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Virus Research 131 (2008) 86–94

Post-translational modifications of Abutilon mosaic virusmovement protein (BC1) in fission yeast

Tatjana Kleinow ∗, Gerlinde Holeiter 1, Marc Nischang, Monika Stein,Miriam Karayavuz, Christina Wege, Holger Jeske

Institute of Biology, Department of Molecular Biology and Plant Virology, University of Stuttgart,Pfaffenwaldring 57, 70550 Stuttgart, Germany

Received 6 June 2007; received in revised form 18 August 2007; accepted 23 August 2007Available online 4 October 2007

bstract

The movement protein (MP) of Abutilon mosaic virus (AbMV, Geminiviridae) exhibited a complex band pattern upon gel electrophoresisndicating its post-translational modification when expressed in AbMV-infected plants or, ectopically, in fission yeasts. High-resolution separationccording to charge and molecular weight in acetic acid/urea polyacrylamide or sodium dodecyl sulphate polyacrylamide gels followed by westernlot analysis revealed a pattern of AbMV MP from infected plants more related to that from fission yeast than from bacteria. For this reason,xpression in fission yeast was established as an experimental system to study post-translational modifications of AbMV MP. Metabolic labelling

ith 32P-orthophosphate confirmed a phosphorylation of all MP variants suggesting multiple phosphorylation sites. Treatment with calf intestinal

lkaline phosphatase removed this label completely, but was unable to eliminate all protein bands with lower electrophoretic mobility. Thus,ultiple phosphorylations contribute to the complex migration behaviour of MP, but additional post-translational modifications may occur as well.2007 Elsevier B.V. All rights reserved.

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eywords: Schizosaccharomyces pombe; In vivo labelling; Geminivirus

. Introduction

Abutilon mosaic virus (AbMV) belongs to the genus Bego-ovirus (Geminiviridae) and has a bipartite genome consisting

f two single-stranded DNA circles (DNA A and B), which areeplicated via double-stranded intermediates in plant cell nucleiRojas et al., 2005; Stanley et al., 2005). DNA A encodes pro-eins necessary for replication, expression and encapsidation,hereas DNA B contains two open reading frames, one for theuclear shuttle protein (NSP, syn. BV1 or BR1) and the otheror the movement protein (MP, syn. BC1 or BL1; Bisaro, 2006;ehnle et al., 2004; Rojas et al., 2005; Stanley et al., 2005;nseld et al., 2004, 2001; Zhang et al., 2001). NSP and MP

re important for cell-to-cell and long-distance transport via thehloem (Brough et al., 1988; Etessami et al., 1988). NSP medi-ted the export of viral DNA from the nucleus to the cytoplasm

∗ Corresponding author. Tel.: +49 711 685 65075; fax: +49 711 685 65096.E-mail address: tatjana.kleinow@bio.uni-stuttgart.de (T. Kleinow).

1 Present address: Institute of Cell Biology and Immunology, University oftuttgart, Allmandring 31, 70550 Stuttgart, Germany.

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168-1702/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2007.08.011

nd import of viral DNA into the nucleus of newly infectedells (Gafni and Epel, 2002; Pascal et al., 1994; Sanderfoott al., 1996; Ward et al., 1988). MP redirected the NSP fromhe cytoplasm to the cell periphery (Sanderfoot and Lazarowitz,995) and viral DNA via the plasmodesmata to adjacent cellsNoueiry et al., 1994). Two transport models are currently undernvestigation. The “relay race model” predicts that NSP mediatesuclear export of viral DNA and passes it to MP, which transfershe DNA via plasmodesmata to adjacent cells (Noueiry et al.,994; Rojas et al., 1998). In the second “couple-skating model”,SP transports viral DNA to the cell periphery and MP acts

s a membrane adaptor for NSP–DNA complexes to facilitateell-to-cell movement (Frischmuth et al., 2004, 2007; Hehnlet al., 2004; Pascal et al., 1994; Sanderfoot and Lazarowitz,995; Zhang et al., 2002, 2001). Data obtained from localizationtudies using transiently expressed NSP:GFP (green fluorescentrotein) and MP:GFP in plant cells supported the couple-skating

odel for AbMV (Zhang et al., 2002, 2001). In vitro assembly of

ouble-stranded supercoiled DNA with AbMV NSP and MP intoonspicuous structures provided evidence for a physical inter-ction of MP, NSP and DNA (Hehnle et al., 2004). However,

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east two-hybrid system failed to detect any direct interactionetween the two proteins, whereas AbMV MP deletion mutantsndicated a homo-oligomerization of the C-terminus in Gal4-ased yeast two-hybrid and Cyto-Trap® analysis confirmed thentimate association of AbMV MP with the cytoplasmic face ofhe plasma membrane (Frischmuth et al., 2004).

Geminiviral MPs turned out to be determinants forathogenicity as well (Duan et al., 1997; Hou et al., 2000;ngham et al., 1995; Pascal et al., 1993; Saunders et al., 2001; vonrnim and Stanley, 1992). Like other begomoviruses, AbMV

xhibited a variation of disease symptoms depending on theost plant (Hou et al., 2000; Wege et al., 2000). The compo-ents underlying these differences are still unknown. Mutationaltudies indicated that the C-terminus of MP might play a rolen symptom development (Duan et al., 1997; Hou et al., 2000;ascal et al., 1993; Saunders et al., 2001).

There is increasing evidence that various viral MPs exists phosphoproteins in plant cells and that their phosphoryla-ion status specifies sets of functions (Waigmann et al., 2004).hosphorylation of MPs has been reported for numerous RNAiruses (Citovsky et al., 1993; Haley et al., 1995; Kawakamit al., 1999; Matsushita et al., 2000, 2002; Seron et al., 1996;okolova et al., 1997; Watanabe et al., 1992). The regulatory rolef phosphorylation on transport activity of MPs has been stud-ed extensively for Tobacco mosaic virus (TMV; Tobamovirus).MV MP harbours three carboxyterminal phosphorylation sites,hich were phosphorylated in vitro and in vivo by cell wall-

ssociated kinases (Citovsky et al., 1993; Waigmann et al.,000). TMV MP mutants mimicking phosphorylation by nega-ively charged amino acid replacement lost their ability to gatelasmodesmata and showed a negative effect on transport in theost plant Nicotiana tabacum (Trutnyeva et al., 2005; Waigmannt al., 2000). A plasmodesmata-associated protein kinase of theasein kinase 1 family was able to phosphorylate the C-terminalesidues of TMV MP in vitro (Lee et al., 2005). An internalhr104 of TMV MP was confirmed to be phosphorylated initro by an ER-associated kinase activity, and phosphorylation-imicking mutants for Thr104 strongly inhibited cell-to-cellovement (Karger et al., 2003). Phosphorylation of MPs may

lay a role in the switch between replication/translation and viralpread as well (Karpova et al., 1997, 1999). TMV RNA coatedn vitro with MP could not be translated or replicated, unless thehosphorylation of MP converted the RNA–protein complex totranslatable substrate.

Circumstantial evidence suggested that geminiviral MPs arelso post-translationally modified (Pascal et al., 1993; von Arnimt al., 1993). Western blot analysis of protein extracts from plantsnfected with the begomoviruses African cassava mosaic virusACMV) or Squash leaf curl virus (SLCV) showed various MPands with altered migration behaviour (Pascal et al., 1993; vonrnim et al., 1993). In the case of ACMV, the nature of theodifications remained to be determined, but glycosylation, or

he association with nucleic acids were excluded (von Arnim

t al., 1993). In vivo labelling experiments of SLCV MP andSP in insect cells have shown phosphorylation, but not gly-

osylation of either protein (Pascal et al., 1994). Cabbage leafurl virus (CaLCuV, begomovirus) NSP interacted specifically

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arch 131 (2008) 86–94 87

ith a proline-rich extensine-like receptor kinase and serveds substrate for phosphorylation (Florentino et al., 2006). Inscherichia coli ectopically expressed AbMV MP exhibited aingle protein band of the expected molecular weight, and wasound to be phosphorylated (Wege and Jeske, 1998). In yeast,rotein bands with slower mobility than expected were observedy western blot analysis (Frischmuth et al., 2004, 2007), andere similar to those from infected plants (Zhang et al., 2001).hese data suggest eukaryote-specific post-translational modifi-ations of the MP. To characterize these modifications in closeretail, we studied the phosphorylation of AbMV MP in fissioneast as an experimental model.

. Methods

.1. Protein expression in bacteria and yeast

AbMV MP was expressed in and purified from E. coli asescribed earlier (Wege and Jeske, 1998). The plasmids pESP1-REP2-BC1 (Leu+) and pESP1-pREP2 (Leu+) (Frischmuth etl., 2007) were transformed into Schizosaccharomyces pombestrain SP-Q01; leu1-32; Stratagene) using LiCl (Ausubel et al.,999). Expression was controlled by the nmt1 promoter (“noessage in thiamine”; Maundrell, 1993). Single yeast colonies

rown on EMMT Leu− plates (Edinburgh Minimal Mediumlus 25 �M thiamine; Ausubel et al., 1999) were inoculated toMMT Leu− medium containing reduced Na2HPO4 concentra-

ion (5.2 mM; EMMT1/3P) and shaken overnight. EMMT1/3Pedium was inoculated with this overnight culture to start withD600 0.2, and shaking was continued for 5 h. Cultures wereivided prior to the start of protein expression. One half wasashed twice with EMM1/3P without thiamine and diluted

nto EMM1/3P medium to a starting OD600 of 0.02. For aepressed control, the second half of the pre-culture was pro-essed similarly, but in the presence of 25 �M thiamine. Theultures were incubated for 18 h with shaking. Cells were har-ested by centrifugation (1 min, 12 000 × g, room temperature),ashed twice with ice-cold PBS buffer (137 mM NaCl, 6.5 mMa2HPO4, 2 mM NaH2PO4) and frozen in liquid nitrogen. Cellsere resuspended in ice-cold PBST-PI buffer (PBS suppliedith 1% Triton×-100 and protease inhibitors: 1 mM phenyl-ethylsulfonyl fluoride [PMSF], 1 mg/ml aprotinine, 1 �M

epstatin A, 100 �M leupeptin and 1 �g/ml chymostatin). Cellsere vortexed together with glass beads at intervals for 5 min

30 s, 30 s on ice). The suspension was transferred to a freshube and centrifuged (4 min, 12 000 × g, room temperature).fter recovering the supernatant to a second tube, the pelletas dissolved in the same volume of PBST-PI buffer as the

upernatant.

.2. In vivo labelling

Pre-cultures were obtained as described above. Following

he release of thiamine repression by transferring the fissioneast to EMM1/3P medium, or EMMT1/3P for repressedontrol, respectively, the cultures were supplemented with25 �Ci/ml 32P-orthophosphate (General Electric [GE] Health-

8 Research 131 (2008) 86–94

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Fig. 1. Accumulation of MP during AbMV infection. Western blot analysis ofthe MP content in upper leaves of Nicotiana benthamiana plants systemicallyinfected with AbMV at different days post inoculation (dpi). An extract of anMm

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are, formerly Amersham Biosciences) and incubated for 18 h.arvesting, washing and protein extraction were done asescribed above. For experiments with calf intestinal alkalinehosphatase (CIAP, New England Biolabs) treatment, the yeastells were washed twice in TBS buffer (137 mM NaCl, 20 mMris–HCl; pH 7.6) and resuspended in NEB buffer 3 (50 mMris–HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT; pH 7.9t 25 ◦C supplemented with protease inhibitors). The pelletractions of the total protein extracts were dissolved in the super-atants volume of NEB buffer 3. Half a volume of supernatantnd resuspended pellet were pre-warmed for 10 min at 30 ◦Cefore 30 U CIAP were added and shaken for further 15 min.or the time course experiments, aliquots were removed from

he reaction at time points as indicated in Fig. 6. All samplesere immediately processed for separation by sodium dodecyl

ulphate polyacrylamide gels (SDS-PAGE).

.3. Protein extracts from plants

AbMV was agro-inoculated on Nicotiana benthamianaOMIN plants as described earlier (Evans and Jeske, 1993;on Arnim et al., 1993). Sink leaves from fully symptomaticr uninfected plants were homogenized at 4 ◦C in 2 ml grindinguffer (GB) per gram plant material (von Arnim et al., 1993) plusrotease inhibitors (protease inhibitor cocktail tablets, EDTA-ree [Roche] and 1 mM PMSF). The crude protein extracts wereither cleared by centrifugation (4 ◦C, 7 min, 20 000 × g) andhe supernatants analysed by western blots (Figs. 1 and 2) or

icrosomes were enriched. For partial purification of micro-omes, the crude extracts were filtered through a nylon mesh,aid on a 60% (w/w) sucrose cushion and centrifuged (4 ◦C,0 min, 20 000 × g) to yield pellet (P20) and supernatant (S20).

20 was resuspended in the same volume GB as for S20. S20as loaded onto a sucrose step gradient (25% (w/w) sucrose,

nd 60% (w/w) sucrose) and centrifuged at 100 000 × g for 2 ht 4 ◦C, resulting in supernatant (S100) and interphase (i100)

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ig. 2. Analysis of MP in acetic acid/urea gels. The protein profile of the samples wbMV MP expressed in Escherichia coli (Ec; lane +), Schizosaccharomyces pombef a non-infected plant (Nb; lane ni) and from non-induced S. pombe (Sp; lane −), oample are rather products of leaky repression than cross-reactive, since vector controigration marker. Brackets mark the positions of MP signals in plant samples and let

on-infected N. benthamiana plant (ni) was used as a control. Arrows indicateP signals and asterisks cross-reacting host proteins. Positions of molecularass (kDa) marker are shown on the left.

ractions. Aliquots from each purification step were analysed byestern blotting (Fig. 3).

.4. SDS-PAGE and western blotting

After adding SDS loading buffer to the samples (final con-entration 2% SDS, 6.5% glycerol, 62.5 mM Tris–HCl, 5%-mercaptoethanol, 0.002% bromophenolblue; pH 6.8) andeating for 5 min at 95 ◦C, 25 �g/�l iodoacetamide were sup-lied. Proteins were separated by 12.5% SDS-PAGE (Laemmli,970). Either small gels (8 cm × 10 cm; Figs. 1, 2 and 4) weresed, or for better resolution large SDS-PAGE (16 cm × 18 cm,igs. 3, 5 and 6). In both cases, electrophoresis was continuedntil the 17 kDa marker protein of a prestained protein ladder

MBI Fermentas; 10–180 kDa) was run out of the gel matrix.

small gel was processed at 17.5 mA for 2.5 h and a large gelt 12 mA overnight. Blotting to nitrocellulose membranes wasone using 50 mM Tris, 50 mM boric acid and 10% methanol as

as revealed by Coomassie blue staining (A) or western blot analysis (B) for(Sp; lane +), and AbMV-infected N. benthamiana plants (Nb; lane i). Extractsr E. coli (Ec; lane −), were used as controls. Bands in the non-induced yeastls usually did not exhibit these signals (see Fig. 5). RNase A protein served asters in yeast and bacteria. The polarity of the electrodes is indicated at the left.

T. Kleinow et al. / Virus Rese

Fig. 3. Comparison of AbMV MP expressed in different cells at high-resolutionin SDS-PAGE. Western blot analysis for MP from purified inclusion bodiesof E. coli (Ec), protein extracts (pellet fraction) of MP-expressing S. pombe(Sp), or from upper leaves of non-infected (ni) and AbMV-infected (i) N. ben-thamiana plants (Nb). Filtrates from plant protein extracts (F) were laid on a60% sucrose cushion and centrifuged at 20 000 × g revealing supernatant (S20)and pellet (P20). S20 was subjected to sucrose step gradient centrifugation at100 000 × g yielding interphase (i100) and supernatant (S100). MP signals inypm

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east and plants are labelled by letters. Cross-reacting non-specific signals inlant extracts (asterisks) and potential proteolytically digested MP (bracket) arearked. Positions of molecular mass (kDa) of marker are indicated.

ransfer buffer. Small gels were semidry-blotted at 20 V, 400 mAor 40 min and large ones at 10 V, 140 mA for 90 min. Westernlot detection using an anti-AbMV MP polyclonal antiserumrom rabbit (1:5000) and an alkaline phosphatase-conjugated

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ig. 4. In vivo phosphorylation of MP in S. pombe. Cells containing pESP1-pREP22P-orthophosphate. Proteins from supernatant (S) and pellet (P) fractions were extraestern blot is shown in (B). MP-specific bands are indicated (a, b and c). MP signals in

o vector controls in Fig. 5). Positions of molecular mass (kDa) marker are labelled.

ig. 5. De-phosphorylation of MP by CIAP treatment. After in vivo labelling with 3

BC1) or pESP-pREP2 (V) with (+) and without induction (−) were treated with (+CIDS-PAGE and analysed for MP by western blot (A) and autoradiography of the sameo signals were detected, the bands in the non-induced pESP1-pREP2-BC1 yeast saositions of molecular mass (kDa) marker are marked.

arch 131 (2008) 86–94 89

ffinity-purified goat anti-rabbit IgG (Rockland Inc.) was per-ormed according to Wege and Pohl (2007).

.5. Acetic acid/urea (AU)-PAGE and western blotting

AU-PAGE loading buffer (5% acetic acid, 3 M urea, 0.01%ethyl green) was added to the samples before heating at 100 ◦C

or 3 min. AU-PAGE (15%) and semidry blotting to nitrocellu-ose membrane were carried out according to Wang et al. (1997).Nase A (isoelectric point 7.8) was used as marker protein.

mmunodetection was carried out as mentioned above.

. Results

.1. Increasing amounts of MP variants during infection oflants with AbMV

To investigate the occurrence of MP band patterns duringhe course of AbMV infection, plants were analysed at differentays post infection (dpi) by western blotting (Fig. 1). Emergingink leaves could be sampled until 38 dpi, shortly after plantstarted flowering. To identify host proteins cross-reacting withhe antiserum, sink leaves from non-infected plants were used as

ontrol. Loading of protein amounts was verified by Serva violettaining of parallel SDS-PAGE (data not shown) and can bevaluated in blots from the cross-reacting bands of host proteinsFig. 1, asterisk). Nineteen days post infection was the earliest

-BC1 with (+) and without (−) induction were cultivated in the presence ofcted and subjected to western blot analysis (A). An autoradiogram of the samethe non-induced samples resulted most likely from leaky repression (compared

2P-orthophosphate, extracts from fission yeast containing pESP1-pREP2-BC1AP) or kept without (−CIAP) CIAP. Samples were subjected to high-resolutionwestern blot (B). Because in the vector controls on the corresponding positionsmples are interpreted as leaky repression. MP-specific bands (a, b and c) and

90 T. Kleinow et al. / Virus Research 131 (2008) 86–94

Fig. 6. Time course of MP de-phosphorylation by CIAP. Pellet proteins were extracted from a fission yeast culture expressing MP after metabolic labelling with3 ts weh s wellP

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2P-orthophosphate. The protein fractions were incubated with CIAP and aliquoigh-resolution SDS-PAGE and analysed subsequent by western blotting (A) aositions of molecular mass (kDa) marker are indicated.

ime point at which MP could be clearly detected in extracts ofnfected sink leaves (data not shown). The amount of proteinxhibiting a higher apparent molecular weight than expectedncreased at 25 dpi and accumulated further up to 38 dpi (Fig. 1).

.2. The migration behaviour of AbMV MP in gels wasimilar if expressed in yeasts and plants but different fromacterial-derived protein

To compare AbMV MP produced in different organisms ando explore if yeast-expressed MP resembles that in plants, wenalysed proteins from various sources by AU-PAGE, a tech-ique which separates proteins according to their charge andolecular weight (Boulikas, 1985; Zweidler, 1978). To avoid

ny effect of tags on protein properties, non-tagged full-lengthP was expressed in bacteria and yeasts. From E. coli trans-

ormants containing the MP expression constructs inclusionodies were enriched (Wege and Jeske, 1998). Total proteinxtracts from MP-expressing yeast cells were separated intoupernatant and pellet fractions upon low speed centrifugation.s a membrane-associated protein, AbMV MP is mainly present

n the pellet fraction, which was therefore used for further anal-sis (Aberle et al., 2002; Frischmuth et al., 2004, 2007; Zhangt al., 2002). The heterologously expressed proteins were com-ared to MP from sink leaves of AbMV-infected plants. As aontrol, non-induced and non-infected samples were analysedn parallel. In all three organisms, a complex band pattern wasound after western blotting (Fig. 2A). Although a single bandf the expected molecular weight was obtained in SDS-PAGEor E. coli-derived MP (compare Fig. 3), AU-PAGE separationevealed two bands (Fig. 2B, z and y) and two blocks each con-isting of four bands with higher mobility (Fig. 2B, x and w),ndicating the presence of more negatively charged MP vari-nts. Compared to E. coli, MP from fission yeast exhibitedifferent migration behaviours. The major MP band (Fig. 2B,) showed a lower mobility reflecting a less positive chargend/or an increased molecular weight. Additionally, two sig-als with a higher mobility than in E. coli (Fig. 2B, s and r)

nd further two bands (Fig. 2B, u and t) were detected. In thebMV-infected plant samples, the identification of the specificP pattern was more difficult and needed accurate comparison

o the non-infected sample due to low MP content and numerous

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re removed at the indicated time points (0, 15, 30, 45 and 60 min), separated byas autoradiography of the same blot (B). MP signals are labelled (a, b and c).

ross-reactive host proteins. Nevertheless, the most prominentP-specific signals of the AbMV-infected plant extract could

e assigned to a range which was more related to that of MProm S. pombe than that from E. coli (Fig. 2B, brackets). Inummary, AU-PAGE analysis indicated the presence of multi-le MP variants, which from bacteria were more different thanhose of plants and yeast.

To further analyse the differentially modified AbMV MP atigh-resolution, SDS-PAGE with a longer separation distanceas used. MP from enriched inclusion bodies of E. coli, total pro-

ein extracts from MP-expressing S. pombe (pellet fraction), orrom AbMV-infected N. benthamiana (28 dpi), and as controls,on-induced or non-infected samples were tested in parallel.ecause MP expression occurs only temporally and is limited

o phloem tissues during AbMV infection, the overall proteinontent in an infected leaf is extremely low (Abouzid et al.,988; Horns and Jeske, 1991; Wege et al., 2001; Zhang et al.,001). To enhance the relative MP amount, microsomes wereartially purified to enrich the membrane-associated MP. E. coli-xpressed MP showed only one single band with the expectedolecular weight of 33 kDa after western blotting (Fig. 3, Ec). In

ontrast, MP derived from plants as well as from yeasts revealeddditional bands with lower mobility in SDS-PAGE (Fig. 3a, b,and d in Sp and Nb). In plants (Fig. 3, Nb) unspecific, cross-

eactive signals were present at the apparent molecular weightsf 24 and 38 kDa, the latter of which could be reduced by partialurification of microsomes leading to better discrimination ofP bands (Fig. 3, Nb, i100). AbMV MP-specific bands with

igher mobility appeared in the S100 fraction (Fig. 3, bracket)nd presumably represent degradation products.

.3. AbMV MP was found to be phosphorylated in fissioneast

Because the band pattern was more similar for MP producedn plants and yeast than in E. coli, we used fission yeast as aodel system to determine the nature of post-translational mod-

fications and tested first for in vivo phosphorylation. After 18 h

f radioactive labelling, fission yeast cells were harvested, totalroteins extracted and subjected to western blotting as well as toutoradiography (Fig. 4). Non-induced yeast cultures were useds control. To investigate a possible difference in MP phosphory-

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ation between supernatant and pellet fractions, we analysed bothn parallel. MP-specific immunodetection with the typical bandattern of various apparent molecular weights (compare Fig. 3)ere obtained within both fractions after induction (Fig. 4A).he autoradiogram (Fig. 4B) of the respective western blot

evealed signals at the positions of all three bands (Fig. 4Ba,and c).To scrutinize whether the radioactive label arose from in

ivo phosphorylation and not from unspecifically associated2P-orthophosphate, we repeated the labelling experiments, butncubated one half of each protein sample with calf intestinallkaline phosphatase (CIAP) for enzymatic de-phosphorylationrior to analysis. CIAP de-phosphorylates phosphoserine, phos-hothreonine and phosphotyrosine residues (Ausubel et al.,999). As an additional control, we included empty vector-arbouring yeast cells in the analysis. Because the first in vivohosphorylation experiments exhibited no difference betweenupernatant and pellet fractions with respect to MP phosphory-ation, but more phosphorylated host proteins in the supernatant,e focused on the pellet fractions for further testing. In west-

rn blots a MP band pattern with the typical diverse migrationehaviour was observed in the untreated samples (Fig. 5A, a, bnd c). In contrast, after CIAP treatment a reduction of MP sig-als with lower mobility (Fig. 5A, b and c) and a complementaryncrease of the band at 33 kDa (Fig. 5A, a) was detected. Theutoradiogram of this western blot (Fig. 5B) showed the cor-esponding disappearance of radioactive label for CIAP-treatedamples, particularly for protein band c.

To test whether a longer CIAP incubation would completelye-phosphorylate and eliminate the MP bands with slowerigration; we performed a time course experiment. The pellet

ractions were incubated with CIAP for 0, 15, 30, 45, and 60 min.liquots from the reaction were removed, subjected to westernlot analysis and autoradiography. The western blot (Fig. 6A)evealed again that CIAP reduced the amount of MP species withower mobility (Fig. 6A, b and c), but did not eliminate themven after 1 h treatment. The radioactive signal for all three MPands (a, b and c) was already absent after 30 min incubation,ndicating their predominant de-phosphorylation (Fig. 6B).

. Discussion

The comparison of AbMV MP expressed in E. coli, S. pombe,r AbMV-infected plants revealed a protein pattern after eukary-tic expression, which was significantly different from that inacteria. With proteins from fission yeast and plants, bands withower mobility reproducibly occurred in SDS-PAGE and west-rn blot analysis and indicated post-translational modificationsn eukaryotes. In contrast, expression in bacteria resulted in aingle MP-specific band corresponding to the expected molec-lar weight of 33 kDa. However, even for this protein a diverseand pattern was detectable in western blots after AU-PAGE,orresponding to differential post-translational modifications in

. coli, for which phosphorylation of MP has been shown before

Wege and Jeske, 1998). The MP migration in AU-PAGE dif-ered between S. pombe- and E. coli-expressed proteins. Theeduced mobility of the majority of MP from yeast may result

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arch 131 (2008) 86–94 91

rom either a decreased positive charge due to multiple phos-horylations, and/or an increased molecular weight. The MPattern in AU-PAGE from infected plants was more closelyelated to that from fission yeast than to the pattern from bacte-ia, indicating several post-translational modifications of AbMV

P in eukaryotes. Because the yeast and plant pattern were notompletely identical, plant-specific modifications may exist inddition.

During AbMV infection, MP expression was transient and theumber of MP-expressing infection sites low due to phloem-imitation, which prohibited a study of post-translational

odifications in planta so far (Abouzid et al., 1988; Horns andeske, 1991; Wege et al., 2001). Thus, we established S. pombes a reasonable experimental system to study post-translationalodifications of the geminiviral MP, since the comparison ofP in gels with different separation principles indicated similarodifications in yeast and plants. Moreover, the first fundamen-

al functional step of viral movement seems to be conserved inssion yeast as well, because AbMV MP and NSP revealed aimilar localization in S. pombe as in plant cells (Aberle et al.,002; Frischmuth et al., 2004, 2007; Zhang et al., 2002, 2001). Inoth systems, NSP appeared in the nucleus, whereas MP local-zed to the plasma membrane. Additionally, in sub-populationsf plant cells MP could be found around the nuclei (Zhang etl., 2002, 2001). Upon co-expression of both proteins in fissioneast, MP redirected NSP from the nucleus to the plasma mem-rane (Frischmuth et al., 2007). A similar redirection combinedith cell-to-cell transport of NSP has been observed in plant

ells using NSP:GFP (Zhang et al., 2001).In this study, metabolic labelling of MP with 32P-

rthophosphate in fission yeast demonstrated the presence ofovalently bound phosphate in the protein. Radioactive labellingccurred for all MP bands, which could be detected by anti-odies supporting the idea of multiple phosphorylation sites.reatment with CIAP eliminated predominantly the label ofP, but did not remove the protein variants with lower mobil-

ty indicating possible other post-translational modifications.he yeast-expressed untagged C-terminus of AbMV MP exhib-

ted in western blots protein bands with lower mobility as well,owever; in vivo labelling assays provided no evidence for phos-horylation (supplementary Fig. S1). These results could eitherndicate the absence of phosphorylation sites in the C-terminalnd or may be attributed to a lack of recognition by the phospho-ylating kinase, caused by changed protein structure, or differentocalizations within the yeast cell leading to spatial separation.n contrast, other putative modification systems recognize the-terminus as substrate leading to protein bands with alteredigration behaviour. This result fits to our previous finding

n full-length MP, which exhibited still protein signals withhanged migration behaviour in immunodetection although therotein was predominantly de-phosphorylated (Fig. 6). AbMVP ectopically expressed in yeast was tested for ubiquitination

y western blot analysis using an ubiquitin-specific antibody,

ut no ubiquitination was detected (unpublished data). Proba-ly AbMV MP is not glycosylated, because ACMV MP fromlants and SLCV MP expressed in insect cells have tested neg-tive for glycosylation (Pascal et al., 1993; von Arnim et al.,

92 T. Kleinow et al. / Virus Research 131 (2008) 86–94

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ig. 7. Potential phosphorylation sites of AbMV MP. In the amino acid sequen999). Putative sites are marked by triangles. Pilot (dotted line) and membraneor oligomerization is indicated by a dashed line (Frischmuth et al., 2004).

993). Thus, other putative modifications of AbMV MP have toe taken strongly into consideration.

Other geminiviral proteins may be phosphorylated as well.he transcriptional activator protein (TrAP) from Tomato goldenosaic virus (TGMV) was shown to occur as phosphopro-

ein if expressed in insect cells (Hartitz et al., 1999; Wang etl., 2003). Correspondingly, the protein exhibited two to threelosely spaced bands. In contrast to AbMV MP, these bandsere converted to a single one by CIAP treatment. Cell frac-

ionation studies supported the conclusions that TrAP localizeso cytoplasm and nucleus, but phosphorylated protein accumu-ated preferential in the nucleus, indicating that this modification

ay be involved in the cellular localization of TrAP.Potential phosphorylation sites in AbMV MP were deter-

ined using Net phos2.0 program (Fig. 7; Blom et al., 1999).n total, 15 sites for serine, three for threonine and one foryrosine phosphorylation were detected. The phosphorylationites were found to be conserved between MPs of differentegomoviruses supporting the importance of these sites. Analy-is of various GFP fusions of AbMV MP deletions expressedransiently in plant cells identified the central portion of therotein as the domain responsible for membrane association,nd the N-terminus determining the dichotomy between mem-rane and peri-nuclear localization (Zhang et al., 2002). Analysisf protein interactions in yeast two-hybrid assay indicated anligomerization via the C-terminal end (Frischmuth et al., 2004).otential sites for phosphorylation are equally distributed over

he functional domains of MP. Therefore, several aspects ofbMV transport might be regulated by phosphorylation, e.g.

ocalization or oligomerization. Due to different kinase activi-ies during physiological changes, developmental stages, or inifferent plant tissues, the phosphorylation status of MP mayary.

MPs need to bind specifically the transport substrate, guidet to the cell periphery and via the plasmodesmata to the nextdjacent cell, where it has to be specifically released. We pre-ume that a prerequisite for this multi-functionality is a change

n its binding affinities and cellular localizations, which have toe based on switching the properties of the existing protein. Forhis task, post-translational modifications by host enzymes mostikely play a central role.

aTvf

AbMV MP, phosphorylation sites were predicted by NetPhos2.0 (Blom et al.,r domain are underlined according to Zhang et al., 2002. The part responsible

TMV MP is a well-studied example for such a regulationf MP function by phosphorylation (Waigmann et al., 2004).he phosphorylation status of its C-terminal phosphorylationites was shown to regulate the transport activity. One phospho-ylation leads to enhancement of transport capability whereasultiple phosphorylations had a negative regulatory effect on

ransport activity (Trutnyeva et al., 2005; Waigmann et al., 2000).onsequently a model was proposed in which MP gets highlyhosphorylated by cell wall-associated kinases upon crossinghe plasmodesmata, leading to inhibition of its transport capabil-ty after the successful transfer to the next cell. Because AbMV

P harbours multiple putative phosphorylation sites, a simi-ar complex mechanism controlling transport activity may beossible.

Post-translationally modifying enzymes conserved betweenlants and yeast are certain kinase families like theNF1/AMPK-related kinases (SnRKs), which are involved intress and glucose signalling (Francis and Halford, 2006; Polgend Thomas, 2006; Rolland et al., 2006). There is a remarkableonservation shown by the fact that the plant enzymes couldomplement the snf1 yeast mutation (Bhalerao et al., 1999;alford and Hardie, 1998; Kleinow et al., 2000). By identifi-

ation of host binding partners of geminiviral proteins, severalinases were found to play a role during the infection processnd could therefore be potential candidates for AbMV MP phos-horylation in planta. For TrAP from TGMV and C2 (syn. L2)rotein from Beet curly top virus (BCTV), interactions with andenosine kinase and an SNF1/AMPK-related kinase (SnRK)rom Arabidopsis thaliana were discovered (Hao et al., 2003;

ang et al., 2005, 2003). In both cases, interactions caused inhi-ition of kinase activity, resulting in weakening of host defenceechanisms. Recently, interactions between the replication-

ssociated proteins (Rep) from CaLCuV and TGMV with the A.haliana and N. benthamiana orthologs of the SNF1 activatinginase were identified (Kong and Hanley-Bowdoin, 2002; Shennd Hanley-Bowdoin, 2006). These plant kinases were able tounction as yeast homologs as well. In Arabidopsis, the kinases

ccumulated after viral infection and in cell cycle-active cells.hese findings support the assumption that during the onset ofiral infection, an activation of the SnRK pathway was help-ul to provide energy resources, but in later stages inhibition by

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rAP or C2 might be needed to repress host antiviral defenceechanisms. As well for the NSP proteins of CaLCuV, TGMV,

nd Tomato crinkle yellow leaf virus (TCrYLV) interactionsith two receptor-like kinases from Arabidopsis were identified

Florentino et al., 2006; Fontes et al., 2004; Mariano et al., 2004).ne was a leucine-rich-repeat receptor-like kinase, whose kinase

ctivity was inhibited by NSP binding. This receptor-like kinaseeemed to mediate antiviral plant defence responses. The secondas a proline-rich extensine-like receptor kinase (PERK), forhich NSP was a substrate for phosphorylation. It has been sug-ested that the interaction was necessary for efficient infectionnd full symptom development.

In conclusion, our findings support a model in which post-ranslational modifications, especially phosphorylation, are anmportant mechanism in the regulation of different steps in theiral life cycle. Particularly, the modifications of MPs may actn the control of transport activities. Future analysis of these

odifications in AbMV MP, with the support of the experimentalystem yeast, will help to understand their regulatory role ineminiviral transport.

cknowledgements

The authors would like to thank our gardeners Diether Got-hardt and Anika Allinger for taking care of the experimentallants, Anan Kadri and Bjorn Krenz for critically reading theanuscript and Katharina Kittelmann for a helpful idea on fur-

her analysis of phosphorylated MP. This work was supported bygrant of the Deutsche Forschungsgemeinschaft (KL 1366/3-1).

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.virusres.2007.08.011.

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