Trimming of the N-linked glycans gave rise to glycosylated Ac45-forms of 61 and 63 kDa that are cleaved to a C-terminal fragment of 42–44 kDa the deglycosylated form is 23 kDa, and a p
Trang 1The 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 that interact with the
V-ATPase may play an important role in controlling the
intracellular localization and activity of the proton pump
The neuroendocrine-enriched V-ATPase accessory 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 to 64 kDa Trimming of the N-linked glycans
gave rise to glycosylated Ac45-forms of 61 and 63 kDa
that are cleaved to a C-terminal fragment of 42–44 kDa (the
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 brefeldin A Both the N- and C-terminal fragment pass the medial Golgi, as they become partially endoglycosidase H resistant The Ac45 cleavage event is a relatively slow process (half-life of intact Ac45 is 4–6 h) and takes place in the early secretory pathway, as it is not affected
by brefeldin A and monensin Tunicamycin inhibited N-linked glycosylation of Ac45 and interfered with the cleavage process, suggesting that Ac45 needs proper folding for the cleavage to occur Together, our results indicate 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, respectively, 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 folding 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 of 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 (V0) and a peripheral sector (V1) [26,27] The V-ATPase V1 sector contains the catalytic site which hydrolyses ATP to translocate protons across the mem-brane by the proton-pore forming V0sector In the ER, the assembly of the V-ATPase starts with the V0-sector and may
be completed in this compartment by the build-up of the V1 onto the V0[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 Fax: + 31 24 3615317, Tel.: + 31 24 3610564,
E-mail: g.martens@ncmls.kun.nl
Abbreviations: Baf, bafilomycin A1; BFA, brefeldin A; EndoH,
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
http://www.kun.nl/molanphys/Homepage/home.htm
(Received 26 October 2001, revised 6 February 2002, accepted 8
February 2002)
Trang 2membrane sector of the V-ATPase in only a subset of
organelles [30] Ac45 was initially isolated from bovine
chromaffin granules and identified as a type I
transmem-brane protein of 45 kDa [30] However, N-terminal
sequencing of the isolated 45-kDa protein and the cloning
of full-length Ac45 cDNA revealed that the isolated protein
represents a cleaved fragment of a larger 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] The melanotrope cells of
the Xenopus intermediate pituitary were used for 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 black
background compared to those of white-adapted animals
[32], suggesting that Ac45 has an important role in the
regulated secretory pathway of 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
M A T E R I A L S A N D M E T H O D S
Animals
South-African clawed toads, Xenopus laevis, were bred and
reared in the aquarium facility of 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 for at least three weeks
at 22°C All experiments were carried out under the
guidelines of the Dutch 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 Pro208–
Ser381 (numbering according to [34]) of Xenopus Ac45
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 ofXenopus neurointermediate lobes and immunoprecipitation analysis
Neurointermediate lobes (NILs) from black-adapted Xen-opus laeviswere dissected and preincubated in methionine-and cysteine-free culture 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 CaCl2, 3 mg bovine serum albumin and 2 mg glucose] for 30 min at 22°C Pulse labeling of newly synthesized proteins was performed by incubating the lobes in methionine/cysteine-free culture medium containing 5 mCiÆmL)1 [35S]Met/Cys (Promix, Amersham, Buckinghamshire, UK) for 1 h at 22°C Subsequent chase incubations were in culture medium supplemented with 5 mM L-methionine, 2.5 mM L-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 lM) 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 serum (Gibco-BRL) For immunoprecipi-tation analysis, lobes were homogenized on ice in lysis buffer (50 mM Hepes pH 7.2, 140 mM NaCl, 10 mM EDTA, 1% Tween-20, 0.1% Triton X-100, 0.1% deoxycholate) containing 1 mM phenylmethanesulfonyl fluoride and 0.1 mgÆmL)1soybean trypsin inhibitor Homogenates were cleared by centrifugation (10 000 g, 7 min at 4°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 dilution) Immune complexes were 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 Xenopus 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 mM
phenylmethanesulfonyl fluoride and 0.1 mgÆmL)1soybean trypsin inhibitor Lysates were cleared by centrifugation (10 000 g, 7 min, 4°C) and used for protein deglycosylation (see below) or immediately denatured in sample buffer at
Fig 1 Antigenic epitopes used to produce Ac45 region-specific antisera Recombinant proteins comprising residues Gly68 to Pro388 and Pro208-Ser381, and a synthetic peptide corresponding to the 12 C-terminal amino-acid residues of Xenopus Ac45 were used to produce rabbit polyclonal antisera 1311N, 1311C, and 1311NC, respectively.
Trang 395°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 mM NaCl; 100 mM
Na2PO4; 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 Diagnostics, Mannheim, Germany) to remove
high-mannose N-glycans from glycoproteins Lysates were boiled
for 10 min in 50 mMNa-citrate buffer (pH 5.5) containing
0.1% SDS, gradually cooled to RT, and incubated
overnight in the absence or presence of 40 mUÆmL)1
EndoH at 37°C Proteins were deglycosylated by
N-glycosidase F (Roche Diagnostics, Mannheim, Germany)
to remove both high-mannose and complex
oligosacchar-ides For this purpose, protein lysates were boiled for
10 min in 10 mM Hepes (pH 7.4) containing 0.1% SDS,
cooled down to RT, supplemented with 0.5% Nonidet P-40,
100 lM phenylmethanesulfonyl fluoride and 100 lgÆmL)1
soybean trypsin inhibitor, and incubated overnight at 37°C
with or without 5 U N-glycosidase F per mL
R E S U L T S
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 of Ac45 and 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, compare 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 for
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 forms of Ac45 are indicated Lane 18 with increased contrast
is depicted in lane 18¢ Note that some of the immunoprecipitates
contain 37-kDa glycosylated or 35-kDa deglycosylated POMC that
bound nonspecifically (asterisk).
Trang 4(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 result of a cleavage event (see below) 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 detection of these forms as their 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 incubation, the
mobility of the 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 modification
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-kDa Ac45 is cleaved to
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 yet However, after optimization of
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, the N-terminal Ac45 fragment contains one potential N-linked glycosylation site (Asn128; numbering according to [34]), and this site appears to be used, as N-glycosidase F treatment of the NIL lysate prior to immu-noprecipitation causes a shift in the mobility 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 the 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 product 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 blotting [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 oligosacoligosac-charides 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 the 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, indicating that intact Ac45 is sensitive to EndoH This finding implies that intact 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 this 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,
Trang 5N-glycans are sensitive to 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 the 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 to
EndoH, as we found a faint band of 22 kDa in 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 EndoH-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 in the
detection of an 23-kDa and an 26-kDa product
(Fig 3, lane 2) The intensity of the 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 glycosylated
42–44-kDa C-terminal cleavage products contain N-linked
glycans that are sensitive to EndoH
Together, these results demonstrate that the cleavage of
intact 61/63-kDa Ac45 occurs before the medial Golgi,
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 protein transport, namely the fungal metabolite brefeldin A (BFA) and the sodium ionophore monensin BFA causes fusion of Golgi mem-branes with the ER and the retention of newly synthesized proteins in a lumenal milieu characteristic of 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 the N-terminal cleavage product is degraded in lysosomes, a number of compounds that interfere with lysosomal function were used Leupeptin is a thiol protease inhibitor that inhibits degradation of proteins
in lysosomes [43] The weak base chloroquine and the V-ATPase-specific inhibitor bafilomycin A1 (Baf) are known to inhibit lysosomal and endosomal enzymes by disturbing the intralumenal pH [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, XenopusNILs were incubated overnight in the absence or presence of a drug, and the lobes were lysed and subjected to Western blotting with the 1311N or 1311C antiserum The N-terminal cleavage fragment of 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 be 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 with no drugs, brefeldin A (BFA, 2.5 lgÆmL)1), chloroquine (Chl, 100 l M ), bafilomycin A1 (Baf, 1 l M ), leupeptin (Leu, 100 lgÆmL)1), or monensin (Mon, 100 n M ) Proteins were extracted from these NILs, separated by SDS/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 Total NIL extracts from
black-adapted Xenopus were incubated overnight with no enzyme (lane 1),
EndoH (lane 2), or N-glycosidase F (lane 3) Reactions were stopped
by adding SDS sample buffer, and the samples were subjected to SDS/
PAGE and immunoblotting, using 1311C.
Trang 6C-terminal Ac45 product (Fig 4B), suggesting that cleavage
of intact Ac45 was not affected
Next, we sought to determine whether not only the steady
state levels but also the 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 the 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-treatment 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 fragment, but does not prevent the degradation process These data indicate that the BFA-induced transport block of proteins out of the ER still allows cleavage of intact Ac45, and support our notion that cleavage of Ac45 occurs in the 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 the antibody
in the presence of BFA A binding candidate may be 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 its normal partner (e.g the 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 the absence of BFA (Fig 6, lane 2), the amount of immunoprecipitated C-terminal cleavage prod-uct is much higher than from NILs chased first 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 in 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 allowed 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 allows immunoprecipitation of the N- and C-terminal Ac45
cleavage fragments NILs from black-adapted Xenopus were pulsed for
1 h with [35S]Met/Cys and subsequently chased for the indicated time
periods in the presence of BFA Ac45 products were
immunoprecipi-tated with both the 1311N and 1311C antibody Precipiimmunoprecipi-tated proteins
were resolved by SDS/PAGE and visualized by fluorography.
Migration positions of intact and processed forms of Ac45 are
indicated Note that some of the immunoprecipitates contain 37-kDa
POMC and 70 kDa prohormone convertase PC2 that bound
nonspecifically (asterisk).
Trang 7from 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 1311N antiserum as the intact glycosylated
62–64-kDa form, with the mobility shifting 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 period (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
D I S C U S S I O N
Acidification of organelles is important for 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 of the 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
1 h with [ 35 S]Met/Cys in the presence of BFA, and chased for two
subsequent periods of 4 h in the absence or presence 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 Note that some of
the immunoprecipitates contain 37-kDa POMC and 70 kDa PC2
that bound nonspecifically (asterisks).
Fig 7 NDGA allows immunoprecipitation of the C-terminal fragment
of Ac45 NILs from black-adapted Xenopus were pulsed for 1 h with [ 35 S]Met/Cys and subsequently chased for the indicated time periods in the presence of NDGA or BFA Ac45 products were immunoprecip-itated with antibody 1311C Precipimmunoprecip-itated proteins were resolved by SDS/PAGE and visualized by fluorography Migration positions of intact and processed forms of Ac45 are indicated Note that some of the immunoprecipitates contain nonspecifically bound 37-kDa POMC and 70 kDa PC2 (asterisks).
Trang 8acidifies 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 enzyme 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 neuroendocrine-enriched subunit of
45 kDa may play a role in targeting or controlling the
activity of the V-ATPase in the regulated secretory pathway
[30,32] Deglycosylation experiments and N-terminal
sequencing of bovine Ac45 showed that the isolated protein
is a proteolytically cleaved fragment [30,31] We have
recently shown that 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 synthesis, 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 ER 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 V0sector 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 it 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 independent 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 Val208/Val209, and that following cleavage the N-terminal portion of the C-product 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], and 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 of V-ATPase mutants, indicating 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, pulsed 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)1tunicamycin Radiolabeled proteins
were immunoprecipitated from lobe extracts 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.
Trang 9Kex2p 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 that Ac45 represents the negative regulator, as
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 possible role of the
Ac45 cleavage event Recently, a model for acidification 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 in 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, we 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
A C K N O W L E D G E M E N T S
We would like to thank Ron Engels for taking care of Xenopus, and
Peter Cruijsen for technical assistance This work was supported 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.
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