113
Biochimica et Biophysica Acta, 1171 (1992) 113-116 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
Short Sequence-Paper
BBAEXP 90421
Cloning and characterization of the secY gene from the cyanobacterium Synechococcus PCC7942 Masato Nakai
a,
Ayako Tanaka a, Tatsuo Omata b and Toshiya Endo
a
a Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya (Japan) and b Solar Energy Research Group, The Institute of Physical. and Chemical Research (RIKEN), Wako, Saitama (Japan) (Received 28 August 1992)
Key words: secY gene; Protein export; Secretion; Ribosomal protein operons; Cyanobacterium; (Synechococcus PCC7942)
The secY gene product is an essential component of the Escherich& coli cytoplasmic membrane, which mediates the protein translocation across the membrane. We found a gene homologous to secY in the genome of the cyanobacterium Synechococcus PCC7942. The deduced amino acid sequence, 439 amino acids long, shows 43% homology with that of the E. coli secY. The hydrophobic profile suggests that the Synechococcus SecY protein is an integral membrane protein containing ten membranespanning segments, which are closely related to the E. coli counterpart. The SecY protein may participate in the protein translocation across the cytoplasmic or thylakoid membrane in Synechococcus PCC7942.
Cyanobacteria are prokaryotic organisms performing oxygenic photosynthesis. Their cell envelopes have structures typical of Gram-negative bacteria: the outer and inner (cytoplasmic or plasma) membranes with a peptidoglycan layer between. In the cytoplasm are the thylakoid membranes, which are the site of light capturing and energy conversion in photosynthesis. Unlike other bacterial intracytoplasmic membrane systems, cyanobacterial thylakoids have no obvious physical connection with the cytoplasmic membrane [1]. Proteins with transmembrane destinations in bacteria are often synthesized in the cytosol as a precursor with an amino-terminal extention (a leader peptide) that carries a targeting signal to direct its mature part to the specific membranes [2,3]. Amino-terminal extentions of thylakoid lumen proteins of cyanobacteria share characteristics with bacterial leader peptides: basic amino acid residues at the amino-terminus followed by a stretch of hydrophobic amino acids [4,5]. This suggests that cyanobacteria employ a protein transport system (a targeting signal of the transported protein and a protein translocation machinery of the target membrane) toward the thylakoid, which is closely re-
Correspondence to: T. Endo, Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan. The sequence data reported in this paper have been submitted to the EMBL/Genebank Data Libraries under the accession number X68056.
lated to that for protein secretion across the bacterial cytoplasmic membrane [6]. In a Gram-negative bacterium, Escherichia coli, both secretory signals and a protein translocation (export) machinery of the cytoplasmic membrane have been extensively investigated [2,3]; genetic and in vitro reconstitution studies have revealed that the secY gene product is an essential component of the export machinery [7-9]. The SecY protein homologues may thus mediate protein translocation across the thylakoid membrane a n d / o r the cytoplasmic membrane in cyanobacteria. Indeed, a gene homologous to secY was found in a putative spc ribosomal operon of the cyanobacterium Synechococcus PCC7942 in the present study. The derived amino acid sequence is compared with those of the SecY proteins identified in other organisms [10-18]. In various organisms the SecY is encoded at the promoter distal region of the spc ribosomal protein operon [11-14]. To clone the secY homologue from the Synechococcus PCC7942, we screened the genomic D N A library, which has been constructed in A DASH II TM (Stratagene), by the Southern hybridization using a DNA fragment derived from plasmid pTS10 containing the T O B A C C O chloroplast spc ribosomal protein genes as a probe [19]. A )t DASH II T M clone containing a 11 kb DNA fragment which resulted in a strong hybridization signal was obtained. Partial sequence analysis of the DNA fragment showed that the cloned fragment contained the s l O / s p c ribosomal protein
114 ..... L I 5 ~
ICCTA~C~CG~CTGTC~GTCTGTC~G~C~T~A~TAC~GACG~AC~CAGAAAA~G~A~AG~TA~ATC~G L G D G E L S V S L S V S A A A F T A T A Q Q K I E A A G i01 C T G G T C T A G A T C A ~ G A ~ A G G T T C C ~ C A A A A ~ C ~ T G A A T G G G G G C A ~ C A C C ~ C C C C T C ~ C G ~ T ~ G C T G G C ~ A G C T C G L
v
*
G
S
I
A
S e c Y ~
201 G ~ C T A G A T G ~ C C C ~ A ~ A T ~ T A T G G T C ~ A G ~ G G G A A G A C C C C ~ C ~ C C ~ G ~ C T T T T T T A C A G A T G G C C C ~ G C C T C G ~ A C M V V S R G K T P N A Q E T F L Q M A Q A S G 301TGA~A~TGA~ACGGTC~CT~C~TGTCGACTGGGCA~AT~C~CCC~AT~A~GA~C~TA~CGA R G R I L I T V G L L I L C R L G I F I P V P G I D R V A F S N D 401 T C ~ A G G G C ~ ~ T C ~ G ~ A T T G G C ~ C T C G A T A T T T T ~ G G G G T ~ T ~ C G C T T T G G G C ~ T T T T G C C C ~ A T ~ G L Q G N A N L G G V I G F L D I F S G G G L S A L G V F A L G I L 501CCCTACA~CTCGA~ATTTT~A~T~C~C~C~TCG~GACC~CAAAAAAATG~GA~A~C~C~GA P Y I N A S I I L Q L L T A A V P A L E D L Q K N E G E A G R R K I 601 T C ~ A ~ C C C ~ T A C G ~ C C T C ~ C T G G G C C ~ G C T C C A G A ~ A ~ A ~ C G T A T G G G T G A C C C ~ T A C G C C G ~ A C A C C C ~ A C C C C T A Q L T R Y V S L G W A L L Q S I V I A V W V T R Y A V T P G P L 701 G ~ A C C A ~ C A G A C ~ C T T G G T G G C C ~ C A ~ A T G T G G A ~ A ~ G A G ~ G A T C A C T G A G C G ~ C A ~ G ~ T G G G G C A T C A F T I Q T A L A L V A G S M F V M W I S E L I T E R G I G N G A S 801 C ~ T G A ~ ~ C A ~ G T T G C G A C C T T ~ C ~ G A T C ~ A ~ A G A C C ~ A G A ~ C C A G A G C G G T G A T C ~ A ~ A C T ~ G T ~ A T T G L L I F L N I V A T L P R S L Q Q T L E L A Q S G D R S T V G G I V 901 T C A ~ C T ~ A T C ~ C ~ C T T C 4 G C C A C G A T T G ~ A T C G ~ C ~ C ~ G A G ~ A C ~ A ~ C C ~ T C G ~ T C ~ T C G T C G ~ G ~ I L L I V F L A T I V G I V F V Q E G T R R I P V V S A R R Q V G 1001G~GCG~TACA~GA~GCA~A~TATCT~C~T~C~CC~TG~AT~CGATCA~T~C~GATTTTGG~T~CC N R V Y S E R S S Y L P L R L N Q G G V M P I I F A S A I L V L P ii01 T ~ C T T G G C T ~ C T T C A C C A G T ~ C G ~ G T T G ~ G G A ~ G C T ~ C T A C C T C A ~ C C C ~ T ~ T C C G A C C C C T T G G A ~ A C ~ C C T T T T C T A C C F S L A N F T S N E V V L R I A N Y L S P N G P T P W I Y A L F Y L 1201 T T G T G T ~ A ~ G T G G C G T T T A ~ T A T ~ C T A C T C C T C C ~ A ~ C T C ~ C C C C G T C G A C ~ A G ~ C C T C A A A A A A A T G G G G T C G A G T A ~ C C T ~ V L I V A F S Y F Y S S L I L N P V D L A Q N L K K M G S S I P G 1301 A G T G C G ~ C T G G T C G A ~ C C A ~ C ~ T A C G T A C A ~ A G T ~ T C ~ C C G ~ T G A C ~ C T G G G G C 4 ~ A G ~ C C T ~ T ~ C A T C A T C C C C V R P G R A T S Q Y V Q G V L N R L T I L G A V F L G L V A I I P 1401AC~T~G~G~CTC~ATTC~ACTTCC~GGGGC~CCTC~A~CT~TGTC~GATCGATACC~C~GCA~ T A V E G A T R I R T F Q G F G A T S L L I L V G V A I D T A K Q V 1501 T ~ C C T A C G ~ A T C T C ~ A ~ T A ~ T G G T G ~ G A C T A G T ~ G A C T G A T T ~ T ~ C G C C T ~ A G G T ~ A C A C ~ Q T Y V I S Q R Y E G M V K D * 1601 G C A ~ G T C ~ C A G C T ~ T T G G C C C A C A ~ G A C ~ G ~ A G C ~ C T ~ C A ~ T G T G A C ~ T C ~ C A C C C C T A ~
L
Fig. 1. Nucleotide and deduced amino acid sequences of the secY gene and flanking regions from Synechococcus PCC7942. The putative fragment of ~115 gene (L15) is also indicated.
various sources [10-18] (Table I). Among them, the SecY encoded by the Cryptomonas @ plastid genome shows the highest degree of similarity (52%). This could perhaps reflect a close link between the cyanobacterium and the Cryptomonas • plastid in the light of evolution [20]. On the other hand, the percent identities of the Synechococcus PCC7942 SecY with the Cyanophora paradoxa cyanelle SecY and Pavlova lutherii plastid SecY are similar to or even lower than those with the bacterial SecY proteins. Two archaebacterial SecY proteins from Methanococcus vannielii [17] and Haloarcula marismortui [18] show lesser identity to the Synechococcus PCC7942 SecY than the others. Fig. 2 shows the amino acid alignment of the SecY proteins from various organisms. A hydrophobic analysis by the procedure of Kyte and Doolittle [21] shows
operons of the Synechococcus PCC7942 (data not shown). The 1.7 kb BlnI DNA fragment locating at the promoter distal region of the putative spc operon, which would presumably bear a secY gene, was completely sequenced. The sequenced region contained an open reading frame of 1317 nucleotides with similarity to secY from E. coli; Fig. 1 shows the nucleotide and deduced amino acid sequences of secY and the flanking regions, including the Y-terminus of rpll5 (the gene encoding ribosomal protein L15). The derived SecY protein consists of 439 amino acids with a molecular weight of 47 150. The predicted amino acid sequence of the Synechococcus PCC7942 SecY shows close similarity to those of the previously determined SecY proteins from
TABLE 1
Percentage of identical amino acids of SecY proteins
Synechococcus PCC7942 (SY) Cryptomonas • (CR) Escherichia coli (ES) Bacilus subtilis (BA) Lactococcus lactis (LA) Cyanophora paradoxa (CY) PauloL,a lutherii (PA) Mycoplasma capricolum (MY) Methanococcus uannielii (ME) a HA, Haloarcula marismortui.
CR
ES
BA
LA
CY
PA
MY
ME
HA"
52
43 37
36 36 41
35 36 38 47
35 38 28 28 29
32 35 31 26 27 30
32 32 31 38 39 25 30
22 17 18 21 19 18 17 19
15 20 21 24 23 25 24 22 38
115 SY CR ES 8A LA
CY PA MY
O OOOO OO eoeooOeO OO 000 MVVSRGKTPNAQETP T,~MAQASGLRGRI LI TVGLLILCRLGIF IPVPGIDRVAF SNDL~GNANLGGVIGF ~DIFSGOGL Nm~T8 I K S I K K ~ D L E D R I V F T L F LIVMBRLGTFLp I PGVDHDAFYQS I ISNP .... LVNF LNVFSGOOF MAKQPGLDFQSAKGGLGELKRRLLFVIGALIVFRIGSP IP IPGIDAAVLAK- LL~0QR -GT I IB3~rh~SGGAL MRVBDIRNKI IPTLLMLIVFRIGAF I P V P Y V N . . . . A E A L Q A Q B ( ~ ( -G V F D L L N T F G G G A L MF F KTLKL~FKVZDVRARI LFT IF I LFVTRLGAH I TAPGVN .... V~NL~VADL -PF L~a~LVSGNAM
1 1 1 1 1
1
MLSRLIISIFIIF~TIYLQIFKPVNKTFKQGEAKLKRTL~TL~BULBEIR~I8TLCLIFLZRIGT~LPIPGTALNFDLR~y~NNBRNRLANILNLL8~
1 1
MKF~%IeVLZGPLVLRLFRT IM I L IFARLQ%~Z I p I PG ITEV - - -BSFYI88FRNTS IYNLSALSGGS MVIKKPANKVDKKSTFKSSNKKKNPFKSSFLTKNKDL IYRI LFTLLALI I IRLGVYITVPGVTLD - - -KRFATDSSRIQFFQLLSTLGGGS ===m====== ooooeoeooeoooeoo
SY CR ES BA LA
CY PA MY
80 65 73 57 66 106 63 90
o
CD-1 o
========m
ooocco
~
o l o o o
TM-1 o
**.
ooo
.****
PD-1
o
o
****.
I
•
00110
SALGV~ALG I L P Y I NAS I I LQLL -TAAVPALZDLQKNEGRAGRRK I A Q L T R Y V S L G W A L L Q 8 I V I A -V W V T R Y . . . . . . . . A V T P G P L F T I Q T -A L A L V A G S M F V AB IGVFALGIVPYINAS I IVQLA -TNS I pSLEKLQ~EGELGR~KIVQLTRYVALVWALIQBIGV8 -FWVRPY ........ VFNWDLNFVFAMSLTLTIGSMLI SRA8 IFALGIMPYISAS I I IQLL -TVVHPTLAZ I -KI~RGESGRRKIS~YTRYGTLVLAIFQSIQIA -TGLPm(- -PGM~GLVINPGFATYFTA -WSLVTGTMFL YQFS IFAMGI TPYZTAS I I IQLL~KFTEWSK -~IVGP.RKLAQFTRYFTIVLGF I ~ -YGFNNL - - -ANGMLIBF.BOVSTYLI I -ALVLTGGTAFL QNYSLFAMGVS PYITAS I IVQLL~I LPKYV~WSK -~W IGRRKI~TRYITLVLA~IGIT -AGFQAM - - -SSLNIVQNPN~SYLMI -GVLLTTGSMVV LE IGFFTLGI LPYMNASFFL~VL -TKI LPSLERFQKEQEDTAQUFKKWTRYLTVIWAF IQ8 IVIBWIWIRPY ........ ALNWDFFL~LKV -W A L T L G A V IV NVIS ILTLGLGPFFSABLAVQFL -VKLYPAFEKLQNEBGE3GRKTIVRYTRI LTVLFC I IEBFFL~ -NSLRSF ........ VFNWN81 SYTVV -AAAVTTG8 LVL GRFS ILALGVS PYITAS I IVQLL- STDVI pVLTRSK -SGERGRKKLDKLTKI IMIPFAL~TIFTL88~GL I IPGDNTNAIANBAFYYI LIPLVMLGGBFF • ** ~ TM-2 ~ = = = = = = = m z z C D - 2 mmmmzm=== ~ TM-3 ~ ****** PD-1 ***** ~ TM-4
OO SY CR ES BA LA CY
PA MY
174, 159 172 156 165 201 157 192
O(~OOeO~oe# 0000 O O O OOO OOO O MWI 8B LIT~RGIGNGA~LL IFLNIVATLPRNL~TLE .... LAQSGDR~TV - -GGIVI LLI - - -VF LATIV -GIVFV~OTRRIPWSAR~VGNRVY ....... MWFSBQIT~KGIGNGPSLL IF INI ISGLPKLLQSQIQ ..... 8TRLNIQAL - - -DIFVLVF - - - IF~I I -GI IF IQ~I~RIP I ISA -P~LG~ ....... MWLGEQIT~RGIGNGI811 IFAGIVR~LPPAIARTIE . . . . Q ~ / ~ G D L H F L - -V L L L V A V L - - -V ~ A V T F F - - V ~ V - ~ G ~ R R I~r%~NYAK~RRVY ....... MWLGEQITSHGVGNOI811 IFAGIVSS IPKTIGQIYETQFVG~ND~LF IHI - -VKVALLVI - - -AI LAVIV -GVI F IO~AVRK IAIQYAF~TGR~ pAG ....... TWMGEQINEKGFGSOVBVI IFAGIVSGIPSAIK~V~DIKFLNVRP83 IPMB - -WIPVIGLI - - -L~AIVI IYVTTFV~QARRKVPIQYTKLT~T ........ MI IAEQITE IGLTNGSSLLIF INI IARIPNSIEQLFN .... SNINWTFPM~ - -SSLILSL8 - - - LSF ITMF -VI IGLOR~RPVPVLIA~A~R~KFNEP ITEA VWLSEVIT~RGIGNG~SLLILIGNL~RF -R~LINKDD .... FDSLNVS~ - -NLYI IYI I - - - ITLVSMLIFSTL~GARKIPWSAY~LIDGV~D .......
MLIAD~ITIKGIGNGI8IvIFIGIII~MP8NLKSTFE---YVSN8GE~ANIFF~LLN~MIYINvFLLVIL-~VVI~A~RKIPIQ~G~GLTD8 = CD-3
- ~
OOO0 sy
CR ES BA LA
CY PA MY
262 244 259 248 257 296 245 284
TM-5 ooeoe~eooo
BA LA CY
PA MY
357 339 355 341 3S2 394 339 380
0
***
PD-3
0
0
***
~
TM-6
~
.........
=z..m....====.. oo
00
•
00
00
CD-4
-= ....
oo
0~0~00
--SERSSYLP~IIFASAILV-LPF8LAN-FT~NEV---VLRIANY-L~P~GPTP~IYALF-YLVLIVAF8YFY~-SLILNPVD~8IP --DNKTSYLP~BGVMPIIFASAVL~-LPAYLA~-L~BN~---LRTVLHL-FDGT8NNKLLYLLF-TPTLILFF8~YT-BLILNPNDV~W~U-~88IY - -AA~STHLP ~GVIPAIFA~811 L -FPATIA~WFGGGTGWN~LTTISLY -L~PG~PL- - -YVLL -YA~AI IFFCI~T -ALV~NPP.~TADNL--~I~GAFVP - -GGQST~LP~VIPVIFAVAPLI -TPRT I£~FFGTNDV- - -TKWIONN-FDNTBPV- -~I -YVALI I A ~ T Y F Y A - F V ~ M A D ~ Y Z P . . . . . S S Y L P L R V N P A ~ V I p V Z F A G 8 1 T T -A P A T I L ~ - F L ~ 8 ~ N V G W L ~ TL -~AL~YTTWT~MLF -Y A L L I V L F T F F Y 8 - F V ~ V N P ~ ] ~ A ~ N L ~ Y lp ERRXTQAYIFP~LLPAOI~PVIFA~TIFD - -LA -LPA -FTNFLL- - -~Y~LIUFPFNSLP~DFCYL ITIMLPSBNY~LTIMINPKTLA~F~NM(NALI P
--DMRRSYIPIP~G~R~VVPII~S88ILLFLTT~IK~-LPNANI---ATRVI---LDBVNL~IFT~T-FLVLIIF~8~FYT-LIILBPBDIA~LEl~M~8VIQ --8~HTPYLPLK--L~NA~IPVI~ABA~I~-TPITI~-II~GVN--PDsGFVIFTRDTL~FNT~GIBI~--GILIVLFTFLY8-~V~INP~KIA~NF~F~TFIP m.= ....... =. ~
sY CR ES
~
TM-7
~
****
*
PD-4
***
** ~
TM-8
---.....
oooee • 0 oo oeooo o o e 0 ooocooo 0000 0 0 0 GV~PGRAT~YV~p~TRLTILGAVFL~LVA~IPTAV~GATR~RTFQGFGAT8LLILVGVAIDTAK~VQTYvIsQR~GMVKD
..
439
GVRPGKATTEYLQKTLNRLTF LGALF LAP IAIVPN I I3 TLTNL~V~KGLGGT8 LL I IVGV~DTBF~ I~'L 18]~I'BT IVR O II%PGE~TAKYI DI~TLVG~LY I TF I C L I P ~ -PF~ -Q~TB LL I ~ I ~ l ~ BQY~BA--.L~GYGR GVRPGEMTQDRI T81 LYRLTFVG~ IF LAVI S I LP IFF I~FAGLPQSA~I GGT8 LL IVVGVAL~ T~L~S~LVXR~RGFMXN SVRP GKGT3 KYV8 RLLMRLATVG8 LF LGL I S I I P IAAQNVWGLPK IVALOGT8 LL I L I~VA I~AV~LBGYLLKRKYAGFMDNP LE~ GVRP GS B TKVYS EQL IHRI~F IGSFVLALVC I LP S IV~RS LGLPKI~ I L8 pV81 S IALGVAVDTTRRI TSYLG~ 8 S p FKRDB BKRE p LKRDF SKRRSAN
DTKPGVATKVYIRE~ILQASFVG~ILLSALILIPSI~PLSISGITBLILSPSII~YRDTRE~LLSB GIKPGE~TTKYLTGIINRLSVVGSVFLAI IALLPYVISKLTQLPSNLAIGGTGLI IC ISVAIOTV~LKGRI I~NF === .... ========== ~ TM-9 ~ **** ~ TM-10 ~ ====== PD-S
CD-5
I E ~ I ~ T S H I CD-6 ======
420
443 423 439 492 419 476
Fig. 2. Alignment of SecY proteins from Synechococcus PCC7942 (SY), Cryptomonas q9 (CR) [10], Escherichia coli (ES) [11], Bacillus subtilis (BA) [13], Lactococcus lactis (LA) [12], Cyanophora paradoxa (CY) [15], Pal,loca lutherii (PA) [16] and Mycoplasma capricolum (MY) [14]. The conserved amino acids are indicated with an closed circle; open circles indicate residues conserved in at least five of the eight SecY proteins. The putative transmembrane regions (TM-1 ~ 10), the cytoplasmic domains (CD-1 ~ 6), and the periplasmic domains (PD-1 ~ 5) which have been derived by the topology analysis of the E. coli SecY [22] are also indicated.
that the Synechococcus PCC7942 SecY protein is an integral m e m b r a n e protein consisting of ten transmembrane segments with the same topology (the cytosolic side as the cis side of the m e m b r a n e topology) as that of the E. coli SecY protein [22] (data not shown). These tentative transmembrane domains are indicated in Fig. 2. Interestingly, the conserved amino acid residues are clustered more extensively in the transm e m b r a n e domains TM-1, 2, 5, 7 and 9 and in the cytoplasmic domains CD-2, 3 and 5 than the remainder of the molecules. Some of the conserved residues may be functionally a n d / o r structurally essential for the SecY proteins. In E. coli, the SecY protein constitutes a part of the export machinery for the protein translocation across the cytoplasmic m e m b r a n e [8,9,23]. Cyanobacteria have the internal thylakoid m e m b r a n e in addition to the cytoplasmic membrane, and these membranes have distinct polypeptide compositions from each other [24]. Therefore, each m e m b r a n e in cyanobacteria should have its own protein translocation machinery. Although the subcellular location of the SecY protein of
Synechococcus PCC7942 remains to be determined, it
probably mediates the protein translocation across the cytoplasmic or thylakoid membranes. The cyanobacterial SecY protein identified in the present study will provide a starting point to gain insight on the mechanism of intracellular protein sorting between the cytoplasmic and thylakoid membranes and also on the relationship of the protein transport systems among cyanobacteria, other bacteria without thylakoids, and perhaps plant chloroplasts. This work was supported in part by a grant from the H u m a n Frontier Science Program Organization, a grant for 'Biodesign Research Program' from R I K E N to T.E. and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We are grateful to Dr. Masahiro Sugiura (Nagoya University, Japan) for plasmid pTS10, and Dr. H. Wada (National Institute for Basic Biology, Japan) for the Synechococcus D N A library. We also wish to thank Dr. Koreaki Ito (Kyoto University, Japan) for plasmid pKY3 containing the E. coli s e c Y gene which was used in the initial stage of this work.
116
References 1 Golecki, J.R. and Drews, G. (1982) in The Biology of Cyanobacteria (Carr, N.G. and Whitton, B.A., eds.), pp. 125-141, Blackwell, Oxford. 2 Model, P. and Russel, M. (1990) Cell 61, 739-741. 3 Driessen, A.J.M. (1992) Trends Biochem. Sci. 17, 219-223. 4 Kuwabara, T., Reddy, K.J. and Sherman, L.A. (1987) Proc. Natl. Acad. Sci. USA 84, 8230-8234. 5 Van der Plas, J., Bovy, A., Kruyt, F., De Vrieze, G., Dassen, E., Klein, B. and Weisbeek, P. (1989) Mol. Microbiol. 3, 275-284. 6 Smeekens, S., Weisbeek, P. and Robinson, C. (1990) Trends Biochem. Sci. 15, 73-76. 7 Ito, K., Wittekind, M., Nomura, M., Shiba, K., Yura, T., Miura, A. and Nashimoto, H. (1983) Cell 32, 789-797. 8 Brundage, L., Hendrick, J.P., Schiebel, E., Driessen, A.J.M. and Wickner, W. (1990) Cell 62, 649-657. 9 Nishiyama, K., Kabuyama, Y., Akimaru, J., Matsuyama, S., Tokuda, H. and Mizushima, S. (1991) Biochim. Biophys. Acta 1065, 89-97. 10 Douglas, S.E. (1992) FEBS Lett. 298, 93-96.
11 Cerretti, D.P., Dean, D., Davis, G.R., Bedwell, D.M. and Nomura, M. (1983) Nucleic Acids Res. 9, 2599-2616. 12 Koivula, T;, Palva, I. and Hemil~i, H. (1991) FEBS Lett. 288, 114-118. 13 Nakamura, K., Nakamura, A., Takamatsu, H., Yoshikawa, tt. and Yamane, K. (1990) J. Biochem. 107, 63-67. 14 Ohkubo, S., Muto, A., Kawauchi, Y., Yamao, F. and Osawa, S. (1987) Mol. Gen. Genet. 210, 314-322. 15 Michalowski, C.B., Pfanzagl, B., L6ffelhardt, W. and Bohnert, H.J. (1990) Mol. Gen. Genet. 224, 222-231. 16 Scaramuzzi, C.D., Stokes, H.W. and Hiller, R.G. (1992) FEBS Lett. 304, 119-123. 17 Auer, J., Spicker, G. and B6ck, A. (1991) Biochimie 73, 683-688. 18 Arndt, E. (1992) Biochim. Biophys. Acta 1130, 113-116. 19 Sugiura, M., Shinozaki, K., Zaita, N., Kusuda, M. and Kumano, M. (1986) Plant Sci. 44, 211-216. 20 Gray, M.W. and Doolittle, W.F. (1982) Microbiol. Rev. 46, 1-42. 21 Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132. 22 Akiyama, Y. and Ito, K. (1987) EMBO J. 6, 3465-3470. 23 Shiba, K., Ito, K., Yura, T. and Cerretti, D.P. (1984) EMBO J. 3, 631 635. 24 Murata, N. and Omata, T. (1988) Methods Enzymol. 167,245-251.
Biochimica et Biophysica Acta, 1171 (1992) 113-116 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
Short Sequence-Paper
BBAEXP 90421
Cloning and characterization of the secY gene from the cyanobacterium Synechococcus PCC7942 Masato Nakai
a,
Ayako Tanaka a, Tatsuo Omata b and Toshiya Endo
a
a Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya (Japan) and b Solar Energy Research Group, The Institute of Physical. and Chemical Research (RIKEN), Wako, Saitama (Japan) (Received 28 August 1992)
Key words: secY gene; Protein export; Secretion; Ribosomal protein operons; Cyanobacterium; (Synechococcus PCC7942)
The secY gene product is an essential component of the Escherich& coli cytoplasmic membrane, which mediates the protein translocation across the membrane. We found a gene homologous to secY in the genome of the cyanobacterium Synechococcus PCC7942. The deduced amino acid sequence, 439 amino acids long, shows 43% homology with that of the E. coli secY. The hydrophobic profile suggests that the Synechococcus SecY protein is an integral membrane protein containing ten membranespanning segments, which are closely related to the E. coli counterpart. The SecY protein may participate in the protein translocation across the cytoplasmic or thylakoid membrane in Synechococcus PCC7942.
Cyanobacteria are prokaryotic organisms performing oxygenic photosynthesis. Their cell envelopes have structures typical of Gram-negative bacteria: the outer and inner (cytoplasmic or plasma) membranes with a peptidoglycan layer between. In the cytoplasm are the thylakoid membranes, which are the site of light capturing and energy conversion in photosynthesis. Unlike other bacterial intracytoplasmic membrane systems, cyanobacterial thylakoids have no obvious physical connection with the cytoplasmic membrane [1]. Proteins with transmembrane destinations in bacteria are often synthesized in the cytosol as a precursor with an amino-terminal extention (a leader peptide) that carries a targeting signal to direct its mature part to the specific membranes [2,3]. Amino-terminal extentions of thylakoid lumen proteins of cyanobacteria share characteristics with bacterial leader peptides: basic amino acid residues at the amino-terminus followed by a stretch of hydrophobic amino acids [4,5]. This suggests that cyanobacteria employ a protein transport system (a targeting signal of the transported protein and a protein translocation machinery of the target membrane) toward the thylakoid, which is closely re-
Correspondence to: T. Endo, Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan. The sequence data reported in this paper have been submitted to the EMBL/Genebank Data Libraries under the accession number X68056.
lated to that for protein secretion across the bacterial cytoplasmic membrane [6]. In a Gram-negative bacterium, Escherichia coli, both secretory signals and a protein translocation (export) machinery of the cytoplasmic membrane have been extensively investigated [2,3]; genetic and in vitro reconstitution studies have revealed that the secY gene product is an essential component of the export machinery [7-9]. The SecY protein homologues may thus mediate protein translocation across the thylakoid membrane a n d / o r the cytoplasmic membrane in cyanobacteria. Indeed, a gene homologous to secY was found in a putative spc ribosomal operon of the cyanobacterium Synechococcus PCC7942 in the present study. The derived amino acid sequence is compared with those of the SecY proteins identified in other organisms [10-18]. In various organisms the SecY is encoded at the promoter distal region of the spc ribosomal protein operon [11-14]. To clone the secY homologue from the Synechococcus PCC7942, we screened the genomic D N A library, which has been constructed in A DASH II TM (Stratagene), by the Southern hybridization using a DNA fragment derived from plasmid pTS10 containing the T O B A C C O chloroplast spc ribosomal protein genes as a probe [19]. A )t DASH II T M clone containing a 11 kb DNA fragment which resulted in a strong hybridization signal was obtained. Partial sequence analysis of the DNA fragment showed that the cloned fragment contained the s l O / s p c ribosomal protein
114 ..... L I 5 ~
ICCTA~C~CG~CTGTC~GTCTGTC~G~C~T~A~TAC~GACG~AC~CAGAAAA~G~A~AG~TA~ATC~G L G D G E L S V S L S V S A A A F T A T A Q Q K I E A A G i01 C T G G T C T A G A T C A ~ G A ~ A G G T T C C ~ C A A A A ~ C ~ T G A A T G G G G G C A ~ C A C C ~ C C C C T C ~ C G ~ T ~ G C T G G C ~ A G C T C G L
v
*
G
S
I
A
S e c Y ~
201 G ~ C T A G A T G ~ C C C ~ A ~ A T ~ T A T G G T C ~ A G ~ G G G A A G A C C C C ~ C ~ C C ~ G ~ C T T T T T T A C A G A T G G C C C ~ G C C T C G ~ A C M V V S R G K T P N A Q E T F L Q M A Q A S G 301TGA~A~TGA~ACGGTC~CT~C~TGTCGACTGGGCA~AT~C~CCC~AT~A~GA~C~TA~CGA R G R I L I T V G L L I L C R L G I F I P V P G I D R V A F S N D 401 T C ~ A G G G C ~ ~ T C ~ G ~ A T T G G C ~ C T C G A T A T T T T ~ G G G G T ~ T ~ C G C T T T G G G C ~ T T T T G C C C ~ A T ~ G L Q G N A N L G G V I G F L D I F S G G G L S A L G V F A L G I L 501CCCTACA~CTCGA~ATTTT~A~T~C~C~C~TCG~GACC~CAAAAAAATG~GA~A~C~C~GA P Y I N A S I I L Q L L T A A V P A L E D L Q K N E G E A G R R K I 601 T C ~ A ~ C C C ~ T A C G ~ C C T C ~ C T G G G C C ~ G C T C C A G A ~ A ~ A ~ C G T A T G G G T G A C C C ~ T A C G C C G ~ A C A C C C ~ A C C C C T A Q L T R Y V S L G W A L L Q S I V I A V W V T R Y A V T P G P L 701 G ~ A C C A ~ C A G A C ~ C T T G G T G G C C ~ C A ~ A T G T G G A ~ A ~ G A G ~ G A T C A C T G A G C G ~ C A ~ G ~ T G G G G C A T C A F T I Q T A L A L V A G S M F V M W I S E L I T E R G I G N G A S 801 C ~ T G A ~ ~ C A ~ G T T G C G A C C T T ~ C ~ G A T C ~ A ~ A G A C C ~ A G A ~ C C A G A G C G G T G A T C ~ A ~ A C T ~ G T ~ A T T G L L I F L N I V A T L P R S L Q Q T L E L A Q S G D R S T V G G I V 901 T C A ~ C T ~ A T C ~ C ~ C T T C 4 G C C A C G A T T G ~ A T C G ~ C ~ C ~ G A G ~ A C ~ A ~ C C ~ T C G ~ T C ~ T C G T C G ~ G ~ I L L I V F L A T I V G I V F V Q E G T R R I P V V S A R R Q V G 1001G~GCG~TACA~GA~GCA~A~TATCT~C~T~C~CC~TG~AT~CGATCA~T~C~GATTTTGG~T~CC N R V Y S E R S S Y L P L R L N Q G G V M P I I F A S A I L V L P ii01 T ~ C T T G G C T ~ C T T C A C C A G T ~ C G ~ G T T G ~ G G A ~ G C T ~ C T A C C T C A ~ C C C ~ T ~ T C C G A C C C C T T G G A ~ A C ~ C C T T T T C T A C C F S L A N F T S N E V V L R I A N Y L S P N G P T P W I Y A L F Y L 1201 T T G T G T ~ A ~ G T G G C G T T T A ~ T A T ~ C T A C T C C T C C ~ A ~ C T C ~ C C C C G T C G A C ~ A G ~ C C T C A A A A A A A T G G G G T C G A G T A ~ C C T ~ V L I V A F S Y F Y S S L I L N P V D L A Q N L K K M G S S I P G 1301 A G T G C G ~ C T G G T C G A ~ C C A ~ C ~ T A C G T A C A ~ A G T ~ T C ~ C C G ~ T G A C ~ C T G G G G C 4 ~ A G ~ C C T ~ T ~ C A T C A T C C C C V R P G R A T S Q Y V Q G V L N R L T I L G A V F L G L V A I I P 1401AC~T~G~G~CTC~ATTC~ACTTCC~GGGGC~CCTC~A~CT~TGTC~GATCGATACC~C~GCA~ T A V E G A T R I R T F Q G F G A T S L L I L V G V A I D T A K Q V 1501 T ~ C C T A C G ~ A T C T C ~ A ~ T A ~ T G G T G ~ G A C T A G T ~ G A C T G A T T ~ T ~ C G C C T ~ A G G T ~ A C A C ~ Q T Y V I S Q R Y E G M V K D * 1601 G C A ~ G T C ~ C A G C T ~ T T G G C C C A C A ~ G A C ~ G ~ A G C ~ C T ~ C A ~ T G T G A C ~ T C ~ C A C C C C T A ~
L
Fig. 1. Nucleotide and deduced amino acid sequences of the secY gene and flanking regions from Synechococcus PCC7942. The putative fragment of ~115 gene (L15) is also indicated.
various sources [10-18] (Table I). Among them, the SecY encoded by the Cryptomonas @ plastid genome shows the highest degree of similarity (52%). This could perhaps reflect a close link between the cyanobacterium and the Cryptomonas • plastid in the light of evolution [20]. On the other hand, the percent identities of the Synechococcus PCC7942 SecY with the Cyanophora paradoxa cyanelle SecY and Pavlova lutherii plastid SecY are similar to or even lower than those with the bacterial SecY proteins. Two archaebacterial SecY proteins from Methanococcus vannielii [17] and Haloarcula marismortui [18] show lesser identity to the Synechococcus PCC7942 SecY than the others. Fig. 2 shows the amino acid alignment of the SecY proteins from various organisms. A hydrophobic analysis by the procedure of Kyte and Doolittle [21] shows
operons of the Synechococcus PCC7942 (data not shown). The 1.7 kb BlnI DNA fragment locating at the promoter distal region of the putative spc operon, which would presumably bear a secY gene, was completely sequenced. The sequenced region contained an open reading frame of 1317 nucleotides with similarity to secY from E. coli; Fig. 1 shows the nucleotide and deduced amino acid sequences of secY and the flanking regions, including the Y-terminus of rpll5 (the gene encoding ribosomal protein L15). The derived SecY protein consists of 439 amino acids with a molecular weight of 47 150. The predicted amino acid sequence of the Synechococcus PCC7942 SecY shows close similarity to those of the previously determined SecY proteins from
TABLE 1
Percentage of identical amino acids of SecY proteins
Synechococcus PCC7942 (SY) Cryptomonas • (CR) Escherichia coli (ES) Bacilus subtilis (BA) Lactococcus lactis (LA) Cyanophora paradoxa (CY) PauloL,a lutherii (PA) Mycoplasma capricolum (MY) Methanococcus uannielii (ME) a HA, Haloarcula marismortui.
CR
ES
BA
LA
CY
PA
MY
ME
HA"
52
43 37
36 36 41
35 36 38 47
35 38 28 28 29
32 35 31 26 27 30
32 32 31 38 39 25 30
22 17 18 21 19 18 17 19
15 20 21 24 23 25 24 22 38
115 SY CR ES 8A LA
CY PA MY
O OOOO OO eoeooOeO OO 000 MVVSRGKTPNAQETP T,~MAQASGLRGRI LI TVGLLILCRLGIF IPVPGIDRVAF SNDL~GNANLGGVIGF ~DIFSGOGL Nm~T8 I K S I K K ~ D L E D R I V F T L F LIVMBRLGTFLp I PGVDHDAFYQS I ISNP .... LVNF LNVFSGOOF MAKQPGLDFQSAKGGLGELKRRLLFVIGALIVFRIGSP IP IPGIDAAVLAK- LL~0QR -GT I IB3~rh~SGGAL MRVBDIRNKI IPTLLMLIVFRIGAF I P V P Y V N . . . . A E A L Q A Q B ( ~ ( -G V F D L L N T F G G G A L MF F KTLKL~FKVZDVRARI LFT IF I LFVTRLGAH I TAPGVN .... V~NL~VADL -PF L~a~LVSGNAM
1 1 1 1 1
1
MLSRLIISIFIIF~TIYLQIFKPVNKTFKQGEAKLKRTL~TL~BULBEIR~I8TLCLIFLZRIGT~LPIPGTALNFDLR~y~NNBRNRLANILNLL8~
1 1
MKF~%IeVLZGPLVLRLFRT IM I L IFARLQ%~Z I p I PG ITEV - - -BSFYI88FRNTS IYNLSALSGGS MVIKKPANKVDKKSTFKSSNKKKNPFKSSFLTKNKDL IYRI LFTLLALI I IRLGVYITVPGVTLD - - -KRFATDSSRIQFFQLLSTLGGGS ===m====== ooooeoeooeoooeoo
SY CR ES BA LA
CY PA MY
80 65 73 57 66 106 63 90
o
CD-1 o
========m
ooocco
~
o l o o o
TM-1 o
**.
ooo
.****
PD-1
o
o
****.
I
•
00110
SALGV~ALG I L P Y I NAS I I LQLL -TAAVPALZDLQKNEGRAGRRK I A Q L T R Y V S L G W A L L Q 8 I V I A -V W V T R Y . . . . . . . . A V T P G P L F T I Q T -A L A L V A G S M F V AB IGVFALGIVPYINAS I IVQLA -TNS I pSLEKLQ~EGELGR~KIVQLTRYVALVWALIQBIGV8 -FWVRPY ........ VFNWDLNFVFAMSLTLTIGSMLI SRA8 IFALGIMPYISAS I I IQLL -TVVHPTLAZ I -KI~RGESGRRKIS~YTRYGTLVLAIFQSIQIA -TGLPm(- -PGM~GLVINPGFATYFTA -WSLVTGTMFL YQFS IFAMGI TPYZTAS I I IQLL~KFTEWSK -~IVGP.RKLAQFTRYFTIVLGF I ~ -YGFNNL - - -ANGMLIBF.BOVSTYLI I -ALVLTGGTAFL QNYSLFAMGVS PYITAS I IVQLL~I LPKYV~WSK -~W IGRRKI~TRYITLVLA~IGIT -AGFQAM - - -SSLNIVQNPN~SYLMI -GVLLTTGSMVV LE IGFFTLGI LPYMNASFFL~VL -TKI LPSLERFQKEQEDTAQUFKKWTRYLTVIWAF IQ8 IVIBWIWIRPY ........ ALNWDFFL~LKV -W A L T L G A V IV NVIS ILTLGLGPFFSABLAVQFL -VKLYPAFEKLQNEBGE3GRKTIVRYTRI LTVLFC I IEBFFL~ -NSLRSF ........ VFNWN81 SYTVV -AAAVTTG8 LVL GRFS ILALGVS PYITAS I IVQLL- STDVI pVLTRSK -SGERGRKKLDKLTKI IMIPFAL~TIFTL88~GL I IPGDNTNAIANBAFYYI LIPLVMLGGBFF • ** ~ TM-2 ~ = = = = = = = m z z C D - 2 mmmmzm=== ~ TM-3 ~ ****** PD-1 ***** ~ TM-4
OO SY CR ES BA LA CY
PA MY
174, 159 172 156 165 201 157 192
O(~OOeO~oe# 0000 O O O OOO OOO O MWI 8B LIT~RGIGNGA~LL IFLNIVATLPRNL~TLE .... LAQSGDR~TV - -GGIVI LLI - - -VF LATIV -GIVFV~OTRRIPWSAR~VGNRVY ....... MWFSBQIT~KGIGNGPSLL IF INI ISGLPKLLQSQIQ ..... 8TRLNIQAL - - -DIFVLVF - - - IF~I I -GI IF IQ~I~RIP I ISA -P~LG~ ....... MWLGEQIT~RGIGNGI811 IFAGIVR~LPPAIARTIE . . . . Q ~ / ~ G D L H F L - -V L L L V A V L - - -V ~ A V T F F - - V ~ V - ~ G ~ R R I~r%~NYAK~RRVY ....... MWLGEQITSHGVGNOI811 IFAGIVSS IPKTIGQIYETQFVG~ND~LF IHI - -VKVALLVI - - -AI LAVIV -GVI F IO~AVRK IAIQYAF~TGR~ pAG ....... TWMGEQINEKGFGSOVBVI IFAGIVSGIPSAIK~V~DIKFLNVRP83 IPMB - -WIPVIGLI - - -L~AIVI IYVTTFV~QARRKVPIQYTKLT~T ........ MI IAEQITE IGLTNGSSLLIF INI IARIPNSIEQLFN .... SNINWTFPM~ - -SSLILSL8 - - - LSF ITMF -VI IGLOR~RPVPVLIA~A~R~KFNEP ITEA VWLSEVIT~RGIGNG~SLLILIGNL~RF -R~LINKDD .... FDSLNVS~ - -NLYI IYI I - - - ITLVSMLIFSTL~GARKIPWSAY~LIDGV~D .......
MLIAD~ITIKGIGNGI8IvIFIGIII~MP8NLKSTFE---YVSN8GE~ANIFF~LLN~MIYINvFLLVIL-~VVI~A~RKIPIQ~G~GLTD8 = CD-3
- ~
OOO0 sy
CR ES BA LA
CY PA MY
262 244 259 248 257 296 245 284
TM-5 ooeoe~eooo
BA LA CY
PA MY
357 339 355 341 3S2 394 339 380
0
***
PD-3
0
0
***
~
TM-6
~
.........
=z..m....====.. oo
00
•
00
00
CD-4
-= ....
oo
0~0~00
--SERSSYLP~IIFASAILV-LPF8LAN-FT~NEV---VLRIANY-L~P~GPTP~IYALF-YLVLIVAF8YFY~-SLILNPVD~8IP --DNKTSYLP~BGVMPIIFASAVL~-LPAYLA~-L~BN~---LRTVLHL-FDGT8NNKLLYLLF-TPTLILFF8~YT-BLILNPNDV~W~U-~88IY - -AA~STHLP ~GVIPAIFA~811 L -FPATIA~WFGGGTGWN~LTTISLY -L~PG~PL- - -YVLL -YA~AI IFFCI~T -ALV~NPP.~TADNL--~I~GAFVP - -GGQST~LP~VIPVIFAVAPLI -TPRT I£~FFGTNDV- - -TKWIONN-FDNTBPV- -~I -YVALI I A ~ T Y F Y A - F V ~ M A D ~ Y Z P . . . . . S S Y L P L R V N P A ~ V I p V Z F A G 8 1 T T -A P A T I L ~ - F L ~ 8 ~ N V G W L ~ TL -~AL~YTTWT~MLF -Y A L L I V L F T F F Y 8 - F V ~ V N P ~ ] ~ A ~ N L ~ Y lp ERRXTQAYIFP~LLPAOI~PVIFA~TIFD - -LA -LPA -FTNFLL- - -~Y~LIUFPFNSLP~DFCYL ITIMLPSBNY~LTIMINPKTLA~F~NM(NALI P
--DMRRSYIPIP~G~R~VVPII~S88ILLFLTT~IK~-LPNANI---ATRVI---LDBVNL~IFT~T-FLVLIIF~8~FYT-LIILBPBDIA~LEl~M~8VIQ --8~HTPYLPLK--L~NA~IPVI~ABA~I~-TPITI~-II~GVN--PDsGFVIFTRDTL~FNT~GIBI~--GILIVLFTFLY8-~V~INP~KIA~NF~F~TFIP m.= ....... =. ~
sY CR ES
~
TM-7
~
****
*
PD-4
***
** ~
TM-8
---.....
oooee • 0 oo oeooo o o e 0 ooocooo 0000 0 0 0 GV~PGRAT~YV~p~TRLTILGAVFL~LVA~IPTAV~GATR~RTFQGFGAT8LLILVGVAIDTAK~VQTYvIsQR~GMVKD
..
439
GVRPGKATTEYLQKTLNRLTF LGALF LAP IAIVPN I I3 TLTNL~V~KGLGGT8 LL I IVGV~DTBF~ I~'L 18]~I'BT IVR O II%PGE~TAKYI DI~TLVG~LY I TF I C L I P ~ -PF~ -Q~TB LL I ~ I ~ l ~ BQY~BA--.L~GYGR GVRPGEMTQDRI T81 LYRLTFVG~ IF LAVI S I LP IFF I~FAGLPQSA~I GGT8 LL IVVGVAL~ T~L~S~LVXR~RGFMXN SVRP GKGT3 KYV8 RLLMRLATVG8 LF LGL I S I I P IAAQNVWGLPK IVALOGT8 LL I L I~VA I~AV~LBGYLLKRKYAGFMDNP LE~ GVRP GS B TKVYS EQL IHRI~F IGSFVLALVC I LP S IV~RS LGLPKI~ I L8 pV81 S IALGVAVDTTRRI TSYLG~ 8 S p FKRDB BKRE p LKRDF SKRRSAN
DTKPGVATKVYIRE~ILQASFVG~ILLSALILIPSI~PLSISGITBLILSPSII~YRDTRE~LLSB GIKPGE~TTKYLTGIINRLSVVGSVFLAI IALLPYVISKLTQLPSNLAIGGTGLI IC ISVAIOTV~LKGRI I~NF === .... ========== ~ TM-9 ~ **** ~ TM-10 ~ ====== PD-S
CD-5
I E ~ I ~ T S H I CD-6 ======
420
443 423 439 492 419 476
Fig. 2. Alignment of SecY proteins from Synechococcus PCC7942 (SY), Cryptomonas q9 (CR) [10], Escherichia coli (ES) [11], Bacillus subtilis (BA) [13], Lactococcus lactis (LA) [12], Cyanophora paradoxa (CY) [15], Pal,loca lutherii (PA) [16] and Mycoplasma capricolum (MY) [14]. The conserved amino acids are indicated with an closed circle; open circles indicate residues conserved in at least five of the eight SecY proteins. The putative transmembrane regions (TM-1 ~ 10), the cytoplasmic domains (CD-1 ~ 6), and the periplasmic domains (PD-1 ~ 5) which have been derived by the topology analysis of the E. coli SecY [22] are also indicated.
that the Synechococcus PCC7942 SecY protein is an integral m e m b r a n e protein consisting of ten transmembrane segments with the same topology (the cytosolic side as the cis side of the m e m b r a n e topology) as that of the E. coli SecY protein [22] (data not shown). These tentative transmembrane domains are indicated in Fig. 2. Interestingly, the conserved amino acid residues are clustered more extensively in the transm e m b r a n e domains TM-1, 2, 5, 7 and 9 and in the cytoplasmic domains CD-2, 3 and 5 than the remainder of the molecules. Some of the conserved residues may be functionally a n d / o r structurally essential for the SecY proteins. In E. coli, the SecY protein constitutes a part of the export machinery for the protein translocation across the cytoplasmic m e m b r a n e [8,9,23]. Cyanobacteria have the internal thylakoid m e m b r a n e in addition to the cytoplasmic membrane, and these membranes have distinct polypeptide compositions from each other [24]. Therefore, each m e m b r a n e in cyanobacteria should have its own protein translocation machinery. Although the subcellular location of the SecY protein of
Synechococcus PCC7942 remains to be determined, it
probably mediates the protein translocation across the cytoplasmic or thylakoid membranes. The cyanobacterial SecY protein identified in the present study will provide a starting point to gain insight on the mechanism of intracellular protein sorting between the cytoplasmic and thylakoid membranes and also on the relationship of the protein transport systems among cyanobacteria, other bacteria without thylakoids, and perhaps plant chloroplasts. This work was supported in part by a grant from the H u m a n Frontier Science Program Organization, a grant for 'Biodesign Research Program' from R I K E N to T.E. and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We are grateful to Dr. Masahiro Sugiura (Nagoya University, Japan) for plasmid pTS10, and Dr. H. Wada (National Institute for Basic Biology, Japan) for the Synechococcus D N A library. We also wish to thank Dr. Koreaki Ito (Kyoto University, Japan) for plasmid pKY3 containing the E. coli s e c Y gene which was used in the initial stage of this work.
116
References 1 Golecki, J.R. and Drews, G. (1982) in The Biology of Cyanobacteria (Carr, N.G. and Whitton, B.A., eds.), pp. 125-141, Blackwell, Oxford. 2 Model, P. and Russel, M. (1990) Cell 61, 739-741. 3 Driessen, A.J.M. (1992) Trends Biochem. Sci. 17, 219-223. 4 Kuwabara, T., Reddy, K.J. and Sherman, L.A. (1987) Proc. Natl. Acad. Sci. USA 84, 8230-8234. 5 Van der Plas, J., Bovy, A., Kruyt, F., De Vrieze, G., Dassen, E., Klein, B. and Weisbeek, P. (1989) Mol. Microbiol. 3, 275-284. 6 Smeekens, S., Weisbeek, P. and Robinson, C. (1990) Trends Biochem. Sci. 15, 73-76. 7 Ito, K., Wittekind, M., Nomura, M., Shiba, K., Yura, T., Miura, A. and Nashimoto, H. (1983) Cell 32, 789-797. 8 Brundage, L., Hendrick, J.P., Schiebel, E., Driessen, A.J.M. and Wickner, W. (1990) Cell 62, 649-657. 9 Nishiyama, K., Kabuyama, Y., Akimaru, J., Matsuyama, S., Tokuda, H. and Mizushima, S. (1991) Biochim. Biophys. Acta 1065, 89-97. 10 Douglas, S.E. (1992) FEBS Lett. 298, 93-96.
11 Cerretti, D.P., Dean, D., Davis, G.R., Bedwell, D.M. and Nomura, M. (1983) Nucleic Acids Res. 9, 2599-2616. 12 Koivula, T;, Palva, I. and Hemil~i, H. (1991) FEBS Lett. 288, 114-118. 13 Nakamura, K., Nakamura, A., Takamatsu, H., Yoshikawa, tt. and Yamane, K. (1990) J. Biochem. 107, 63-67. 14 Ohkubo, S., Muto, A., Kawauchi, Y., Yamao, F. and Osawa, S. (1987) Mol. Gen. Genet. 210, 314-322. 15 Michalowski, C.B., Pfanzagl, B., L6ffelhardt, W. and Bohnert, H.J. (1990) Mol. Gen. Genet. 224, 222-231. 16 Scaramuzzi, C.D., Stokes, H.W. and Hiller, R.G. (1992) FEBS Lett. 304, 119-123. 17 Auer, J., Spicker, G. and B6ck, A. (1991) Biochimie 73, 683-688. 18 Arndt, E. (1992) Biochim. Biophys. Acta 1130, 113-116. 19 Sugiura, M., Shinozaki, K., Zaita, N., Kusuda, M. and Kumano, M. (1986) Plant Sci. 44, 211-216. 20 Gray, M.W. and Doolittle, W.F. (1982) Microbiol. Rev. 46, 1-42. 21 Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132. 22 Akiyama, Y. and Ito, K. (1987) EMBO J. 6, 3465-3470. 23 Shiba, K., Ito, K., Yura, T. and Cerretti, D.P. (1984) EMBO J. 3, 631 635. 24 Murata, N. and Omata, T. (1988) Methods Enzymol. 167,245-251.
