311C. Precipitated proteins were resolved by
SDS/PAGE and visualized by fluorography. Migration positions o f
intact and processed forms of Ac45 are indicated. Note t hat some of
the immunoprecipitates contain nonspecifically bound 37-kDa POMC
and  70 kDa PC2 (asterisks).
1850 V. Th. G. Schoonderwoert et al. (Eur. J. Biochem. 269) Ó FEBS 2002
acidifies from the ER to Golgi to secretory granules.
Responsible for the acidification is the activity of the
multimeric V-ATPase enzyme complex that translocates
protons across membranes at the expense of ATP [26,48].
Several mechanisms have been proposed that may explain
how the lumen of an organelle acquires its specific pH. The
membranes of the organelles may differ in their permeability
for protons, the composition or assembly state of the

V-ATPase e nzyme itself may vary between the different
organelles, or organelle-specific proteins/factors may regu-
late the V-ATPase. Evidence has been presented for all of
these mechanisms (reviewed in [4,27,49]), suggesting that
they may work simultaneously or in a cell type-specific
manner. In the chromaffin cells of the bovine adrenal
medulla, secretory granules have been found to contain a
V-ATPase that is associated with the accessory subunit
Ac45 [30]. This n euroendocrine-enriched subunit of
 45 kDa may play a role in targeting or contro lling the
activity of the V-ATPase in the reg ulated secretory pathway
[30,32]. Deglycosylation experiments and N-terminal
sequencing of bovine Ac45 s howed that the isolated protein
is a proteolytically cleaved fragment [30,31]. We have
recently shown t hat Xenopus Ac45 is synthesized as an
N-glycosylated intact protein which is subsequently
processed to a C-terminal cleavage product of  40 kDa
[34]. The results obtained in the present study allow us to
propose the following more detailed model for the s ynthesis,
processing and transport of Ac45 in Xenopus intermediate
pituitary cells. Ac45 is synthesized as an intact protein of
 46 kDa that is N-linked glycosylated to  62- and
 64-kDa products. Trimming of the N -glycans in the E R
gives rise to products of  61 and  63 kDa. As most
oligomeric complexes are assembled in the ER [50], the
association of Ac45 with the V-ATPase V
0
sector may well
be established already in this compartment. The intact
glycosylated  61/63-kDa Ac45 protein was found to be

cleaved to an  22-kDa N-terminal and a 42 to 44-kDa
C-terminal product. The cleavage takes place in the ER or
cis-Golgi, as i t is not inhibited by BFA, and occurs before
the cleavage products acquire EndoH resistance in the
medial Golgi. When N-linked glycosylation was prevented
by tunicamyin, the cleavage of Ac45 was inhibited,
suggesting that the protein needs proper folding or associ-
ation with the pump before cleavage can occur. However,
we can not exclude the possibility that tunicamycin inter-
fered with the activity of the elusive Ac45 cleavage enzyme.
The extensive time between glycosylation and cleavage
of Ac45 may indicate that its folding and assembly with
the V-ATPase is a complex process. Following cleavage
of intact glycosylated Ac45, both cleavage products pass
the medial Golgi, as the single N-linked glycan on the
N-terminal fragment and some of the N-glycans on the
C-terminal fragment acquire resistance to EndoH. Subse-
quently, the N-terminal cleavage fragment is degraded by a
mechanism that is i ndependent of the endosomal-lysosomal
system, as the degradation process is not affected by drugs
that disturb the acidification of these compartments or that
inhibit hydrolytic lysosomal enzymes. The C-terminal
cleavage fragment increases  1 kDa in size by an unknown
type of modification and is likely transported to secretory
granules, as in bovine Ac45 has been found to be associated
with the chromaffin granular V-ATPase [30]. The bovine
Ac45 C-terminal fragment (222 amino-acid residues) starts
with Val209, suggesting that the intact molecule is proteo-
lytically cleaved between Val208 and Val209 (numbering
according to [34]) [31]. Remarkably, this presumptive

cleavage site is not conserved in Ac45 of Xenopus and other
species [34]. Therefore, we hypothesize that the site of
cleavage in Ac45 is located in a more conserved region
N-terminally of Val2 08/Val209, and t hat f ollowing cleavage
the N-terminal portion of the C-produ ct is subjected to
exoproteolytic processing. Exoproteolytic trimming would
explain why in Xenopus the size of the (deglycosylated)
C-terminal cleavage fragment ( 23 kDa) is smaller than
expected on the basis of the sizes of intact (deglycosylated)
Ac45 ( 46 kDa) and the (deglycosylated) N-terminal
cleavage product ( 20 kDa). Exoproteolytic processing is
not unusual, as it has also been described for several
cathepsins and the light chain of myeloperoxidase [51], a nd
for lactase-phlorizin hydrolase (LPH) [52].
Thus far, the only indication of a possible involvement
of a cleavage enzyme in the regulation of V-ATPase
activity comes from yeast mutant studies. A yeast mutant
for the endoprotease Kex2p shows phenotypic character-
istics similar to those o f V -ATPase m utants, in dicating
that the Kex2p endoprotease is necessary for V -ATPase
activity in vivo. A model has been proposed in which
Fig. 8. Tunicamycin inhibits the glycosylation and cleavage of intact
Ac45. Lobes dissected from black-adapted Xenopus were preincubated
overnight, pu lsed for 1 h with [
35
S]Met/Cys and chased for the indi-
cated time periods. The incubations were performed either in the
absence or presence of 10 lgÆmL
)1
tunicamycin. Radiolabeled protein s

were immunoprecipitated from lobe extrac ts using antibody 1311N.
Immunoprecipitates were resolved by SDS/PAGE and visualized by
fluorography. Migration positions of glycosylated (61–64 kDa) and
unglycosylated (46-kDa) intact Ac45, as well as the 22-kDa N-terminal
Ac45 cleavage product are indicated.
Ó FEBS 2002 The fate of Ac45 in the secretory pathway (Eur. J. Biochem. 269) 1851
Kex2p would cleave a negative regulator of the
V-ATPase, thereby activating the pump. Ac45 has been
suggested to be this negative regulator [53] and, in a
region just N-terminal of the N-terminus of the bovine
C-terminal cleavage product, Ac45 contains a conserved
sequence (Arg183-Pro-Ser-Arg186; numbers refer to
Xenopus [34]); that could act as a recognition site for
furin (consensus of furin cleavage site is RX(K/R)R [54]);
[55], the vertebrate Kex2p ortholog [56,57]. However, it is
unlikely t hat Ac45 r epresents the negative regulator, a s
yeast does not seem to contain an Ac45 ortholog [26].
Furthermore, Kex2p cleaves proproteins in the late Golgi,
whereas we found that Xenopus Ac45 is cleaved in the ER
or cis-Golgi.
The question arises concerning the possib le role of the
Ac45 cleavage event. Recently, a model for acidifi cation in
the regulated secretory pathway has been proposed [10]. In
this model, the gradual decrease in the pH value of the
organelles of the secretory pathway is attributed to a
decrease in the proton permeability from the ER to the
mature secretory granules, concomitant with a gradual
increase in the number of active V-ATPases from the ER to
the Golgi. How the number of active H
+

-pumps increases
from the ER to the Golgi is not clear from this model. Ac45
could be a key player in this process. Intact glycosylated
Ac45 may interact with the V-ATPase and thereby keeping
the pump inactive i n the ER. Following Ac45 cleavage, the
V-ATPase would become active, whereby the cleavage may
have allowed the dissociation of the (inhibiting) N-terminal
cleavage fragment.
Altogether, w e conclude that N-linked glycosylated intact
Ac45 is cleaved to an  22-kDa N-terminal and a 42–44-
kDa C-terminal cleavage fragment in the ER or cis-Golgi,
where activation of the V-ATPase is necessary. Following
passage through the Golgi, the N -terminal fragment is
degraded and, together with the V-ATPase, the C-terminal
fragment is targeted to secretory granules.
ACKNOWLEDGEMENTS
We would like to thank Ron Engels for ta king care of Xenopus,and
Peter C ruijsen f or technical assistance. This work was suppo rted by
grant 805-33-212 from the Netherlands Organization for Scientific
Research-Earth and Life Sciences (NWO-ALW), and by European
Union-Training and Mobility Researchers network ERBFMR-
XCT960023.
REFERENCES
1. Mellman, I ., Fuchs, R. & Helenius, A . (1986) Acidification of the
endocytic and exocytic pathways. Annu. Rev. Bioc hem. 55,663–
700.
2. Arai, K., Shimaya, A., Hiratani, N. & Ohkuma, S. (1993) Puri-
fication and characterization of lysosomal H(+)-ATPase. An
anion-sensitive v-type H(+)-ATPase from rat liver lysosomes.
J. Biol. Chem. 268, 5649–5660.

3. Clague, M.J., Urbe, S., Aniento, F. & Gruenberg, J. (1994)
Vacuolar ATPase activity is required for endosomal carrier vesicle
formation. J. Biol. Chem. 269, 21–24.
4. Schoonderwoert, V.T.G. & Martens, G.J.M. (2001) Proton
pumping in the secretory pathway. J. Membr. Biol. 182, 1 59–169.
5. Seksek, O., Biwersi, J. & Verkman, A.S. (1995) Direct measure-
ment of trans-Golgi pH in living cells and regulation by second
messengers. J. Biol. Chem. 270, 4967–4970.
6. Kim, J.H., Lingwood, C.A., Williams, D.B., Furuya, W.,
Manolson, M .F. & Grinstein, S. (1996) Dynamic measurement of
the pH of the Golgi complex in living cells using retrograde
transport of the verotoxin r eceptor. J. Cell Biol. 134, 1387–1399.
7. Demaurex, N., Furuya, W., D’Souza, S., Bonifacino, J.S. &
Grinstein, S. (1998) M echanism of acidification of the trans-Golgi
network (TGN). In situ measurements of pH using retrieval of
TGN38 a nd furin from the cell su rfac e. J. Biol. Chem. 27 3, 2044–
2051.
8. Miesen bock, G., De Angelis, D .A. & Roth man, J.E. (1 998)
Visualizing secretion and synaptic transmission with pH-sensitive
green fluorescent proteins. Nature 394, 192–195.
9. Wu, M.M., Llopis, J., Adams, S., McCaffery, J.M., Kulomaa,
M.S.,Machen,T.E.,Moore,H.P.&Tsien,R.Y.(2000)Organelle
pH studies using targeted avidin and fluorescein-biotin. Chem.
Biol. 7, 197–209.
10. Wu,M.M.,Grabe,M.,Adams,S.,Tsien,R.Y.,Moore,H.P.&
Machen, T.E. (2001) Mechanisms of pH regulation in the regu-
lated secretory pathway. J. Biol. Chem. 276, 3 3027–33035.
11. Wilson, D.W., Lewis, M.J. & P elham, H.R. (1993) pH-dependent
binding of KDEL to its receptor in vitro. J. Biol. Chem. 268, 7465–
7468.

12. Scheel, A.A. & Pelham, H.R. (1996) Purification and character-
ization of the human KDEL receptor. Biochemistry 35, 10203–
10209.
13. Yilla, M., Tan, A., Ito, K., Miwa, K. & P loegh, H.L. (1993)
Involvement o f the vacuolar H (+)-ATPases in the secretory
pathway of HepG2 cells. J. Biol. Chem. 268, 19092–19100.
14. Xu, H. & Shields, D. (1994) Prosomatostatin processing in
permeabilized cells. Endoproteolytic cleavage is mediated by a
vacuolar ATPase that generates an acidic pH in the trans-Golgi
network. J. Biol. Chem. 269, 22875–22881.
15. Burgess, T.L. & Kelly, R.B. (1987) Constitutive and regulated
secretion of proteins. Annu. Rev. Cell Biol. 3, 243–293.
16. Chanat, E. & Huttner, W.B. (1991) Milieu-induced, selective
aggregation of regulated secretory proteins in the trans-Golgi
network. J. Cell Biol. 115, 1505–1519.
17. Kim, T., Tao-Cheng, J., Eiden, L.E. & Loh, Y.P. (2001) Chro-
mogranin A, an Ôon/offÕ switch controlling dense-core secretory
granule biogenesis. Cell 106, 499–509.
18. Cool, D.R., Normant, E., Shen, F.S., Chen, H.C., Pan nell, L.,
Zhang, Y. & L oh, Y.P. (1997) Carboxypeptidase E i s a regulated
secretory pathway sorting receptor: ge netic obliteration leads to
endocrine disorders in Cpe (fat) mice. Cell 88, 73–83.
19. Urbe,S.,Dittie,A.S.&Tooze,S.A.(1997)pH-dependentpro-
cessing of secretogranin II by the endopeptidase PC2 in isolated
immature se cre tory g ra nule s. B ioche m. J . 32 1, 65–74.
20. Laine, J. & Lebel, D. (1999) Efficient binding of regulated secre-
tory prote in a ggregates to membrane ph osph olipids a t a c idic p H.
Biochem. J. 338, 289–294.
21. Kuliawat, R. & Arvan, P. (1994) Distinct molecular mechanisms
for protein sorting within immature secretory granules of pan-

creatic beta-cells. J. Cell Biol. 126, 77–86.
22. Tooze, J. & Tooze, S.A. (1986) Clathrin-coated vesicular transport
of secretory proteins during the formation of ACTH-containing
secretory granules in AtT20 ce lls. J. Cell Biol. 103, 839–850.
23. Dittie, A.S., Hajibagheri, N. & Tooze, S.A. (1996) The AP-1
adaptor complex binds to immature secretory granules from PC12
cells, an d is regulated by ADP-ribosylation factor. J. Cell Biol .
132, 523–536.
24.Klumperman,J.,Kuliawat,R.,Griffith,J.M.,Geuze,H.J.&
Arvan, P. (1998) Mannose 6-phosphate receptors are sorted
from imm ature secretory granules via adaptor protein AP-1,
clathrin, and syntaxin 6-positive vesicles. J. Cell Biol. 141,
359–371.
25. Halban, P.A. & Irminger, J.C. (1994) Sorting and processing of
secretory proteins. Biochem. J. 29 9 , 1–18.
1852 V. Th. G. Schoonderwoert et al. (Eur. J. Biochem. 269) Ó FEBS 2002
26. Nelson, N. & Harvey, W.R. (1999) Vacuolar and plasma mem-
brane proton -adenosinetripho sphatases. Physiol. Rev. 79, 361–
385.
27. F orgac , M. (2 000) S tructu re, me chan ism a nd regu lation of the
clathrin-co ated vesicle and yeast vacuolar H(+)-ATPa ses. J. Exp.
Biol. 203, 71–80.
28. Myers, M. & Forgac, M. (1993) Assembly of the peripheral
domain of the bovine vacuolar H(+)-adenosine triphosphatase.
J. Cell Physiol. 156, 35–42.
29. Graham, L.A., Hill, K.J. & Stevens, T.H. (1998) Assembly of the
yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum
and r equires a Vma12p/Vma22p assembly complex. J. Cell Biol.
142, 39–49.
30. Supe k, F., S up ekova, L., Mandiyan, S., Pan , Y.C., Ne lson, H. &

Nelson, N. (1994) A novel accessory subunit for vacuolar H (+) -
ATPase from chromaffin granules. J. Biol. Chem. 269, 24102–
24106.
31. Getlawi, F., Laslop, A., Schagger, H., Ludwig, J., Haywood, J. &
Apps, D. (1996) Chromaffin gra nule membrane glycoprotein I V is
identical with Ac45, a membrane-integral subunit o f the granule’s
H
+
-ATPase. Neurosci. Lett. 219, 13–16.
32. Holthuis, J.C., Jansen, E.J., van-Riel, M.C. & Martens, G.J.
(1995) Molecular probing of the secretory pathway in peptide
hormone-produc ing cells. J. Cell Sc i. 108, 3295–3305.
33. Jenks, B.G.J.L.H., Martens, G.J.M. & Roubos, E.W. (1993)
Adaptation physiology: the fu nctioning of pituitary melanotrope
cells during background adaptation of the amphibian Xenopus
laevis. Zoo. Sci. 10, 1–11.
34. Holthu is, J.C., Jansen, E.J., Schoonderwoert, V.T., B urbach, J.P.
& Martens, G.J. (1999) Biosynthesis of the vacuolar H
+
-ATPase
accessory subunit Ac45 in Xenopus pituitary. Eur. J. Biochem. 262,
484–491.
35. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
36. Maley,F.,Trimble,R.B.,Tarentino,A.L.&Plummer,T.H.Jr
(1989) Characterization of glycoproteins and their associated
oligosaccharides through the use of endoglycosidases. Anal.
Biochem. 180, 195–204.
37. Tarentino, A.L., Plummer, T.H. Jr & Maley, F. (1974) The release
of intact oligosaccharides from specific glycoproteins by endo-

beta-N-acetylglucosaminidase H. J. Biol. Chem. 249, 818–824.
38. Plummer, T.H. Jr & Tarentino, A.L. (1981) Facile cleavage of
complex oligosaccharides from glycopeptides by almond emulsin
peptide: N-glycosidase. J. Biol. C hem . 256, 10243–10246.
39. Lip pincott- Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., Orci,
L. & Klausner, R.D. (1991) Brefeldin A’s effects on endosomes,
lysosomes, and the TGN suggest a general mechanism f or regu-
lating or ganelle structure and m embrane traffic. Cell 67 , 601–616.
40. Thorens, B., Gerard, N. & Deriaz, N. (1993) GLUT2 surface
expression and intracellular transport via t he constitutive pathway
in pancreatic beta cells and insulinoma: evidence for a block i n
trans-Golgi network exit by brefeldin A. J. Cell Biol. 123, 1687–
1694.
41. Schoonderwoert, V .T., Holthuis, J .C., Tanaka, S ., Tooze, S.A. &
Martens, G.J. (2000) Inhibition of the vacuolar H
+
-ATPase per-
turbs the transport, sorting, processing and release of regulated
secretory proteins. Eur. J. Biochem. 267, 5646–5654.
42. Tartakoff, A.M. (1983) Perturbation of vesicular traffic with the
carboxylic ionophore monensin. Cell 32, 1026–1028.
43. Ascoli, M. (1979) Inhibition of the degradation of receptor-bound
human choriogonadotropin by leupeptin. Biochim. Biophys. Acta.
586, 608–614.
44. Doms, R.W., Russ, G. & Yewdell, J.W. (1989) Brefeldin A
redistributes resident and itinerant Golgi proteins to the endo-
plasmic reticulum. J. Cell Biol. 109, 61–72.
45. Donaldson, J.G., Lippincott, S.J., Bloom, G.S., Kreis, T.E. &
Klausner, R.D. (1990) Dissociation of a 110-kD pe ripheral
membrane protein from the G olgi apparatus is an early event in

brefeldin A action. J. Cell Biol. 111, 2295–2306.
46. Orci, L., Tagaya, M., Amherdt, M., Perrelet, A., Donaldson,
J.G., Lippincott-Schwartz, J., Klausner, R.D. & Rothman, J.E.
(1991) Brefeldin A, a drug that b locks secretion, prevents t he
assembly of non-clathrin-coated buds on G olgi cisternae. Cell 64,
1183–1195.
47. Fujiwara, T., Takami, N., Misumi, Y. & Ikehara, Y. (1998)
Nordihydroguaiaretic acid blocks protein transport in the secre-
tory pathway causing redistribution of Golgi proteins into the
endoplasmic reticulum. J. Biol. Chem. 273, 3068–3075.
48. Stevens, T.H. & Forgac, M. (1997) Structure, function and reg-
ulation o f the vacuolar (H+) -ATPase. Annu. Rev. Cell Dev. Biol.
13, 779–808.
49. Futai, M., Oka, T., S un-Wada, G., M oriyama, Y., Kanazawa, H.
& Wada, Y. (2000) Luminal acidification of d iverse organelles by
V-ATPase in animal cells. J. Exp. Biol. 203, 107–116.
50. Hurtley, S.M. & Helenius, A. (1989) P rotein oligomerization in t he
endoplasmic reticulum. Annu.Rev.CellBiol.5, 277–307.
51. Hasilik, A. (1992) The early and late processing of lysosomal
enzymes: proteolysis and compartmentation. Experientia 48,
130–151.
52. Mesonero, J.E., Gloor, S.M. & Semenza, G. (1998) Processing of
human intestinal prolactase to an intermediate form by furin or by
a furin-like proprotein con vertase. J. Biol. Chem. 273, 29430–
29436.
53. Oluwatosin, Y.E. & Kane, P.M. (1998) Mutations in the yeast
KEX2 gene cause a Vma(–)-like p henotype: a possible role for the
KEX2 endoprotease in vacuolar acidification. Mol. Cell. Biol. 18,
1534–1543.
54. Molloy, S.S., Bresnahan, P.A., Leppla, S.H., Klimpel, K.R. &

Thomas, G. (1992) Human furin is a calcium-dependent serine
endoprotease that recognizes the sequence Arg-X-X-Arg and
efficiently cleaves anthrax toxin protective antigen. J. Biol. C he m.
267, 16396–16402.
55. Matthews,D.J.,Goodman,L.J.,Gorman,C.M.&Wells,J.A.
(1994) A survey o f furin substrate specificity u sing substrate phage
display. Protein Sci. 3, 1197–1205.
56. Roebroek, A.J., Schalken, J.A., Leunissen, J.A., Onnekink, C.,
Bloemers, H.P. & Van de Ven, W.J. (1986) Evolutionary con-
served close linkage of the c-fes/fps proto-oncogene and genetic
sequences encoding a receptor-like protein. EMBO J. 5, 2197–
2202.
57. Barr, P.J., Mason, O.B., Landsberg, K.E., Wong, P.A., Kiefer,
M.C. & Brake, A.J. (1991) cDNA a nd gene structure for a human
subtilisin-like protease with cleavage specificity for paired basic
aminoacidresidues.DNA Cell Biol. 10, 319–328.
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