Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 in Xenopus pituitary

Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 inXenopus pituitary

Joost C. M. Holthuis1, Eric J. R. Jansen1, Vincent Th. G. Schoonderwoert1, J. Peter H. Burbach2 and

Gerard J. M. Martens1

1Department of Animal Physiology, University of Nijmegen, Toernooiveld, the Netherlands; 2Rudolf Magnus Institute for Neurosciences,

Department of Medical Pharmacology, Utrecht University, the Netherlands

Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including

secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes

coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus

intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I

transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian

Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot

analysis showed that immunoreactive Ac45 represents a <40-kDa product that is expressed predominantly in

neuroendocrine tissues; deglycosylation resulted in a <27-kDa immunoreactive Ac45 product which is smaller than

predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an

N-glycosylated protein of <60 kDa; the nonglycosylated, newly synthesized form is <46 kDa which is

similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly

expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45

protein is synthesized as an N-glycosylated <60-kDa precursor that is intracellularly cleaved to an <40-kDa

product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory

granules.

Keywords: vacuolar ATPase; organellar acidification; prohormone processing; pituitary gland; Xenopus.

Acidification of organelles connected with the vacuolar systemof eukaryotic cells is of crucial importance in numerous cellularprocesses, including the targeting of newly synthesized proteinsto lysosomes and secretory granules, maintenance of acidhydrolase activity in lysosomes, proteolytic processing ofprohormones in secretory granules, coupled transport ofbiogenic amines across secretory vesicle membranes, and theunloading of cargo during receptor-mediated endocytosis [1].This acidification is accomplished by a unique class ofATP-driven proton pumps called vacuolar H+-ATPases(V-ATPases) [2,3]. Through genetic and biochemicalanalyses in yeast and mammals, 10 different subunits of theV-ATPase have been identified. The subunits are distributed overtwo distinct moieties, a hydrophobic membrane sector (V0) anda hydrophilic catalytic sector (V1) [2,3]. The membrane sector isresponsible for proton translocation, while the peripherallyattached catalytic sector faces the cytoplasm and forms the ATPhydrolytic centre of the pump (reviewed in [4,5]) V-ATPaseshave been purified from a number of organelles, includingGolgi, lysosomes, endosomes, secretory granules and the plasma

membrane [3]. How V-ATPases are directed to, and acquire theirspecialized activities in, such a large variety of organelles andmembranes is still unknown. Possible mechanisms for generat-ing this topological and functional diversity may arise fromV-ATPases assembled with isoforms of one or more subunits, orthrough subtle changes in the lipid composition of themembrane [6±11]. Alternatively, the presence of organelle-specific membrane proteins may play a role in the sorting andregulation of the pump [10]. In the search for organelle-specificproteins in purified preparations of V-ATPase from bovinechromaffin granules, Supek et al. [12] identified a novel 45-kDaprotein, named Ac45, which copurified with the membranesector of the pump. Ac45 was detected at relatively high levelsin the adrenal medulla, in various parts of the brain and in thepituitary gland. When compared to the amount of Ac45 found inthe chromaffin granular membranes, V-ATPases purified fromkidney microsomes contained reduced amounts of Ac45 andkidney membrane V-ATPase preparations were devoid of theprotein [12]. From their analyses, Supek et al. [12] and Getlawiet al. [13] concluded that the isolated bovine Ac45 product isnot the intact protein but may represent a proteolytically cleavedfragment. A study on the biosynthesis of Ac45 may clarify thispoint.

We recently isolated an intermediate pituitary cDNA encod-ing a partial sequence of the Xenopus ortholog of bovine andhuman Ac45. This was achieved by a differential screeningstrategy designed to identify genes coexpressed with theprohormone proopiomelanocortin (POMC) in the melanotropecells of Xenopus intermediate pituitary [14]. These neuro-endocrine cells can be stimulated to produce and release large

Eur. J. Biochem. 262, 484±491 (1999) q FEBS 1999

Correspondence to G. J. M. Martens, Department of Animal Physiology,

University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, the Netherlands.

Fax: +31 24 3652714, Tel.: +31 24 3652601, E-mail: [email protected]

Abbreviations: DAB, 3,3 0-diaminobenzidine tetrahydrochloride; ER, endo-

plasmic reticulum; FITC, fluoresceine isothiocyanate; NIL, neurointer-

mediate lobe; PAP, peroxidase-antiperoxidase; NaCl/Pi, phosphate-buffered

saline; POMC, proopiomelanocortin; TGN, trans-Golgi network; V-ATPase,

vacuolar H+-ATPase.

(Received 7 December 1998, accepted 10 March 1999)

q FEBS 1999 Biosynthesis of Ac45 in Xenopus pituitary (Eur. J. Biochem. 262) 485

amounts of POMC-derived, melanophore-stimulating peptidesby placing the toad on a black background [15]. In the presentstudy, we determined the complete primary structure of the firstnonmammalian Ac45 protein, and monitored its biosynthesisand immunolocalization in Xenopus pituitary. We found thatAc45 is highly expressed and proteolytically processed in thebiosynthetically active Xenopus intermediate pituitary cells.

EXPERIMENTAL PROCEDURES

Animals

South-African clawed toads, Xenopus laevis, were adapted to ablack or white background by keeping them in black or whitebuckets, respectively, under constant illumination for at least3 weeks at 22 8C.

Cloning and sequence analysis of Xenopus Ac45 cDNA

A Xenopus neurointermediate lobe (NIL) cDNA library fromblack background-adapted toads was differentially screenedwith cDNA probes derived from NIL mRNA of black-andwhite-adapted animals as described previously [14]. Thisscreening resulted in the isolation of a partial 1.3-kb cDNA(clone X1311) encoding Xenopus Ac45. To isolate a full-lengthcDNA, the X1311 cDNA insert was random prime-labeled with[a-32P]dATP (3000 Ci´mmol21, ICN Radiochemicals, CostaMesa, CA, USA) and used to screen 6 £ 105 plaque-formingunits from an amplified Xenopus hypothalamus cDNA library inlambda uni-ZAP XR [16]. Seven positively hybridizing plaqueswere purified and the pBluescript SK2 phagemids were rescuedby in-vivo excision according to the manufacturers instructions(Stratagene, La Jolla, CA, USA). One of these clones, X1311±4,contained a 2.0-kb cDNA insert encoding the entire Ac45 openreading frame. Sequencing on both strands and with pBluescriptsubclones or specific primers was performed with single-and double-stranded DNA using T7 DNA polymerase(LKB-Pharmacia, Uppsala, Sweden) and the dideoxy chaintermination method [17].

Production of Ac45 antigens and generation of antisera

Polyclonal antisera were raised in rabbits against Xenopus Ac45.A synthetic peptide comprising the 12 C-terminal amino-acidresidues of Xenopus Ac45 with an additional cysteine at theN-terminus (-CPKGPSIAVPQTE-) was coupled to KeyholeLipet hemocyanin with m-maleimidobenzol-N-hydroxy-succinimide ester (Pierce, Rockford, USA). For the productionin Escherichia coli of recombinant protein composed ofXenopus Ac45 amino-acid residues Gly68 to Pro388 with ahexahistidine tail at its N-terminus, a 1.0-kb SacI/SmaI cDNAfragment of Xenopus hypothalamus cDNA clone X1311-2(1.7-kb insert) was ligated into the SacI/SmaI sites of the vectorpQE-30 (Qiagen Inc., Chatsworth, CA, USA). Followingpurification by SDS/PAGE, the protein was electroeluted fromthe gel. Rabbits were immunized with 500 mg of coupledpeptide or recombinant protein in Freund's complete adjuvant.Four weeks later, and at 3-week intervals thereafter, rabbits wereboosted with antigen in Freund's incomplete adjuvant. Theproduction of specific anti-Ac45 antibodies was monitored byenzyme-linked immunosorbent assay. The antisera against thepeptide and recombinant antigens were named 1311C and1311N, respectively.

Metabolic labeling of Xenopus NIL proteins andimmunoprecipitation analysis

NILs from black-adapted Xenopus were dissected and incubatedovernight in Xenopus culture medium (6.7 mL L-15 medium(Gibco-BRL, Gaithersburg, MD, USA), 3 mL milli-Q water,10 mg´mL21 kanamycin and 1% antibiotic-antimycotic solution(Gibco-BRL), 8 mg CaCl2, 3 mg bovine serum albumin and2 mg glucose) supplemented with 10% fetal bovine serum(Gibco-BRL) in the absence or presence of 10 mg´mL21

tunicamycin. Pulse labeling of newly synthesized proteins wasperformed by incubating lobes in methionine- and cysteine-freeculture medium containing 1.7 mCi´mL21 Tran[35S]-label (ICNRadiochemicals) in the absence or presence of tunicamycinfor the indicated time periods at 22 8C. Subsequent chaseincubations were in culture medium supplemented with 5 mml-methionine and 2.5 mm l-cysteine. Lobes were homo-genized on ice in lysis buffer (50 mm Hepes pH 7.2, 140 mmNaCl, 1% Tween 20, 0.1% Triton X-100, 0.1% deoxycholate,1 mm phenylmethanesulfonyl fluoride and 0.1 mg´mL21

soybean trypsin inhibitor). Homogenates were cleared bycentrifugation (10 000 g, 7 min at 4 8C), supplemented with0.1 volume of 10% SDS and diluted 10-fold with lysisbuffer before addition of anti-Ac45 antisera (1 : 500dilution). To remove possible anti-Ac45 antisera epitopeblocks, NILs were either lyzed in 50 mm Tris/HCl (pH 7.5),1% SDS, 50 mm 2-mercaptoethanol and 2 mm EDTA, andboiled for 5 min, or prepared in 8 m urea or 6 m guanidinehydrochloride. Following clearance by centrifugation, thelysate was diluted 15-fold with 50 mm Tris (pH 7.5), 1 mmEDTA, 0.1% Triton X-100, 0.5% NP-40 and 0.9% NaCl ordiluted with lysis buffer to 0.1 m urea or 0.1 m guanidinehydrochloride and immunoprecipitated. Immune complexeswere precipitated with protein-A Sepharose (LKB-Pharmacia)and resolved by SDS/PAGE. Radiolabeled proteins werevisualized by fluorography.

Cell transfection and metabolic labeling

Cell culture media were obtained from Gibco-BRL andsupplemented with 10% (v/v) fetal bovine serum. Green monkeyCV-1 kidney fibroblasts were cultured in Iscoves-modifiedEagle's medium and mouse anterior pituitary-derived AtT20cells in high glucose Dulbecco's modified Eagle's medium.Transfection of CV-1 and AtT20 cells was accomplished by thecalcium phosphate precipitation method [18]. A 1.7-kb EcoRIfragment of Xenopus hypothalamus cDNA clone X1311-4encoding the entire Ac45 protein, was subcloned downstreamof the cytomegalovirus promoter into the EcoRI site of theeukaryotic expression vector pcDNA3 (Invitrogen, San Diego,CA, USA). AtT20 cells were transfected with the XenopusAc45-pcDNA3 construct and after 48 h selected for stableexpression of Ac45 in medium containing 750 mg´mL21

neomycin (Boehringer, Mannheim, Germany). For transientexpression studies, CV-1 cells were plated in 20-mm culturedishes, grown until 30% confluency and transfected with 2.5 mgDNA per construct per dish. CV-1 cells (48 h after transfection)and stably transfected AtT20 cell lines were starved for 60 minin methionine- and cysteine-free Dulbecco's modified Eagle'smedium (ICN Biochemicals) supplemented with 10% dialyzedfetal bovine serum (Gibco-BRL) and subsequently pulsedfor 180 min in the same medium with 0.2 mCi´mL21

Tran[35S]-label. Cells were lysed on ice in lysis buffer and thecell lysates and incubation media were processed for immuno-precipitation analysis as described above.

486 J. C. M. Holthuis et al. (Eur. J. Biochem. 262) q FEBS 1999

Primary cultures of Xenopus intermediate pituitary cells

Primary cultures of intermediate pituitary cells were establishedfrom NILs of black-adapted Xenopus. To eliminate blood cellsfrom the lobes, animals were perfused intracardially withRinger's solution (112 mm NaCl, 2 mm KCl, 2 mm CaCl2,15 mm Hepes, pH 7.4, and 2 mg´mL21 glucose) for 10 min.Lobes were dissected, washed in sterile Xenopus culturemedium, transferred to Ringer's solution containing 0.25%(w/v) trypsin and incubated for 30 min at 20 8C. Lobes weresuspended by 10 passes through a siliconized Pasteur's pipet,transferred to a syringe and filtered through a nylon filter (poresize 150 mm). Cells were washed in Xenopus culture mediumsupplemented with 10% fetal bovine serum, collected bycentrifugation and cultured on poly-l-lysine coated coverslipsat 20 8C for 3 days.

Immunofluorescence microscopy

For steady-state immunofluorescence localization of Ac45, cellswere fixed in 2% paraformaldehyde/NaCl/Pi, pH 7.4, for 1 hat 4 8C, incubated in 100 mm glycine/NaCl/Pi for 30 min at4 8C, permeabilized in ice-cold 0.1% Triton X-100/NaCl/Pi

(NaCl/Pi-TX), and incubated with antiserum 1311C (1 : 300)in NaCl/Pi-TX containing 2% (w/v) bovine serum albumin

(NaCl/Pi-TXB) for 2 h at 4 8C. To visualize bound 1311Cantibodies, permeabilized cells were incubated with fluoresceineisothiocyanate (FITC) conjugated goat anti-rabbit antibodies(1 : 100; Nordic, Tilburg, the Netherlands) in NaCl/Pi-TXB for2 h at 4 8C. Immunostained cells were mounted in Citifluor(Agar Scientific Ltd, Stanstead Essex, UK) and viewed underepifluorescence optics with a Leica DMRB/E microscope(Leica, Heerbrugg, Switzerland) and a vario-orthomat camerasystem.

Western blot analysis

Xenopus tissues were dissected and immediately homogenizedin lysis buffer containing 10 mm EDTA. Lysates were cleared bycentrifugation (10 000 g, 7 min, 4 8C) and diluted with samplebuffer. Deglycosylation of proteins was achieved beforedilution by incubating the lysates with 0.2 U N-glycosidase F(Boehringer Mannheim). Proteins were separated by SDS/PAGEand electrotransferred to nitrocellulose. The membrane wasblocked and washed with blocking buffer (100 mm NaCl;100 mm Na2PO4; 1% Tween-20) containing 5% low-fatpowdered milk. Further washing steps and incubations withprimary and secondary antibodies were performed with blockingbuffer containing 2% low-fat powdered milk. The secondary

Fig. 1. Nucleotide sequence and deduced

amino-acid sequence of cDNA clone X1311-4

encoding Xenopus Ac45. The signal peptide

sequence is underlined and the putative signal

peptide cleavage site is indicated by an arrow.

Positive numbering of amino acids starts in the

mature protein. Double underlined amino acids

are predicted to span the membrane. Potential

Asn-linked glycosylation sites are marked by

black dots. Overlined text shows the consensus for

polyadenylation. The nucleotide sequence of

cDNA clone X1311-4 is available from the EMBL

database under accession number X82421.


antibody used was a peroxidase-conjugated anti-(rabbit IgG)antibody at a dillution of 1 : 3000, which was detected with anelectrochemilumescence kit (ECL; Amersham, Buckingham-shire, UK).

Immunocytochemistry

Pituitary glands of Xenopus were fixed in Bouin's fixative (70%picric acid; 25% formaldehyde; 5% acetic acid) for 16 h.Paraffin sections (5 mm) were pretreated with 50 mm Tris/NaCl(pH 7.6) containing 150 mm NaCl and 0.3% Triton X-100(Tris/NaCl/TX). The sections were blocked with 20% normalgoat serum and incubated with antiserum 1311C (1 : 500).Incubation with the secondary antibody goat anti-(rabbit IgG)(1 : 150; Nordic), was followed by an incubation withrabbit peroxidase-antiperoxidase (PAP; 1 : 1000; Nordic).Tris/NaCl/TX was used for all dillutions and washing steps.Immunocomplexes were visualized by rinsing the sections in50 mm Tris/HCl (pH 7.6) and subsequent incubation in 0.04%3,3 0-diaminobenzidine tetrahydrochloride (DAB, Sigma, 0.25%nickel ammonium sulphate and 0.015% H2O2 in 50 mmTris/HCl (pH 7.6). The reaction was terminated by rinsing in50 mm Tris/HCl (pH 7.6). Specificity of the immunoreactionwas demonstrated by preabsorption of the antiserum 1311C withthe antigen.

RESULTS

Cloning and sequence analysis of the Xenopus ortholog ofAc45

During a search for genes coordinately expressed with POMC inXenopus melanotrope cells [14], we isolated a 1.3-kb cDNA

(clone X1311) representing a highly regulated transcript of<2.3 kb. Northern blot and RNase protection analyses revealedthat this transcript is particularly abundant in neuroendocrinetissues with over 10-fold higher expression levels in brain andpituitary than in liver, heart or kidney [14]. In order to obtain thecomplete nucleotide sequence of this transcript, a Xenopushypothalamus cDNA library was screened with X1311 cDNA asa probe. The largest of the hybridization-positive clonesisolated, clone X1311-4, contained a cDNA insert of 2002 bp,excluding the poly(A) tail, whose nucleotide and deducedamino-acid sequence is presented in Fig. 1. The 621-bp3 0-untranslated region carries two polyadenylation signals,15- and 54-bp upstream of the poly(A) tail. A potentialtranslation initiation site was found at nucleotide positions3±11 (CAGAGATGG; [19]). Translation from this site wouldproduce a protein of 458 amino acids with a calculatedmolecular mass of 50 214 Da. The putative initiator methionineprecedes a hydrophobic signal sequence of 24 amino acids witha cleavage site conforming to the 21, 23 rule [20]. When thissignal sequence is cleaved off, a protein with a calculatedmolecular mass of 47 756 Da remains. A hydrophobicity plotindicated the presence of a potential transmembrane segmentclose to the C-terminus. The region between the signal peptideand the transmembrane domain contains seven consensus sitesfor N-linked glycosylation. A database search revealed a highdegree of structural similarity with the bovine protein Ac45, anaccessory subunit of V-ATPase from adrenal medulla chromaffingranules [12]. An additional match was found with a ratsequence (AF035387) and a human sequence of Ac45, compiledfrom an EST (AA489800), a genomic fragment (L44140) and apartial cDNA (clone CF2; D16469) isolated during a search forexpressed sequences encoded by the Xq terminal region of thehuman X-chromosome [21] (Fig. 2). We therefore conclude that

Fig. 2. Alignment of the amino-acid sequences

of Xenopus, bovine, human and rat Ac45. The

single-letter amino-acid code is used and identical

residues are boxed. Gaps are introduced for

optimal alignment. The signal peptide (SP) are

indicated and the transmembrane (TM) region is

boxed. Positive numbering of residues starts at the

first residue following the predicted signal peptide

cleavage site (arrow). Potential Asn-linked gly-

cosylation sites are shown in white letters. The

cleavage site of bovine Ac45 is indicated by an

arrow head. The bovine Ac45 amino-acid

sequence was taken from [12]. The human

sequence was compiled from an EST

(AA489800), a genomic fragment (L44140) and a

partial cDNA (clone CF2 [21]; D16469), and the

rat sequence was deduced from cDNA AF035387.


the protein encoded by clone X1311 is the Xenopus ortholog ofbovine, human and rat Ac45.

Evolutionary conservation of Ac45

Figure 2 shows an alignment of the amino-acid sequences ofXenopus, bovine, human and rat Ac45. With the signal peptidesequences excluded, Xenopus and bovine Ac45 share 60%amino-acid sequence identity over 427 matched residues (78%similarity), Xenopus and human 60% identity over 427 matchedresidues (77% similarity), and Xenopus and rat 61% identityover 426 residues (78% similarity). Bovine and human Ac45share 86% identity over 429 matched residues (92% similarity),bovine and rat 86% identity over 428 residues (90% similarity),and human and rat 92% identity over 428 residues (95%similarity). The positions of the transmembrane domains andfour of the potential N-linked glycosylation sites are conservedamong the four species. Two of the four cysteine residues inXenopus Ac45 are also present in mammalian Ac45. The Ac45sequence does not contain a conserved pair of basic amino-acidresidues (a potential proprotein processing site [22]).

Western blot analysis of Ac45

In order to determine the size, cellular fate and localization ofAc45, we raised two polyclonal antisera, one directed against a

recombinant protein comprising Xenopus Ac45 residues 68±388(antiserum 1311N) and the other against a synthetic peptidecomprising the C-terminal 12 amino-acid residues of Ac45(antiserum 1311C). Western blot analysis of Xenopus NIL usinganti-Ac45 antiserum 1311N showed no immunoreactive protein,while staining with the 1311C antiserum revealed an <40-kDaproduct (Fig. 3A). In view of the predicted size (see above), thisproduct presumably represents processed Ac45, as it reacts withthe 1311C antiserum, a C-terminal cleavage product of Ac45.The fact that with the 1311N antiserum the <40-kDa C-terminalAc45 cleavage fragment was not found, suggests that thisantiserum is not directed towards this portion of Ac45. It is, atpresent, not clear why the N-terminal Ac45 cleavage product(expected size of <20 kDa, see below) was not detected withthe 1311N antiserum. The level of immunoreactive Ac45 was<40% lower in the NILs of white-adapted Xenopus than inthose of black-adapted animals. A <40-kDa product was alsofound in the pituitary pars distalis and the brain, albeit in smalleramounts. No immunoreactive Ac45 was detected in liver, heart,pancreas, gut, kidney, lung and oocytes. Stainings with thepreimmune sera were negative (data not shown). Treatment ofthe NIL lysate with N-glycosidase-F prior to Western blotanalysis caused a shift in the migration from <40 kDa to<27 kDa (Fig. 3B).

Fig. 3. Western blot analysis of glycosylated and deglycosylated Ac45

from Xenopus neurointermediate lobes. (A) Proteins in total lysates of

neurointermediate lobes from black- and white-adapted Xenopus were

separated by SDS/PAGE, transferred to nitrocellulose and probed with the

anti-Ac45 antiserum 1311C. (B) Deglycosylation with N-glycosydase F

increases the mobility of the <40-kDa form of Ac45 to an <27-kDa form.

Fig. 4. Biosynthesis of Ac45 in Xenopus neurointermediate lobes. Lobes

dissected from black-adapted animals were preincubated overnight in the

absence (±) or presence (+) of 10 mg´mL21 tunicamycin and subsequently

pulsed for 3 h with Tran[35S]-label. Radiolabeled proteins were immuno-

precipitated from lobe extracts (three lobes per lane) and incubation media

using preimmune serum (lane 1) or 1311N antiserum (lanes 2±5).

Immunoprecipitates were resolved by SDS/PAGE and visualized by

fluorography. Protein markers for molecular mass (M) and migration

positions of immunoprecipitated Ac45 are indicated on the left. Migration

positions of POMC and a POMC-derived cleavage product in total protein

extracts (0.04 lobe per lane) are indicated on the right. Asterisks indicate the

migration postitions of the nonglycosylated proteins.


Biosynthesis of Ac45 in Xenopus intermediate pituitary

As the size of the intact Xenopus Ac45 protein predicted fromthe cDNA sequence (48 kDa, unglycosylated form) is differentfrom that observed on Western blot (unglycosylated form of<27 kDa, Fig. 3B), we examined the biosynthesis of XenopusAc45. Both anti-Ac45 antisera gave a newly synthesizedprotein of <60 kDa in immunoprecipitation studies onTran[35S]-labeled NILs from black-adapted animals; thisprotein was not recognized by the preimmune sera (Fig. 4,lanes 1 and 2, and data not shown). Pretreatment of NILs withtunicamycin prior to pulse labeling and immunoprecipitationanalysis resulted in a dramatic increase in the mobility of theprotein, corresponding to a decrease in its size from <60 kDa to<46 kDa (Fig. 4, lane 3). Ac45-related products were not foundin the chase media of the pulse-incubated NILs (Fig. 4, lanes 4and 5). Electrophoresis under reducing and nonreducingconditions gave similar mobilities for Ac45 (data not shown),suggesting that no intramolecular disulfide bonds were formedduring its biosynthesis. The above results indicate that (a) theprotein detected by our antisera represents newly synthesizedintact Ac45; (b) Ac45 undergoes N-linked glycosylation atmultiple sites; (c) the N-terminal, major portion of Ac45containing the potential N-linked glycosylation sites is trans-located into the lumen of the endoplasmic reticulum (ER) wherethis type of glycosylation is known to occur; (d) Ac45 ispresumably anchored in the membrane by the hydrophobic

segment found in its C-terminal region; (e) the intact <60-kDaAc45 product observed during the biosynthetic studies iscleaved to the immunoreactive <40-kDa C-terminal productfound on Western blots. As during our metabolic pulse-labelingstudies we did not detect an <40-kDa Ac45 cleavage product,we performed pulse-chase experiments with both anti-Ac45antisera. Although during the chase the amount of <60-kDaintact Ac45 gradually decreased, even long chase periods (up to24 h) did not give a <40-kDa product. Furthermore, extensivebiosynthetic studies using a variety of tissue extraction andimmunoprecipitation conditions to unmask any possible epitopeblocking did not lead to the precipitation of an <40 kDaradiolabelled Ac45 product. Also, the expected newly syn-thesized <20-kDa N-terminal Ac45 cleavage fragment was notfound during the biosynthetic studies (data not shown).

Biosynthesis of Ac45 in transfected cells

As an alternative approach to examine the biosynthesis of Ac45,we transfected anterior pituitary-derived AtT20 cells with aeukaryotic expression construct encoding Xenopus Ac45 underthe control of the cytomegalovirus promoter. Ten stable celllines expressing Xenopus Ac45 were selected by immuno-precipitation analysis of radiolabelled proteins; these cell linesproduced an N-glycosylated newly synthesized protein of<60 kDa not observed in control (untransfected) AtT20 cells.Because our antisera did not recognize endogenous Ac45 inAtT20 cells, the degree of overexpression in the transfected celllines could not be determined. When CV-1 fibroblast cells weretransiently transfected with the Xenopus Ac45 expressionconstruct and subjected to immunoprecipitation analysis, againa newly synthesized protein of <60 kDa was detected. Themobility of this protein shifted to <46 kDa when cells werepretreated with tunicamycin (data not shown). Like in theXenopus intermediate pituitary cells, we never detected an<40-kDa C-terminal or <20-kDa N-terminal newlysynthesized Ac45 cleavage product in the transfected cells.When analysed by immunofluorescence microscopy, transfectedCV-1 cells gave a strong and punctate Ac45 staining in thejuxtanuclear region with numerous labeled vesicular structuresdistributing in the cell periphery (Fig. 5A). In contrast,tunicamycin-treated cells displayed a reticular staining, typicalfor the ER, throughout the cytoplasmic compartment (Fig. 5B).These results indicate that N-glycosylation of Ac45 isindispensible for its proper export out of the ER, a phenomenonwhich applies to many other membrane glycoproteins [23].

Immunolocalization of Ac45 in Xenopus pituitary

Immunocytochemical analysis of Xenopus pituitary using the1311C antiserum revealed an intense staining in the cytoplasmof the melanotrope cells of the intermediate pituitary (Fig. 6).Like on Western blots, the 1311N antiserum gave noimmunocytochemical staining and essentially no staining wasfound with preabsorbed antisera. The difference in the amountof immunoreactive Ac45 detected on the Western blot of NILsfrom black-and white-adapted toads (Fig. 3) was reflected bythe intensities of the immunocytochemical staining patterns inthe melanotrope cells of black and white animals (Fig. 6). In theblack animals, the high biosynthetic rate of the melanotropecells is presumably responsible for the relatively high levels ofAc45, whereas the presence of the Ac45 protein in the cells ofwhite animals is probably due to stored protein. When applied inimmunofluorescence studies on primary cultures of NILsdissected from black-adapted animals, the 1311C antiserum

Fig. 5. Immunofluorescence localization of Ac45 in transfected CV-1

fibroblasts. Cells were transiently transfected with a Xenopus Ac45

expression construct and then incubated overnight in the absence (A) or

presence (B) of 10 mg´mL21 tunicamycin. After fixation and permeabiliza-

tion, cells were incubated with the anti-Ac45 antiserum 1311N and bound

antibodies were visualized with FITC-conjugated secondary antibodies.


gave a bright punctate staining in the cytoplasm of themelanotrope cells. Other cell types encountered in the culturedNILs (e.g. fibroblasts, endothelial cells, stellate cells) weredevoid of Ac45 immunostaining (data not shown). These resultsdemonstrate that the melanotrope cells constitute the primarysite of Ac45 production in Xenopus pituitary, whereas the non-neuroendocrine cells contain only a minor amount, if any, of theprotein.

DISCUSSION

V-ATPases are responsible for the acidification of a number oforganelles, such as endosomes, lysosomes, the Golgi complexand several classes of secretory vesicles, and consequentlyparticipate in a diverse range of cellular processes [2,24].Because in the various organelles the major subunits ofV-ATPases are highly similar, if not identical, it is conceivablethat accessory polypeptides are necessary for organelle-specificactivity. An accessory subunit of the V-ATPase is Ac45, a type Itransmembrane protein initially copurified with V-ATPase frombovine chromaffin granules [12]. In the present study, we clonedthe first nonmammalian Ac45 protein, and examined its

biosynthesis and immunolocalizaton in Xenopus pituitary.Comparison of the amino-acid sequence of Xenopus Ac45with its bovine, human and rat counterparts revealed that theoverall degree of conservation is moderate and only specificregions in Ac45 are well conserved. The comparative analysismay thus contribute to a rational design of future structure±function studies aimed at identifying functionally importantregions in the Ac45 protein.

Our biosynthetic labeling experiments with Xenopus inter-mediate pituitary cells revealed that Ac45 is synthesized in theER as a type I transmembrane protein of <46 kDa andsubsequently most of the seven lumenally located potentialN-linked glycosylation sites are used, resulting in thebiosynthesis of a <60 kDa Ac45 product. This result togetherwith the detection of an <40-kDa glycosylated (<27-kDaunglycosylated) Xenopus Ac45 immunoreactive C-terminalfragment on Western blots indicates that Ac45 is proteolyticallyprocessed. Despite of the use of two anti-Ac45 antisera (directedagainst the lumenal and cytoplasmic portions) in our metabolicpulse-chase labeling experiments with long chase periods andthe attempts to unmask possible Ac45 epitope blocking, itremains unclear why we did not detect the <40-kDa Ac45

Fig. 6. Immunocytochemical analysis of Ac45

in Xenopus pituitary. Paraffin sections of

pituitaries from (A) black- and (B) white-adapted

Xenopus were incubated with 1311C antiserum.

Bar, 100 mm.


cleavage product as a newly synthesized protein. The reasonwhy the <20-kDa N-terminal Ac45 cleavage fragment was notobserved, either on Western blots or during the pulse-chasebiosynthetic studies, is also a subject of future research.Considering the sizes of the unglycosylated intact and cleaved(C-terminal) forms of Xenopus Ac45 (<46 kDa and <27 kDa,respectively), one would expect the cleavage site in Ac45 to bearound amino-acid residue 180 in the Ac45 sequence, inreasonable agreement with the site found for bovine Ac45(namely between Val208 and Val209 [13]).

We find that Xenopus Ac45 is produced mainly inneuroendocrine cell types, suggesting that Ac45 is not acommon subunit of V-ATPases but contains sorting signalswhich direct the protein to neuroendocrine-specific intracellularorganelles harbouring a V-type proton pump. Alternatively,Ac45 may assist in the assembly of a functional enzymecomplex or regulate pump activity in specific organelles such asthe trans-Golgi network (TGN), the earliest compartment of thesecretory pathway shown to have an acidic pH [25], or thesecretory granules. An intriguing possibility for the function ofAc45 is that in the early stages of the secretory pathway, Ac45associates with, and inhibits the activity of, V-ATPase until it iscleaved and the pump is activated, allowing acidification of theTGN and the (immature) secretory granules. In the TGN/secretory granules of neuroendocrine cells, acidification isnecessary for prohormone processing [26,27]. In this context itis of note that Xenopus Ac45 cDNA was isolated during adifferential screening approach that resulted in the identificationof cDNAs encoding proteins that are upregulated in coordinationwith prohormone production. Intermediate pituitary cells ofanimals adapted to a black background contain <30 times morePOMC mRNA and <10 times more Ac45 transcripts than thoseof white-adapted animals [14]. The present finding of the highlevels of the Ac45 protein expressed in the biosyntheticallyactive melanotrope cells of black-adapted animals is in line withthis result. Together, these observations suggest a role for Ac45in the V-ATPase-mediated acidification of secretory compart-ments in which prohormone processing is initiated.

ACKNOWLEDGEMENTS

We gratefully acknowledge Tony Coenen for technical assistance and Ron

Engels for animal care. This work was supported by the Netherlands

Organization for Scientific Research (NWO; grant number 805-33.212) and

EU-TMR Research Network ERBFMRXCT960023.

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Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 in Xenopus pituitary