Volume 308. number 3, 258-260 FEBS 11439 August 1992 O 17,92 Federation of Earol.w.an Uioehemieal Societies 0014:~793/92/$~.00

Tobacco chloroplast ribosomes contain a homologue of E. coli ribosomal protein L28

Fumiaki Yokoi and Masahiro Sugiura

Center for Gone l~exeareh. Nagoxa Univer#lO', Nagoya 464-01. $ttpa~#

Received 12 May 1992; revi~d version received 7 July 1992

The ~nes for ribosomal proteins I~ and L33 constitute an opcron (rpm/~63 in ~'. chit. but in plant ~loroplasts L33 is en¢od~ by tl'm chloroplast DNA and L28 s~ms to be e~oded by the nuclear Ilenom¢, A 15 kDa protein was i~iated from the ~0 S subunit of ~bae.¢o chloroplast ribo~rrmc and its N.t©mfinal amino acid sequence was determined, A eDNA for this protein was cloned and analyzed, The eDNA enood¢~ a 151 amino add protein consistinil of a predicted transit.pcptide of 74 amino acids and a mature protein of 77 amino acids, Tlu~ mature protein is homolog,ou~ to E. cull L28, hen~ we named it chloroplast I..2tt (CL28). This is the first report on the preu:n~ ofnn B, ¢oli L.2Z.like protein in another ori~nism.

Chloroplast Ribosomal protein: CI.28; Tobacco

I. INTRODUCTION 2. MATERIAI.S AND METHODS 2.1. [xalalimt of rilmsm.,I prateitt CL28 Chloroplast ribosomes are 70 S in size similar to those Chloroplast ribosomal subunits *.,.'ere prepared from mature to. of E. coli and contain 3-5 different rgNAs and about bacco ieav~ (Hlrolia.tt ralmr~m!vat'. Bright Yellow 4) by a l~,~viourJy 60 ribosomal protein species (reviewed in [1,2]), Deter- reported method [9]. Ribosomal proteins were fractionated by reverse. pha.~ chromatography (Pharnmei.'t FPLC PRO.RPC HRS/10) fol. mination of the entire chloroplast DNA sequen~ and iowr.d by SDS.polyacrylamidc l~l el¢ctrophore~h; (details will I~: pub. analysis of chloroplast ribosomal proteins revealed the lishcd elsewhere). Alter being ¢l¢¢troblotted on a polpAnylidene di. pre~ence of 21 chloroplast loc". for r:bosomal proteins fluoride membrane, tl'm proteins ~:ra stain©d with Coomaraie R3S0 in tobacco [3-5], including rp/33 encoding the ribosomal stain (Pharmacia). A 15 kDa protein band was cut out and its N. protein L33 (CL33), This gone has been 1o :alized in the terminal amino acid sequeru:¢ was deterrninexi with a gas.phat¢ pro. large single copy region of tobacco chloroplast DNA. rein ~¢quen~r (Applied IliosyRemz 490A.120A). based on its homology to E. coli L33 [3]. However no ORF corresponding to £. coli ribosomal protein L28 has been found in tobacco chloroplast DNA, although t ¢4 A.4~4~¢~ GAT'rTf */'(k4AC CA,~4 A ~,Q A't~ A A ~ ~ A~ ~¢ CA A A A A~ in E, coli the genes encoding L28 and L33 o~ur to- H A Z gether in a single operon O'pmBG) [6]. Since E. coil O L CAACAATGGTGGCAGGT&TA^GCCTC&G^C;GACCAGT'qA"~AGT~CAG~C&~ mutants missing L28 are viable [.7], it is possible that " T H V A G I | L it G P V H S • tl R T F S l| | I ~ ¢AGTI0A¢ AA ~ A AI~GGCTTI:'T TTACCA~i~r CGAA A ~C AG'/~ ~ ~-~'rTG AG~A A chloroplast ribosomes lack an E. coil L28-type protein. v T It # A s L P tD • g L it It It I. It V v I" 41 | • k cT'r CCCA ATT~=.AG7GGTC'r ~U4r,~A T'r TC6.A~.'~ C¢¢ h t ~ i # e4TATC'/'TCATCAQCTCCAC The primary structure of L28 has only been reported in S Q ~ II G 6 K | S • T It F | | S S A I' Sl E. coil [8]. Z4 t ~/1~-~T~r~9r~`~A~i-~f~1~G~G1~`~t~A1~`~¢L~c~eA~Ta To ch~k whether the gone encoding chloroplast L28, I V P F It It I. Q • V AAI~ m I C P It, T O O! 30 It GGAAtqAAGTC ~AAC A GGGCAA.ACAA 0 G'~6"~CJ~'reA KA ¢¢A ¢ A~ e ACAA A AAA~rTG¢ if there is one, is encoded by the nuclear genome, we K K It 11 R & K Y S II It X H K T K K I. Q iO| have attempted to isolate the corresponding chloroplast 31], AAT'TT~TA A A¢¢ T'r¢'.,AATA¢ A A G.41:~ATT'TaG~(~.*C A~¢ AA~'.C~rTA'~9~'TGAAA¢ ribosomal protein from tobacco leaves and to clone its F V rl I. Q Y R I ~ ~ g A G K It Y Y It t, |2| 4 | ~ TGA~¢AA~4AA~CATAJ~4~CA~rGAGA~AAATG~A4.*~T'~A?G~rGTA~A eDNA. Sequence analysis revealed in fact the presence m 6 It T g ,4 K T g K N O L D A v h 1.41 of an E, coli L28-type protein in tobacco chloroplast 4il AGAAGG~GGGAT~G~TC'~AGCAA~A.AGTAG~ACCT*fG*~ACATTATAGeATC~T~ ribosomes, suggesting that L28 has a function in ribo- K A G I D I. X It tSi i4 ~CACT~AT'~TT~ATGCC~CAAAAA~AC~AC~AA~A~ ]; ]i~CA~'~G~r~TT~eA~ somes. i

lilt eA~GA'~'TAAAA Fig, 1, Nucleotid¢ and deri~l amino acid sequences of a eDNA encoding tobacco chloroplast ribosomal protein CL28. The triangle Correspondence address: M, Sugiara, Center for Oene R~earch, Na. indicates the proem.sing site. The asterisk rcprc~:nts the stop eodon, goya University, Chikusa, Nagoya, 464.01, Japan, Fax: (81) (52) 782 The conrer,'ed sequen¢.¢ flanking the start ¢odon in dlcot plant,~ is 3849, uad:rlined,

258 Publiahed by Elset,/¢r $ci¢nc¢ Pul)llshers B, i/, Volume 308, nurn~r 3 FEBS LETTERS Ausutt 1992

l ~,o iO $O 40 60 (IO 'tO I?B ZR~XPlMrOI~SNRANKV|HSH ¢LIB RRI CFicTGIf0KINRAI~IwgHgNW'rlU~LGiCVNIAWI¢~; WWlCAGKg~/I~LRLS?g/LI KTI gKNOLDAVASICAGZ D~|KI¢ It it IIII • al Illl Illl 4 1 Ill lit" It Ill • I It II I II Ill I} gLiB $ IWP,..QVTOKIqlPVTO~ N fi IflA LNA?I~I~P,~ LI~N LI'I I1"1R 7t~/18 B g,Jf W I,RV flAgOl'l RV I DIt~ i DTVLA gLBAliOglg't Fil, 2, Comparison of the predicted amino acid ~.qucnc¢ of tobacco chloroplast ribosomal protein CI.2~ (CL28) with that of E, coal ribolmrnal protein L28 (EL28), Asteri~a indicate identical a~ino acidl. The N.tcrmina! amino acid u~;iuenra of the tTB protein ~ ~own above C!.,28.

2,2, Cteni~&and ~¢qu¢;leh~ of the eDNA encodin~ CL2¢ sizcr based on the amino acid sequence (KIXPFTGKK). Screcniol of A H, ta&tt'um leaf eDNA library wai ¢otlttt'ucled by Dr, R,F, the libra~ was carried out accordinl to the instruction manual from Whittier usin8 at eDNA synth~is kit in ,,!,8t10 (Amertham), An ol- Amcnharn (eDNA synthesis kit ABtI0)and [10], TI~ hybridization illon;tfl¢otide probe ($'.TT(TC)TTICCIGT(AG)AAIGGI(GC)(AT)- tempemtt~'~ was 41PC, Recombinant ADNA ~ pmiatrcd by a mini- IATIC-Y) was syntht~i~..d usin8 an Appli~ Bios~tems 318A synthe. lynte method [l 1], The inert was di~ted with E¢oRi and subeloncd

3 E.coli

O

Q,-3 O

3:

0

-3 0 40 80 Residue Number Fitl, 3, Hydropathy plots of tobacco CL2B and E, coli L28, Hydropathy was ~lculatcd with an 1 I.point window according to Kyte and Doolittle [t6].

259 Volume 308. numl~r 3 FEBS LETTERS August 1992 into the Bluescript plasmid. SK+ (Stratallen¢). Th¢ nucleotid¢ r~- structure of L28 has only been reported in E. coil [8]. qucn¢¢ was d¢temlined by the did¢oxy ¢hain termination method [12] The tobacco CL28 sequence is the second report of'this using dca~a.lcqucnu~ sequencing kits (United Slat~ Biochemical). protein. Rat and yeast l~Ss have no homology with E, carl L28. Based on the conserved structure between E, coil I28 and tobacco CL28. L28 is thought to have 3, RESULTS AND DISCUSSION a function as yet unclear ir~ ribosomes. In E, coil the L28 3,1, Isolation of ribosomal proteh~ CL,28 gen¢ (rpmB) is cotranscril:~d with the L33 gent (rpmG) Reverse-phase chromatography of 50 S ribosomal [6]. in land plants the CL33 sene is located in the chic- proteins from tobacco leaves produced at least 12 sharp roplast genome ill while the CL28 gene is in the nucleus, peaks (details will be published elsewhere), The fourth This suggests a chloroplast to nucleus gene relocation peak was resolved t'urther into 4 bands by SDS-PAGE. during evolution (e,8, [l 5D, A 15 kDa protein (band 17B) ca the membrane was subjected to protein microsequencing. Twenty-one ..lrk,~,'lcdj~¢,w,ls: We thank Dis. M. Sugita. T. Waka~.~gi -~nd Y. Li amino acids of the N.terminus were analyzed and 19 of I'or discussions and u~ful SUSllmtions. Ms. C. Sugi|a for techni~l these residues were clearly identified (Fig. 2), assistance :rod Dr, R.F. Whilti¢~ for the tobacco leaf eDNA library construction. We al~ express our fltank,: to Dr, R. l.[¢rrmafm for critical rcadin~ of 1h¢ m,,nuscrip|, 3.2. Sequ¢,ce of'the CL2t] eDNA A eDNA clone was isolated using an oligonuclcotide. which was synthesized based on the N-terminal amino REFERENCES acid sequence of the 15 kDa protein. This eDNA was sequenced and contains an insert of 671 bp with a read- [ll Susiurn, M. (19119) Annu, Rev. Cell i~;,a~. S. $1-70. ing frame of 151 amino acid residues (Fig. l), This [2l M.ch¢. R, (1990) Plant $ci. 72, 1-12. [3] Shinozaki, R., Chine. M,. Tanaka, M,, Wakasugi. T,. H.yashida, reading frame contains three possible initiation codons N,. Mutsubayashi, T,, Zaita, N,, Cltunwongse, J.. Obokata, J,, (residues l. 4 and 15). The first ATG codon occurs in Shinoxaki. K.Y.. Ohio, C,. Toruz,'twa, K., Mcns, O,Y.. Sugita, the sequence AAAAATGGC, which matches the con- M., Deno, H.. Kamotmshira, T,. Yamada. g,, Kusuda, L, sensus sequence flanking functional start ¢odons in Takaiwa, F., Kate, A,. Tohdoh, N,. Shimuda. H, and SUlliura. M. 0986) EMBO J S, 2043-2049. dicot plants [ 13]. This suggests that the first ATG codon [4] Yokel, F.. VaHilcvu, A.. Hayashida, N,. Torazawa, K,, Wa- is the functional initiation codon. The 21 amino acids kasugi. T. and SuBiuPa. M. (1990) FEBS Left. 2"/6. I~K-g0, in the 15 kDa protein which were determined by amino iS} Sugiura. M.. Tora=wa. K. and Wak-SulBi, T., in: Translational acid sequencing match the predicted sequence of resi- Apparatus of Phn|osynth¢fi¢ Organcll=s (R. Mach¢, E, Stutz and dues 75-95. Thus, the mature protein is 77 amino acids A.R. Subramanian. Eds,), Springer, Berlin, 1991. pp, $9-69. [6] Late, J,S., An, G,, Frie,',en, J,D, and l~no, K, (19~1)Mot, Gen, long. The preceding peptide is probably 74 amino acids G=net. I~, 21~-223. in length, of which 38 are hydrophobic (51%) and 9 ar¢ [7] Dobbs. E.R. ¢1979)J. l.]actcriol. 140, 7341-737, basic (12%). This motif is similar to the amino acid [tq Wittmann.Liehold, B, and Marzinzig, E, 09?7) FEllS l.¢tt, xl, composition of other transit pcptidcs [14], suggesting 214-217. [9] Cap¢l, M.S. and Bourque. D.P, (1982).I, Uiol. Chore, 257, "/746- that the pre~ding sequen~ is a transit peptid=. '/?$3. The size of the predicted mature protein is the same [10] Wallace. R.B. nnd Miyada. C.G. 098-/) Methods Enzymol. 152. as that of E. coli L28 (77 amino acids). It is a basic 432-442. protein (12 lysinc residues out of 77) and has a calcu- [I I] Manfioletti, G, and Schneider, C. (1988) Nucleic Acids Rcs. 16. lated molecular weight of 8924. Discrepancies between 21173-211~1, [12] Sangar. F.. Nicklcn. S. and Coulson, A.R. (19-/7) Prec. Natl. observed and calculated molecular weights are often Acad. Sci, USA 74. 54~3-5467, found in ribosomal proteins (e.g. [4]), Th= mature pro- [13] Cavener. D.R.. :rod Ray. S.C. (Ig91) Nucleic Acids Rcs. 19.318.% tein has 36% amino acid identity to E, cots L28 (Fig. 2) 3192, and their hydropathy profiles arc similar (Fig. 3). [14] Hcijn=, G,. Hirai, T,, Kl~sgen, R,13. Stcppuhn..I,, Bruce. B., Keclpltra. K. ,rod Hcrrm:,nn. R. (1991) Plant Mol. Biol. Rcp, 9, Therefore, we conclude that the chloroplast protein is 104-126, a homologue of E.coli L28 and designate it CL28. [15] Smookcr, P.M,, Sdu;nidt, J, and Subramani:m, A,E. U991) Bio. The function of L28 is not clear because mutants ¢himi¢ 73. 845-851. which lack L28 are viable in E. co[i [7]. The primary [161 Kyt¢. J. and Doolittle. R.F. (1982) J. Mol. Biol. 157. 10S-132.

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