14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall.

The Plan Cell, Vol. 15, 1399–1413, June 2003, www.plan cell.o g © 2003 Ame ican Socie y o Plan Biologis s 14-3-3 P o eins A e Cons i uen s o he Insoluble Glycop o ein F amewo k o he Chlamydomonas Cell Wall Jü gen Voig a,1 and Ronald F ank b a Physiologisch-Chemisches Ins i u , Ebe ha d-Ka ls-Uni e si ä Tübingen, Hoppe-Seyle -S asse 4, D-72076 Tübingen, Ge many b Gesellscha ü Bio echnologische Fo schung, Masche ode Weg 1, D-38124 B aunschweig, Ge many The cell wall o he unicellula g een alga Chlamydomonas einha d ii consis s p edominan ly o Hyp- ich glycop o eins, which also occu in he ex acellula ma ix o mul icellula g een algae and highe plan s. In addi ion o he Hyp- ich polypep ides, he insoluble glycop o ein amewo k o he Chlamydomonas cell wall con ains mino amoun s o 14-3-3 p o- eins, as e ealed by immunochemical s udies and mass spec oscopic analysis o yp ic pep ides. Polypep ides immuno- logically ela ed o he 14-3-3 p o eins also we e ound in he cul u e medium o Chlamydomonas. The le els o wo o hese 14-3-3– ela ed polypep ides we e dec eased in he cul u e medium o he wall-de icien mu an cw-15 . These indings indi- ca e ha 14-3-3 p o eins a e in ol ed in he c oss-linking o Hyp- ich glycop o eins in he Chlamydomonas cell wall. INTRODUCTION Cell walls o highe plan s consis no only o cellulose and o he polysaccha ides bu also con ain insoluble, Hyp- ich glycop o- eins and hei soluble p ecu so s, he ex ensins (Kieliszewski and Lampo , 1994; Knox, 1995; Lampo , 2001). Hyp- ich gly- cop o eins a e he p edominan cons i uen s o he ex acellula ma ix o ol ocine algae, which ange om unicellula Chla- mydomonas einha d ii o mul icellula o ganisms in he genus Vol ox (Woessne and Goodenough, 1994). The mul ilaye ed cell wall o he unicellula g een alga Chlamydomonas consis s o an insoluble, Hyp- ich glycop o ein amewo k and se e al chao ope-soluble, Hyp-con aining glycop o eins (Robe s, 1974; Monk e al., 1983; Goodenough and Heuse , 1985; Imam e al., 1985; Robe s e al., 1985; Voig , 1988; Voig e al., 1991). The insoluble wall ac ion can be isola ed om in ac cells by successi e ex ac ions wi h di e en de e gen -con aining bu e s (Voig , 1984; Vogele e al., 1990). A e chemical de- glycosyla ion wi h hyd o luo ic acid (HF)/py idine, he “sac-like” mo phology o he highly pu i ied insoluble wall componen was des oyed and a leas pa o i s polypep ide cons i uen s became soluble in SDS-con aining bu e s (Vogele e al., 1990). Isodi y osine has been de ec ed in hyd olysa es o Chlamydomonas cell walls (Wa enschmid e al., 1993), indi- ca ing ha pe oxidase-ca alyzed c oss-linking o cell wall gly- cop o eins ia Ty side chains, which has been obse ed o he ex ensins o highe plan s (F y, 1982; Eps ein and Lampo , 1984; Biggs and F y, 1990), also occu s in Chlamydomonas. Fu he mo e, i has been epo ed ha a ansglu aminase also ca alyzes he c oss-linking o cell wall p o eins in Chlamydomo- nas (Wa enschmid e al., 1999). The size o he Chlamydomonas cell is limi ed by he size o he insoluble glycop o ein amewo k o he wall, which com- ple ely su ounds he p o oplas as a sac-like s uc u e (Voig , 1984; Imam e al., 1985). The e o e, enla gemen o his insolu- ble glycop o ein amewo k is a p e equisi e o cell g ow h. As e ealed by pulse-labeling and pulse-chase expe imen s wi h 3 H-P o and 35 S-Me , u no e o he insoluble amewo k o he Chlamydomonas cell wall akes place du ing he cell enla ge- men pe iod accompanied by a elease o la ge polypep ide agmen s in o he cul u e medium (Voig , 1985, 1986). The e- sul ing “holes” in he glycop o ein amewo k a e epai ed by he simul aneous inco po a ion o soluble p ecu so s. The pulse-labeling and pulse-chase expe imen s also indica ed ha hese soluble p ecu so s o he insoluble amewo k o he Chlamydomonas cell wall a e cons i uen s o he sal -soluble wall ac ion (Voig , 1986). This conclusion was co obo a ed by he inding ha an an ibody aised agains he deglycosyla ion p oduc s o he highly pu i ied insoluble cell wall componen e- ac ed speci ically wi h a 150-kD glycop o ein, a majo cons i u- en o he chao ope-soluble wall ac ion o Chlamydomonas (Voig e al., 1996). Sc eening o a Chlamydomonas cDNA exp ession lib a y wi h he an ibody agains he deglycosyla ion p oduc s o he pu i ied insoluble cell wall componen o Chlamydomonas (Voig e al., 1996) esul ed in he isola ion o ou cDNA clones ha encode a Chlamydomonas 14-3-3 p o ein (Liebich and Voig , 1995). The e o e, we ha e in es iga ed he c oss- eac i - i y o he an i–cell wall an ibody wi h his 14-3-3 p o ein. Pep- ide scan analysis (F ank, 1992) using 83 pen adecapep ides de i ed om he open eading ame (ORF) o he p e iously cloned Chlamydomonas 14-3-3 cDNA (Liebich and Voig , 1995) e ealed ha his pa icula cell wall an ibody ecognized se - e al epi opes dis ibu ed o e almos he en i e amino acid se- quence o he 14-3-3 p o ein. The conclusion ha he insoluble componen o he Chlamydomonas cell wall con ains a poly- pep ide ha is ela ed immunologically o he 14-3-3 p o eins was u he co obo a ed by immunochemical s udies using an- 1 To whom co espondence should be add essed. E-mail jue gen. oig @ medizin.uni-leipzig.de; ax 49-341-9722109. A icle, publica ion da e, and ci a ion in o ma ion can be ound a www.plan cell.o g/cgi/doi/10.1105/ pc.010611. 1400 The Plan Cell ibodies aised agains indi idual polypep ides eleased om he highly pu i ied insoluble ac ion o he Chlamydomonas cell wall du ing chemical deglycosyla ion and by mass spec o- scopic analyses o yp ic pep ides o he insoluble wall ac- ion. He e, we epo ha 14-3-3 p o eins also occu ex acellu- la ly. Fu he mo e, ou da a p o ide e idence ha 14-3-3 p o eins a e in ol ed in he o ma ion o he insoluble glycop o- ein amewo k o he cell wall. RESULTS Cha ac e iza ion o Cell Wall An ibodies As epo ed p e iously, de ined polypep ides a e eleased om he highly pu i ied, insoluble cell wall ac ion o Chlamydomo- nas by ea men wi h anhyd ous HF/py idine (Vogele e al., 1990). Polyclonal an ibodies we e aised agains he mix u e o hese deglycosyla ed cell wall polypep ides (an i-dICW) and agains wo p ominen cons i uen s o his ac ion wi h appa - en molecula masses o 64 and 45 kD (an i-dICW-64kDa and an i-dICW-45kDa). The speci ici y o hese an ibodies was es ed by p o ein gel blo analysis o cy osol, mic osomes, and LiCl ex ac s o in ac cells. LiCl ex ac s o in ac wild- ype cells con ain soluble p ecu so s o he insoluble cell wall ac- ion (Voig e al., 1991, 1996). The cell wall an ibodies did no c oss- eac wi h cons i uen s o he cy osol (Figu es 1A o 1D, lanes 1) bu ecognized high-molecula -mass componen s (  120 kD) p esen in bo h he mic osomes and he LiCl ex- ac s (Figu es 1A o 1D, lanes 2 and 3). The amoun s o he high-molecula -mass polypep ides ecognized by hese an i- bodies we e conside ably highe in he LiCl ex ac s o wild- ype cells (Figu es 1B o 1D, lanes 4) han in he LiCl ex ac s o wall-de icien cw-15 cells (Figu es 1B o 1D, lanes 3), indica ing ha hese polypep ides a e cons i uen s o he chao ope-solu- ble cell wall laye s and p esumably p ecu so s o he insoluble glycop o ein amewo k o he Chlamydomonas cell wall. These high-molecula -mass polypep ides did no c oss- eac wi h an ibodies aised agains a ecombinan 14-3-3 p o ein (an i-  -Gal/14-3-3; da a no shown). A polyclonal an ibody (an i-dGP100) aised agains he 100-kD componen o he chemically deglycosyla ed, LiCl-soluble cell wall ac ion (Fig- u e 2, lane 2, as e isk) exclusi ely ecognized a 150-kD compo- nen p esen in he LiCl ex ac s o in ac wild- ype cells (Figu e 1E, lane 4) bu absen in he LiCl ex ac s o he wall-de icien mu an cw-15 (Figu e 1E, lane 3). Howe e , when p o ein gel blo s con aining he deglycosyla ion p oduc s o he pu i ied LiCl-soluble, high-molecula -mass cell wall glycop o eins (Fig- u e 2, lane 2) we e p obed wi h his an i-dGP100 an ibody, all o he polypep ides showed c oss- eac i i y (Figu e 2, lane 4). The same esul s also we e ob ained when hese p o ein gel blo s we e p obed wi h an i-dICW, an i-dICW-65kDa, and an i- dICW-45kDa (da a no shown). C oss- eac i i y o an i-dGP100 wi h he chemically deglycosyla ed p ecu so s o he insoluble cell wall ac ion appa en ly is a ibu able o he high Hyp con- en o hese cell wall polypep ides (Voig e al., 1991), espe- cially gi en he ac ha his c oss- eac i i y was no obse ed in he case o he glycosyla ed cell wall p ecu so s (Figu e 2, lane 3). An i-dICW-65kDa and an i-dICW-45kDa exclusi ely ecognized LiCl-soluble p ecu so s o he insoluble cell wall ac ion (Figu es 1C and 1D, lanes 4) whose appa en molecula masses we e subs an ially highe han hose o he co espond- ing cell wall an igens (Table 1). The deglycosyla ion p oduc s o hese LiCl-soluble cell wall glycop o eins (Figu e 2, lane 2) also we e conside ably la ge han he 64- and 45-kD polypep ides eleased om he insoluble cell wall ac ion. The e o e, hese 64- and 45-kD componen s mus be de i ed om he la ge p ecu so s by p o eoly ic p ocessing. As epo ed p e iously, pa ial p o eolysis o he insoluble cell wall ac ion is a p e equisi e o cell enla gemen (Voig , 1985, 1986; Voig e al., 1996) and is accompanied by a elease o cell wall mac omolecules in o he cul u e medium (Voig , 1985, 1986). The size o he polypep ide agmen s e ained in he in- Figu e 1. SDS-PAGE and P o ein Gel Blo Analyses o Chlamydomonas Cy osol, Mic osomes, and LiCl Ex ac s o In ac cw-15 and Wild-Type Cells. Samples co esponding o 30 g o p o ein we e ac iona ed by SDS-PAGE on slab gels con aining 12.5% (w/ ) ac ylamide. A e elec opho esis, he gels we e ei he s ained o p o ein wi h Coomassie B illian Blue (A) o blo ed on o poly inylidene di luo ide (PVDF) memb anes ([B] o [E]). The p o ein gel blo s we e p obed wi h an i-dICW (B), an i-dICW-64kDa (C), an i-dICW-45kDa (D), o an i-dGP100 (E). Lanes 1, cy osol; lanes 2, mic oso- mal ac ion; lanes 3, LiCl ex ac o in ac cw-15 cells; lanes 4, LiCl ex ac o in ac wild- ype cells. 14-3-3 P o eins in he Chlamydomonas Cell Wall 1401 soluble cell wall laye ob iously is no de e mined exclusi ely by he dis ibu ion o clea age si es because he N- e minal amino acid sequences o he 64- and 45-kD cons i uen s we e ound o be a he he e ogenous (Table 2). Mix u es o amino acid esidues we e clea ed o du ing each ound o Edman deg ada ion, in con as o hei LiCl-soluble p ecu so s (Table 2). These indings indica e ha he 64- and 45-kD cons i uen s o he chemically deglycosyla ed insoluble cell wall ac ion do no ep esen single molecula en i ies bu a e mix u es com- posed o polypep ide agmen s de i ed by p o eoly ic p o- cessing o a ious p ecu so s (Figu e 2, lane 2). The insoluble glycop o ein amewo k o he Chlamydomo- nas cell wall con ains polypep ide cons i uen s ha a e ela ed immunologically o he 14-3-3 p o eins. The highly pu i ied in- soluble cell wall componen e ealed immuno luo escence no only wi h an i-dICW, an i-dICW-64kDa, and an i-dICW-45kDa (Figu es 3D, 3H, and 3L) bu also wi h he an ibodies agains he 100-kD deglycosyla ion p oduc o he 150-kD chao ope- soluble cell wall glycop o ein (an i-dGP100; Figu e 3P) and, o a lesse ex end, wi h an i-  -Gal/14-3-3 (Figu e 3T). No immuno- luo escence was obse ed wi h any o he co esponding p e- immune se a unde he same condi ions (Figu es 3B, 3F, 3J, 3N, and 3R). Immuno luo escence s udies wi h o maldehyde- ixed wild- ype cells e ealed ha he di e en cell wall an i- bodies, bu no he an ibody aised agains he ecombinan 14-3-3 p o ein (an i-  -Gal/14-3-3), eac ed wi h he cell su ace o Chlamydomonas wild- ype cells (da a no shown). Dis inc polypep ides a e eleased om he pu i ied insoluble glycop o ein amewo k o he Chlamydomonas cell wall by chemical deglycosyla ion wi h anhyd ous HF/py idine (Voig e al., 1996). When hese deglycosyla ion p oduc s we e sub- jec ed o p o ein gel blo analyses, all o he solubilized polypep ides (Figu e 4A, lane 1) we e ecognized by he cell wall an ise a an i-dICW, an i-dICW-64kDa, and an i-dICW-45kDa (Figu e 4A, lanes 3, 5, and 7). The p ominen 64- and 45-kD componen s (Figu e 4A, lane 1) c oss- eac ed wi h he an ibody aised agains he ecombinan Chlamydomonas 14-3-3 p o- ein (Figu e 4A, lane 9). Addi ionally, he la e an ibody eac ed s ongly wi h a 107-kD componen (Figu e 4A, lane 9) ha was conside ably less eac i e owa d he di e en cell wall an i- bodies (Figu e 4A, lanes 3, 5, and 7). None o he polypep ides eleased om he insoluble wall ac ion by ea men wi h HF/ py idine (Figu e 4A, lane 1) eac ed wi h any o he a ious p e- immune se a (Figu e 4A, lanes 2, 4, 6, and 8). As epo ed e- cen ly, Chlamydomonas cells con ain ou 14-3-3 iso o ms whose subuni s ha e appa en molecula masses o 32, 30, 27, and 24 kD (Voig e al., 2001). In his ange, no polypep ides we e de ec ed in he deglycosyla ion p oduc s o he insoluble Chlamydomonas cell wall ac ion by an i-  -Gal/14-3-3 Ig (Fig- u e 4A, lane 9). The c oss- eac ing 107-, 64-, and 45-kD com- ponen s a e in insic cons i uen s o he insoluble amewo k o he Chlamydomonas cell wall. This conclusion was u he co obo a ed by analyses o Figu e 2. SDS-PAGE and P o ein Gel Blo Analyses o Pu i ied, LiCl- Soluble High-Molecula -Mass Cell Wall Glycop o eins and Thei Degly- cosyla ion P oduc s. Un ea ed and chemically deglycosyla ed cell wall glycop o eins (30 g o p o ein) we e sepa a ed by SDS-PAGE on gel slabs con aining 7% (w/ ) ac ylamide. A e elec opho esis, he gels we e ei he s ained o p o ein wi h Coomassie B illian Blue (lanes 1 and 2) o blo ed on o PVD F memb anes (lanes 3 and 4). The p o ein gel blo s we e p obed wi h an i- dGP100 (lanes 3 and 4). Lanes 1 and 3, un ea ed cell wall glycop o eins; lanes 2 and 4, cell wall glycop o eins ea ed o 3 h wi h ime hylsilyl i- luo ome hanesul ona e a oom empe a u e. The deglycosyla ed poly- pep ide ha was used as an an igen o inc ease he an i-dGP100 an i- body is ma ked wi h an as e isk. Table 1. Polyclonal An ibodies Used in This S udy An ibody An igen Re e ence An i-  -Gal/14-3-3 a Recombinan ( E. coli )  -Gal:(Chlamydomonas) 14-3-3 usion p o ein Voig e al. (2001) An i-dGP100 100-kD deglycosyla ion p oduc o he 150-kD chao ope-soluble cell wall glycop o ein o Chlamydomonas Voig e al. (1996) An i-dICW Mix u e o polypep ides eleased om he pu i ied insoluble glycop o ein amewo k o he Chlamydomonas cell wall by chemical deglycosyla ion wi h anhyd ous HF/py idine Voig e al. (1996) An i-dICW-64kDa 64-kD polypep ide pu i ied om he mix u e o polypep ides eleased om he pu i ied insoluble glycop o- ein amewo k o he Chlamydomonas cell wall by chemical deglycosyla ion wi h anhyd ous HF/py idine This epo An i-dICW-45kDa 45-kD polypep ide pu i ied om he mix u e o polypep ides eleased om he pu i ied insoluble glycop o- ein amewo k o he Chlamydomonas cell wall by chemical deglycosyla ion wi h anhyd ous HF/py idine This epo a Deple ed om an i-( E. coli )  -galac osidase (  -Gal) Igs by p eincuba ion wi h PVDF memb ane–bound ( E. coli )  -Gal. 1402 The Plan Cell polypep ides eleased om he insoluble glycop o ein ame- wo k o he Chlamydomonas cell wall by incomple e deglyco- syla ion wi h ime hylsilyl i luo ome hanesul ona e unde mild condi ions. The pa e n o polypep ides eleased unde hese condi ions (Figu e 4B, lane 1) di e ed conside ably om ha obse ed a e ea men wi h HF/py idine (Figu e 4A, lane 1). The p edominan po ion o he eleased polypep ides was ob- se ed in he s acking gel (da a no shown). Fu he mo e, p ominen polypep ides wi h appa en molecula masses o 38, 47, 59, 64, 67, 74, 79, 100, and  300 kD we e de ec ed by SDS-PAGE analysis (Figu e 4B, lane 1). Mos o hese compo- nen s we e ecognized by he cell wall an ibodies an i-dICW, an i-dICW-64kDa, and an i-dICW-45kDa (Figu e 4B, lanes 3, 5, and 7). The ela i e in ensi ies o hese bands, howe e , we e a he di e en (Figu e 4B, lanes 1, 3, 5, and 7). The an ibodies agains he ecombinan Chlamydomonas 14-3-3 p o ein e- ac ed s ongly wi h a 63-kD componen (Figu e 4B, lane 9) ha c oss- eac ed weakly wi h he di e en cell wall an ibodies (Fig- u e 4B, lanes 3, 5, and 7). In addi ion o he 63-kD componen , an i-  -Gal/14-3-3 ecognized polypep ides wi h appa en mo- lecula masses o  300 kD (Figu e 4B, lane 9). These indings indica e ha he polypep ides ha a e ela ed immunologically o he 14-3-3 p o eins a e c oss-linked o he insoluble glyco- p o ein amewo k o he Chlamydomonas cell wall. Reac i i ies o Di e en Cell Wall An ibodies owa d Epi opes o a Chlamydomonas 14-3-3 P o ein Sc eening o a Chlamydomonas cDNA exp ession lib a y wi h an i-dICW (Voig e al., 1996) esul ed in he isola ion o ou cDNA clones ha encode a Chlamydomonas 14-3-3 p o ein (Liebich and Voig , 1995). By con as , a polyclonal an ibody aised agains he co esponding ecombinan 14-3-3 p o ein (an i-  -Gal/14-3-3) was shown o eac speci ically wi h he in- soluble cell wall ac ion o Chlamydomonas, as e ealed by immuno luo escence s udies (Figu e 3T), indica ing ha he gly- cop o ein amewo k o he Chlamydomonas cell wall con ains a leas one polypep ide ha is ela ed immunologically o his pa icula 14-3-3 p o ein. The e o e, we compa a i ely analyzed he eac i i ies o an i-  -Gal/14-3-3 and an i-dICW, an i-dICW- 64kDa, and an i-dICW-45kDa owa d epi opes o his pa icula Chlamydomonas 14-3-3 p o ein by pep ide-scan analyses (Fig- u e 5). To his end, 83 o e lapping pen adecapep ides (Figu e 5A) we e gene a ed by spo syn hesis on a cellulose memb ane (F ank, 1992) ha , oge he , ep esen ed he en i e amino acid sequence o he ORF de i ed om he p e iously cloned Chlamydomonas 14-3-3 cDNA (Liebich and Voig , 1995). The an i-  -Gal/14-3-3 ecognized se e al pen adecapep ide se- quences (Figu e 5B) dis ibu ed o e almos he en i e 29.5-kD ORF o he cloned Chlamydomonas 14-3-3 cDNA (Figu e 5A). A a he simila pa e n was obse ed o he an i-dICW an i- body used o sc een he cDNA exp ession lib a y, which e- sul ed in he isola ion o 14-3-3 clones. The ela i e eac i i ies o bo h an ise a owa d hese pep ides, howe e , we e a he di e en (Figu e 5B). Pep ides 7, 10, 31 o 34, 54 o 57, 63, 74, 75, and 81 eac ed mo e s ongly wi h an i-dICW (Figu e 5B), whe eas pep ides 11 o 13, 28, 29, and 76 o 78 e ealed weake signals wi h his pa icula an ise um han wi h an i-  -Gal/14-3-3 (Figu e 5B). The an i-dICW-64kDa an ise um ec- ognized pep ides 14 o 16, 25, 26, 38, 39, 41, 48 o 50, 72, 74, and 75 (Figu e 3B). The an i-dICW-45kDa se um (Figu e 5B), on he o he hand, c oss- eac ed wi h se e al pen adecapep ides de i ed om he ORF o he p e iously cloned Chlamydomo- nas 14-3-3 cDNA (Liebich and Voig , 1995), which also we e ecognized by an i-dICW (Figu e 4B) and by an i-  -Gal/14-3-3 (Figu e 4B). These da a clea ly show ha he insoluble cell wall ac ion o Chlamydomonas and i s 64- and 45-kD deglycosyla- ion p oduc s con ain polypep ides ha a e ela ed immunolog- ically o he 14-3-3 p o eins. Di e en ial Reac i i ies o he Va ious Cell Wall An ibodies owa d he 14-3-3 Iso o ms o Chlamydomonas When he mix u e o 14-3-3 p o eins pa ially pu i ied om Chlamydomonas cells was analyzed by SDS-PAGE (Figu e 6, lane 1), wo p edominan polypep ides wi h appa en molecula masses o 30 and 27.5 kD we e obse ed. P o ein gel blo s o he same 14-3-3 p epa a ion p obed wi h he an i-  -Gal/14-3-3 (Figu e 6, lane 11) showed he p esence o wo addi ional 14-3-3 iso o ms wi h appa en molecula masses o 32 and 25 kD and a ain band a 63 kD ha migh be a esidual 14-3-3 dime . None o hese polypep ides eac ed wi h an i-dGP100 aised agains he 100-kD deglycosyla ion p oduc o a 150-kD glyco- p o ein (Figu e 6, lane 9), which was shown o be a p ecu so o he insoluble ac ion o he Chlamydomonas cell wall (Voig e Table 2. N-Te minal Amino Acid Sequences o Polypep ide Cons i uen s o he Insoluble Cell Wall F ac ion o Chlamydomonas and I s LiCl-Soluble P ecu so s N-Te minal Amino Acid Sequence Cell Wall F ac ion Appa en Molecula Mass 1 2 3 4 5 Insoluble 64 kD AGDSTILM GNALR VLGIEQA DVNQV PNESG Insoluble 45 kD GSAMLVI QNAGV DSTEL STQILD GENR LiCl soluble 200 kD S X X X X LiCl soluble 135 kD ? a LiCl soluble 100 kD I N I P N a The N e minus is blocked. 14-3-3 P o eins in he Chlamydomonas Cell Wall 1403 al., 1996). An i-dICW exclusi ely ecognized he 27.5-kD 14-3-3 iso o m (Figu e 6, lanes 2 and 3). An i-dICW-45kDa eac ed p e e en ially wi h he 32-kD 14-3-3 iso o m bu also ecog- nized he 27.5-kD iso o m (Figu e 6, lane 7). An i-dICW-64kDa ecognized he 27.5-kD iso o m bu no he o he 14-3-3 iso- o ms and c oss- eac ed s ongly wi h con amina ing polypep- ides wi h appa en molecula masses o 58 and 45 kD (Figu e 6, lane 5) ha we e no ecognized by he o he an ibodies (Fig- u e 6, lanes 2, 3, 7, 9, and 11). Howe e , conside ably inc eased incuba ion imes in he p esence o 5-b omo-4-chlo o-3-indolyl phospha e and ni oblue e azolium chlo ide we e equi ed o i- sualize hese bands compa ed wi h he o he an ibodies, indica - ing ha he eac i i y o his pa icula an ibody was a he low. P ecu so s o he Insoluble Cell Wall F ac ion Ou indings ha he insoluble glycop o ein amewo k o he Chlamydomonas cell wall con ains polypep ides ha a e e- la ed immunologically o he 14-3-3 p o eins (Figu es 3 o 6) p omp ed us o sea ch o hei soluble p ecu so s. Cell wall p ecu so s, like o he sec e ed polypep ides, mus pass he en- doplasmic e iculum (ER) and he dic yosomes. The e o e, he c ude mic osomes we e ac iona ed by suc ose g adien cen- i uga ion o ob ain he ER, he dic yosomes, and he plasma memb ane, which we e de ec ed by ma ke enzyme ac i i ies. Again, he co esponding polypep ides (Figu e 7, lanes 1 o 3) we e subjec ed o compa a i e p o ein gel blo analyses using Figu e 3. Immuno luo escence Analysis o he Pu i ied, Insoluble Cell Wall Componen o Chlamydomonas. (A), (C), (E), (G), (I), (K), (M), (O), (Q), and (S) Phase-con as mic og aphs. (B), (D), (F), (H), (J), (L), (N), (P), (R), and (T) Fluo escence mic og aphs. (A) and (B) An i-dICW p eimmune se um. (C) and (D) An i-dICW Igs. (E) and (F) An i-dICW-64kDa p eimmune se um. (G) and (H) An i-dICW-64kDa Igs. (I) and (J) An i-dICW-45kDa p eimmune se um. (K) and (L) An i-dICW-45kDa Igs. (M) and (N) An i-dGP100 p eimmune se um. (O) and (P) An i-dGP100 Igs. (Q) and (R) An i--Gal/14-3-3 p eimmune se um. (S) and (T) An ibodies agains he ecombinan Chlamydomonas 14-3-3 p o ein (an i--Gal/14-3-3). Ba in (Q)  50 m o all panels. 1404 The Plan Cell di e en cell wall an ibodies (Figu es 7B o 7E) and he an i-  -Gal/14-3-3 an ibody (Figu e 7F). The di e en cell wall an i- bodies (an i-dICW, an i-dICW-64kDa, an i-dICW-45kDa, and an i-dGP100) eac ed p e e en ially wi h high-molecula -mass componen s wi h appa en molecula masses o  120 kD (Fig- u es 7B o 7E). The inding ha he same pa e ns o c oss- eac ing high-molecula -mass polypep ides we e ob ained wi h all o hese an ibodies (Figu es 7B o 7E) was as onishing be- cause he co esponding an igens we e a he di e en (Table 1). A 150-kD componen was p edominan in he ER (Figu es 7B o 7E, lanes 3) as in he c ude mic osomal ac ion (Figu es 1B o 1D, lanes 2). Inc eased amoun s o o he high-molecula - mass polypep ides we e de ec ed in he dic yosomes (Figu es 7B o 7E, lanes 2) and in he plasma memb ane ac ion (Fig- u es 7B o 7E, lanes 1), which p esumably a e gene a ed by pos - ansla ional modi ica ion in he dic yosomes. Almos he same pa e ns o high-molecula -mass polypep- ides we e de ec ed by he a ious cell wall an ibodies in he dic yosomes (Figu es 7B o 7E, lanes 2), in he plasma mem- b ane ac ion (Figu es 7B o 7E, lanes 1), and in he LiCl ex- ac s o in ac cells (Figu es 7B o 7D, lanes 3 and 4). None o hese high-molecula -mass polypep ides c oss- eac ed wi h he an i-  -Gal/14-3-3 an ibody, which ecognized only he known 14-3-3 iso o ms (Figu e 7F, lanes 1 o 3). As epo ed e- cen ly (Voig e al., 2001), he 27.5-kD componen was p edom- inan in he ER ac ion, which also con ained he 30-kD iso- o m and, o a lesse ex en , he 24-kD iso o m (Figu e 7F, lane 3). Di e en pa e ns o 14-3-3 iso o ms we e de ec ed in he plasma memb ane (Figu e 7F, lane 1) and dic yosome ac ions (Figu e 7F, lane 2), as epo ed ecen ly (Voig e al., 2001). An addi ional 64-kD componen , howe e , was de ec ed in all o hese ac ions (Figu e 7F, lanes 1 o 3), which migh be a esid- ual 14-3-3 dime . The indings desc ibed abo e clea ly show ha he p edomi- nan p ecu so s o he insoluble glycop o ein amewo k o he Chlamydomonas cell wall a e high-molecula -mass polypep- ides (glycop o eins) ha a e modi ied pos - ansla ionally in he dic yosomes and a e no ela ed immunologically o he 14- 3-3 p o eins. The e o e, he cons i uen s o he insoluble glyco- p o ein amewo k o he Chlamydomonas cell wall ha a e im- munologically ela ed o he 14-3-3 p o eins mus be mino componen s o his cell wall ac ion, whose soluble p ecu so s p esumably a e in insic 14-3-3 iso o ms associa ed wi h he mic osomal memb anes. Cha ac e iza ion o 14-3-3–Rela ed P o eins by Analysis o T yp ic F agmen s o he Insoluble Cell Wall F ac ion o Chlamydomonas The da a desc ibed abo e indica e ha 14-3-3– ela ed poly- pep ides a e c oss-linked o he insoluble cell wall ac ion o Chlamydomonas. The e o e, we used mass spec ome y o analyze yp ic agmen s o he highly pu i ied, insoluble cell wall ac ion o he p esence o 14-3-3– ela ed pep ides. No yp ic agmen s o he p e iously desc ibed Chlamydomonas 14-3-3 p o ein (Voig e al., 2001) we e de ec ed in his pep ide ac ion by ma ix-assis ed lase -deso p ion ioniza ion ime-o - ligh analysis. Howe e , some o he pep ide agmen s ob- Figu e 4. SDS-PAGE and P o ein Gel Blo Analyses o Polypep ides Released om he Pu i ied Insoluble Cell Wall F ac ion o Chlamydomo- nas by T ea men wi h Anhyd ous HF/Py idine and T ime hylsilyl T i luo- ome hanesul ona e in he Cold. The eeze-d ied, highly pu i ied insoluble wall ac ion was ea ed wi h anhyd ous HF/py idine o ime hylsilyl i luo ome hanesul ona e as de- sc ibed in Me hods. Twen y mic og ams (lane 1) o 2 g (o he lanes) o solubilized polypep ides was ac iona ed by SDS-PAGE on slab gels con aining 12.5% (w/ ) ac ylamide. A e elec opho esis, he gels we e ei he s ained o p o ein wi h Coomassie B illian Blue o blo ed on o PVDF memb anes. (A) Polypep ides eleased by ea men wi h anhyd ous HF/py idine. Lane 1, gel s ained o p o ein; lanes 2 and 3, p o ein gel blo s p obed wi h an i-dICW (lane 3) and he co esponding p eimmune se um (lan e 2); lanes 4 and 5, p o ein gel blo s p obed wi h an i-dICW-64kDa (lane 5) and he co esponding p eimmune se um (lane 4); lanes 6 and 7, p o ein gel blo s p obed wi h an i-dICW-45kDa (lane 7) and he co esponding p eimmune se um (lane 6); lanes 8 and 9, p o ein gel blo s p obed wi h an ibodies agains he ecombinan 14-3-3 p o ein (lane 9) and he co - esponding p eimmune se um (lane 8). (B) Polypep ides eleased by ea men wi h ime hylsilyl i luo- ome hanesul ona e. Lane 1, gel s ained o p o ein; lanes 2, 4, 6, 8, and 10, p es ained p o ein molecula mass ma ke s (appa en molecula masses a e gi en a igh ); lane 3, p o ein gel blo p obed wi h an i- dICW; lane 5, p o ein gel blo p obed wi h an i-dICW-64kDa; lane 7, p o ein gel blo p obed wi h an i-dICW-45kDa; lane 9, p o ein gel blo p obed wi h an ibodies agains he ecombinan 14-3-3 p o ein. 14-3-3 P o eins in he Chlamydomonas Cell Wall 1405 ained e ealed amino acid sequences ha a e simila o se- quence mo i s o he known 30-kD 14-3-3 iso o m (Table 3). The pep ides VAVLANEQELSVEER and NLLSVSYK ma ch po- si ions 31 o 45 and 46 o 53, espec i ely, o he amino acid sequence o he 30-kD iso o m (Table 3). The scan pep ides 11 o 16 (Figu e 5A) con ain eigh o mo e o hese amino acid es- idues. All o hese scan pep ides eac ed wi h an i-  -Gal/14-3- 3, bu only he scan pep ides 14 o 16 we e ecognized by he di e en cell wall an ibodies (Figu e 5B). This di e ence appa - en ly is a ibu able o he subs i u ion o he sequence VHD (posi ions 35 o 37 o he 30-kD iso o m) by ANE (Table 3), indi- ca ing ha he 30-kD 14-3-3 iso o m is no con ained in he cell wall. The pep ide YLVPSASTTEAAVFYLK ma ches posi ions 110 o 126 o he 30-kD iso o m (Table 3). This sequence was con- ained la gely in he scan pep ides 37 o 41 (Figu e 5A). All o Figu e 5. Compa a i e Epi ope Analyses o he Polyclonal An ibody Raised agains he Recombinan 14-3-3 P o ein and Di e en Polypep ides o he Insoluble Cell Wall F ac ion. (A) Eigh y- h ee o e lapping pen adecapep ides de i ed om he ORF o he p e iously cloned Chlamydomonas 14-3-3 cDNA and ep esen ing he en i e amino acid sequence we e syn hesized by spo syn hesis using cellulose pape as a solid suppo (F ank, 1992). (B) A e ea men wi h blocking solu ion, he cellulose-bound scan pep ides we e incuba ed wi h he di e en an ibodies. A e ex ensi e washing, bound IgGs we e de ec ed by incuba ion wi h alkaline phospha ase–coupled an i- abbi IgG and subsequen isualiza ion o he indi ec ly bound al- kaline phospha ase as desc ibed in Me hods. 1406 The Plan Cell he an ibodies eac ed s ongly wi h he scan pep ides 38, 39, and 41 (pa icula ly wi h pep ide 39) and only weakly wi h pep ide 40 (Figu e 5B). The pep ide AFDEAISDLDSLGEDSY ma ches po- si ions 201 o 218 o he 30-kD iso o m (Table 3). This amino acid sequence was mo e o less con ained (app oxima ely six amino acid esidues) in he scan pep ides 65 o 71 (Figu e 5A). Only scan pep ide 68 eac ed wi h an i-  -Gal/14-3-3, an i- dICW, and an i-dICW-45kDa (Figu e 5B). The pep ide DNLTL- WTSEM ma ches posi ions 229 o 238 o he 30-kD iso o m (Table 3). Mo e han i e o hese amino acid esidues we e con ained in he scan pep ides 74 o 78 (Figu e 5A), which we e ecognized by all o he an ibodies (Figu e 5B). All o hese pep- ides ma ch a single 14-3-3 iso o m ha is being sequenced a p esen (J. Voig , S. S e ano ic, M. Schi le, M. Fausel, J. Maie , K.-H. Adam, and O. Ma qua d , unpublished esul s). The o he pep ides do no ma ch any p o ein sequence p esen in he da- abases. P esumably, hey a e agmen s o he Hyp- ich cell wall glycop o eins. Some o hese pep ides ma ch pa ial amino Figu e 6. SDS-PAGE and Compa a i e P o ein Gel Blo Analyses o he Chlamydomonas 14-3-3 P o eins wi h Polyclonal An ibodies Raised agains Di e en Polypep ides o he Insoluble F ac ion o he Chlamydomonas Cell Wall. Twen y (lane 1), 0.2 (lane 2), o 2 g (o he lanes) o pu i ied 14-3-3 p o eins we e ac iona ed by SDS-PAGE on slab gels con aining 12.5% (w/ ) ac ylamide. A e elec opho esis, he gels we e ei he s ained o p o ein wi h Coomassie B illian Blue (lane 1) o blo ed on o PVDF memb anes. Lanes 4, 6, 8, 10, and 12, p es ained p o ein molecula mass ma ke s (appa en molecula masses a e gi en a igh ); lanes 2 and 3, p o ein gel blo s p obed wi h an i-dICW; lane 5, p o ein gel blo p obed wi h an i-dICW-64kDa; lane 7, p o ein gel blo p obed wi h an i-dICW-45kDa; lane 9, p o ein gel blo p obed wi h an i-dGP100; lane 11, p o ein gel blo p obed wi h an ibodies agains he ecombinan 14-3-3 p o ein. Figu e 7. Compa a i e SDS-PAGE and P o ein Gel Blo Analyses o Chlamydomonas Memb anes. Samples co esponding o 30 g o p o ein we e ac iona ed by SDS-PAGE on slab gels con aining 12.5% (w/ ) ac ylamide. A e elec opho esis, he gels we e ei he s ained o p o ein wi h Coomassie B illian Blue (A) o blo ed on o PVDF memb anes ([B] o [F]). The p o ein gel blo s we e p obed wi h an i-dICW (B), an i-dICW-64kDa (C), an i-dICW-45kDa (D), an i-dGP100 (E), and an ibodies agains he ecombinan Chlamydomonas 14-3-3 p o ein (F). Lanes 1, plasma memb ane; lanes 2, dic yosomes; lanes 3, ER. 14-3-3 P o eins in he Chlamydomonas Cell Wall 1407 acid sequences o LiCl-soluble cell wall glycop o eins (J. Voig and M. Kiess, unpublished esul s). Analyses o Mac omolecules Accumula ed in he Cul u e Medium As epo ed p e iously, he cul u e medium o Chlamydomonas wild- ype s ains con ains agmen s o he insoluble glycop o- ein amewo k o he Chlamydomonas cell wall ha a e e- leased bo h du ing he cell enla gemen pe iod (Liebich and Voig , 1995; Voig e al., 1996) and du ing he libe a ion o zoospo es by deg ada ion o he mo he cell wall (Schlösse , 1966; Miha a and Hase, 1975; Wa enschmid e al., 1988). The cul u e medium o wall-de icien s ains such as cw-15, how- e e , con ains cons i uen s o he chao ope-soluble wall laye s and componen s no mally localized in he pe iplasm (Loppes and Del ou , 1975; Voig , 1986; Voig e al., 1997). Acco dingly, he pa e ns o polypep ides accumula ed in he cul u e me- dium o he wall-de icien s ain cw-15 (Figu e 8A, lane 1) and he Chlamydomonas wild- ype s ain (Figu e 8A, lane 2) we e a he di e en . Apa om he high-molecula -mass compo- nen s (120 kD), he cul u e medium o he wild- ype s ain con ained a p edominan polypep ide o 45 kD (Figu e 8A, lane 2) ha was absen om he cul u e medium o he wall-de i- cien s ain (Figu e 8A, lane 1) and was ecognized by all o he an ibodies aised agains he di e en deglycosyla ed cell wall polypep ides (Figu es 8B o 8E, lanes 2) bu did no c oss- eac wi h he an ibodies aised agains he ecombinan Chlamydo- monas 14-3-3 p o ein (Figu e 8F, lane 2). On he o he hand, he cul u e medium o he wall-de icien s ain cw-15 con ained g ea e amoun s o polypep ides wi h appa en molecula masses o 65 and 150 kD (Figu e 8A, lane 1) compa ed wi h he wild ype (Figu e 8A, lane 2), which also we e ecognized by all o he an ibodies aised agains he di - e en deglycosyla ed cell wall polypep ides (Figu es 8B o 8E, lanes 1) bu did no c oss- eac wi h he an i--Gal/14-3-3 se- um (Figu e 8F, lane 1). The an i--Gal/14-3-3 an ibody ecog- nized ou polypep ides accumula ed in he cul u e medium o bo h he wall-de icien s ain cw-15 (Figu e 8F, lane 1) and he wild ype (Figu e 8F, lane 2) wi h appa en molecula masses o 25, 33, 59, and 64 kD. The ela i e amoun s o he 25-kD com- ponen and especially he 33-kD componen we e dec eased conside ably in he cul u e medium o he wall-de icien s ain cw-15 (Figu e 8F, lane 1) compa ed wi h he wild ype (Figu e 8F, lane 2). DISCUSSION The cell wall o Chlamydomonas consis s o an insoluble Hyp- ich glycop o ein amewo k and se e al chao ope-soluble gly- cop o eins (Robe s, 1974; Monk e al., 1983; Goodenough and Heuse , 1985; Imam e al., 1985; Robe s e al., 1985). In addi- ion o he Hyp- ich glycop o eins, 14-3-3 p o eins a e mino cons i uen s o he insoluble cell wall ac ion, as e ealed by immunochemical s udies (Figu es 3 o 6) and mass spec o- scopic analysis o yp ic pep ides (Table 3). The 14-3-3 p o eins we e disco e ed du ing a sys ema ic s udy o b ain-speci ic p o eins (Moo e and Pe ez, 1967). The name 14-3-3 o igina es om hei mig a ion pa e n on DEAE- cellulose ch oma og aphy and subsequen elec opho esis o b ain p o eins (Moo e and Pe ez, 1967). In he mean ime, hey ha e been ound in all euka yo ic o ganisms s udied o da e (Ai ken e al., 1992; Wang and Shakes, 1996; Fe l e al., 2002). Mammalian cells and highe plan s con ain up o 15 di e - en 14-3-3 genes (Wang and Shakes, 1996; Ai ken, 2002; Fe l e al., 2002). In he yeas Saccha omyces ce e isiae and Schizosaccha omyces pombe, which con ain only wo 14-3-3 genes, inac i a ion o bo h genes is le hal (Fo d e al., 1994; an Heusden e al., 1995). In animal cells and highe plan s, mos s udies ha e concen a ed on he unc ions o 14-3-3 p o eins in he cy osol, including hei e ec s on me abolic enzymes (Hube e al., 2002; Compa o e al., 2003) and signal ansduc ion (Ai ken, 1996, 2002; Robe s e al., 2002; Sehnke e al., 2002). In highe plan s, membe s o his p o ein amily ha e been shown o be cons i uen s o he G-box binding complex in- ol ed in ansc ip ional con ol (De Ve en e al., 1992; Lu e al., 1992; Bihn e al., 1997). P e iously, 14-3-3 p o eins we e de ec ed in he nuclei o human cells (Todd e al., 1998). Some b ain iso o ms we e ound o be associa ed wi h he plasma memb ane and synap ic esicles (Jones e al., 1995). A ole in esicula a icking has been obse ed o he budding yeas (S. ce e isiae) 14-3-3 homologs (Gelpe in e al., 1995). The ou 14-3-3 iso o ms o Chlamydomonas in e ac di e en ially wi h he ER, dic yosomes, and plasma memb ane (Voig e al., 2001). Plan plasma memb ane pump H-ATPases a e ac i- a ed by a complex o he ungal oxin usicoccin wi h 14-3-3 Table 3. Analysis o 14-3-3–Rela ed Pep ides Released by T ypsin om he Insoluble Cell Wall F ac ion o Chlamydomonas Pep ides o he Insoluble Cell Wall F ac ion Co esponding Pep ide F agmen s o he 30-kD 14-3-3 Iso o m Posi ion in he Amino Acid Sequence o he 30-kD 14-3-3 Iso o m VAVLANEQELSVEER VAKLVHDQELSVEER 31 o 45 NLLSVSYK NLLSVAYK 46 o 53 YLVPSASTTEAAVFYLK HLIPTASTGESKVFYLK 110 o 126 AFDEAISDLDSLGEDSYK AFDEAIAELDSLGEESYK 201 o 218 DNLTLWTSEM DNLTLWTSDM 299 o 238 VVLYSAFAAR No iden i ied – MITAIGLVK No iden i ied – EINPNR No iden i ied – EQFDTFGDTAAIR No iden i ied – ESILNALLSK No iden i ied – AGVDPSADPAVVR No iden i ied – MLFTPALAFSSTILR No iden i ied – ETNSAFYPAYTMLGVK No iden i ied – EVSYGLYLSGIAGVFLR No iden i ied – EVILPNSDK No iden i ied – A e diges ion o he pu i ied insoluble cell wall ac ion, pep ides we e examined by ma ix-assis ed lase -deso p ion ioniza ion ime-o - ligh analysis and elec osp ay ioniza ion andem mass spec ome y. Se- quences a e shown in one-le e code. The 30-kD 14-3-3 iso o m is he same as ha shown in Figu e 5. The unde lined esidues a e di e en om he p e iously cloned 30-kD 14-3-3 iso o m.