FEBS 10714 Volume 298, number I. 93-96 Q 1992 Federation of European Biochemical Societies 00145793/92/$5.00

A secY homologue

February 1992

is found in the plastid genome of Cryptomonas

0

Susan E. Douglas Ittsrirure jbr Muric~e Bioscicrtces, Nurimul Reseurc~t Council, 111 I Oxford Sweet, Hui$ts,

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B3H 321, CunrpJu

Received 25 November 1991 An open reading frame with significant similarity to the WC-Ygene or E.sc/tcrk/tb cufi has been found within a ribosomal protein opuon on the plastid gcnomc of the chlorophyll c-containing alga Cr.vpkmmus @. The gene encodes a protein of 420 amino acids (molecular weigh1 46,906 daltons) and conlains ten potential membrane-spanning domains, as in the E. coii homologuc. This report of a srrY homologue in a plastid gcnomc provides preliminary evidence that a prokaryotic-like protein exporl system may be operating in plastids. Algae: Plastid: Ribosomal protein operon; Secretion; Chloroplast cndoplasmic reticulum

1. INTRODUCTION In Gram-negative Eubacteria, at least six proteins (&A, B, D, E, F and Y) are required for targeting and protein translocation across the plasma membrane (see [I]). Many of these proteins have been isolated and their genes cloned and sequenced. The cytosolic Sect3 protein functions as a chaperonin and as part of a receptor cascade [2], the membrane-associated SecA protein functions as an ATPase [3] and the integral membrane proteins SccY and SecE mediate translocation by interacting with SecA [4]. Integral membrane proteins SecD and SecF may be required for the later stages of protein export or for interaction of precursors with signal peptidases [S]. Recently, set homologues have also been found in the Gram-positive Eubacterium, Buciilus subrilis [G-8]. In land plants and green algae, translocation across plastid membranes (see [9],[10-121) and targeting of nuto the plastid envelope clear-encoded proteins membrane of [13,14] have been studied. Cleavage sites for plastid transit peptides [IS] and thylakoid lumen leader peptides [16,17] have also been investigated. Mowever, protein targeting and translocation has not been investigated in the rhodophyte, chromophyte or cryptophyte algae. Chromophyte and cryptophyte algae present especially interesting systems for studying protein translocation since the plastids of these organisms are surrounded by an extra pair of membranes, termed the chloroplast endoplasmic reticulum, or CER [18]. The outermost membrane of the CER, which bears eukaryotic ribosomes and is usually continuous with the nuclear Cmrcspondaxc oddxx S.E. Dou&1as, !ns:itute For Msrine Biosciences.National Research Council, I41 1 Oxford Street. Halifax. Nova Scotia B3H 321, Canada. Fax: (I) (902) 4269413.

Publishrdby Efsevier Science Pubhhers

B. V.

membrane, is probably derived from the cndomembrane system of the host, while the innermost membrane is probably derived from the plasma membrane of the eukaryotic endosymbiont which gave rise to the plastid [19,20]. Ctyptomonads are unique among the CER-containing algae in retaining a nucleomorph (the vestigial nucleus of the eukaryotic endosymbiont) in the periplastidal space between the CER and plastid envelope [21]. Presumably in an alga as complex as Crypromonas @, gene products encoded by the nuclear or nucleomorph genomes that are targeted to the plastid, contain distinctive N-terminal extensions which serve as signals in intracellular sorting and assist these proteins to traverse the additional membranes of the CER. Those gene products destined for the thylakoid of the plastid may contain additional signals. However, sequence analysis of Cryptuntonns @ plastid cpeI3 gene, which encodes the p subunit of phycoerythrin (a lumenal protein), does not predict an N-terminal extension [22]. On the other hand, the sequences of genomic clones of cpeA from the cryp tomonad Cltroo~~o~s, do predict a leader sequence [23]. The mechanism of protein translocation across ei.ther the thylakoid, plastid or CER membranes is unknown although Gibbs [24] has suggested that vesicles carrying nucleus-encoded proteins destined for the plastid bud off from the inner CER membrane and then fuse with the outer plastid envelope membrane. In both B. subriiis and E. co/i, the integral membrane protein SecY is encoded at the promoter distal region of the spc ribosomal protein operon. Although the secY gene is not present on any of the three land plant plastid genomes which have been completely sequenced [25271, it is possible that it might reside on the plastid genome of a less advanced alga. it appears that gene transfer from the plastid progenitor to the host genome has not proceeded as far in some algae as in land plants 93

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[28,29] and in cryptomonids in particular, there appear to be many genes which are encoded on the plastid r&z; than the nuclear genome [30,31]. For example, sequence analysis of the scr operon from Crypromoms 0 shows the presence of ribosomal protein genes which are not found on plastid genomes of land plants 1321.In order to see if a srcY homologue is present on the plastid genome of Crypmmonm @, the promoter distal region of the spc ribosomal protein operon was scq uenced. 2. MATERIALS

AND METHODS

Isolation ofplastid DNA from Cr+rpfoj)ronus@ and construction of Bclo~lcbank has been dcscrikd [33]. Ribosomal protein opcrons were mapped on the plirstid genome by hybridisation with huterologous probes and by sequencing [32]. The promoter dislal region of the spc operon which potentially would encode se& was located on two adjacent 1.9 kb Sufl fragments. Both strands of the DNA from this region were complctcly sequenced using the dideoxy chain termination method [34] and synthetic oligonucleotide primers. Analysis of the DNA sequence and the derived amino acid sequence was performed using the DNA Strider version I, I program [35] and databank scarchin& was performed using the FastP program [36] on a Macintosh II computer (Apple Computers).

3. RESULTS AND DISCUSSION A physical map of the plastid genome of Cryptmoms 0 is shown in Fig. la. The positions of the SIO, spc, alpha and sfr ribosomal protein operons are indicated by solid bars. The promoter distal region of the spc operon is shown in Fig. 1b and the locations of coding regions are indicated by stippled bars. An open reading

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frame of 1,260 nucleotides with similarly to secY from E. co/i was found which spanned the two 1.9 kb SaEI fragments. The nucleotide sequence of secY and the flanking upstream and downstream regions, including the 5’.terminus of rpi36 (the gene encoding ribosomal protein L36), are shown in Fig. 2. The’derived amino acid sequence (420 residues, 46,906 Da) is given below the nucleotide sequence. As with other Cryptornonas @ plastid genes [33,35], there is very little space between the two genes (only 27 nucleotides). An alignment of the secY gene products from Cryprornonas 0.5. subtilis and E. co0 is shown in Fig. 3. The two proteins are essentially colinear, with the exception of N- and C-terminal extensions in the E. co/i sequence relative to those from Crypmmas @ and B. drills. There are several other places where small insertions or deletions occur. Excluding the N- and C-terminal extensions, the amino acid sequences from Cryptomonas @ and E. co/i are 38.7% identical and 60.1% similar (when conservative replacements are included). The similarity to the SecY sequence from 5. strbriiis is only slightly lower (35.3 and 60.7%, respectively). In E. coli, SecY is an integral membrane protein which contains ten transmembrane segments, five periplasmically exposed regions and six cytoplasmicaliy exposed regions [37]. It is part of the membrane-bound translocase which also contains the SecE [4] and possibly SecD and SecF proteins [5]. The hydropatbic profiles of the SecY proteins from E. co/i, B. mbtilis and Cryptornonas 0 are virtually superimposable (Fig. 4) and the ten putative membrane-spanning domains are present in all three molecules. The fact that the secY’ gene is found within the transcriptionally active spc ribosomal operon (Wang, Liu and Douglas, in preparation) indicates that it is not an inactive pseudogene and that the SecY polypeptide is functional in the cell. Also, the conservation in primary amino acid sequence and, more importantly, the hydropathy profile, argues for a functional gene product. The discovery of a gene for a component of a prokaryotic protein export system in a plastid genome is intriguing and may shed light on possible mechanisms of protein translocation in plant cells. Thus far, no set genes have been reported in any plant system, possibly because they are present in nuclear genomes rather than the extensively studied plastid genomes. The plastid genome of Cryptomonas @ contains genes not found on other plant plastid genomes and therefore is a useful system for studies of plastid functions which are normally dependent on the products of nuclear-encoded genes. The possibility that other set genes are also plastid-encoded in Crypfomorras 4 is currently under investigation. The subre!lular location of the SecY from Crypromoms 0 is unknown. The inner membrane of the plastid envelope is the homologue of the plasma membrane of E. cofi and it is possible that SecY is involved

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Fig, 2. Nuclcotidc sequence of the s&i gene and flanking regions from Cr_~~forrronrrs @. The deduced amino acid sequence is shown beneath the nucleotide sequence. The positions or putative trammembrane s&men& (see Fig. 4b) are indicated by ronkin numerdls I-X.

in protein translocation across this membrane. However, most of the protein translocation in plastids is in the reverse direction, i.e. from the cytoplasm into the plastid. SecY may be involved in protein translocation across the thylakoid membranes or even across the CER, although the latter is unlikely given that the outer membrane of this pair is derived from the host cndomembrane system rather than the endosymbiont. Possible mechanisms of protein targeting and translocation in Cryptomows 0 are currently under investigation. The author wishes to thank Colleen Murphy for technical assistance and Ron MacKay for critical reading of this manuscript. This is NRCC publication number 33009.

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Fig. 3. Alignmenr of S&r proteins from Cryptumonus &. Buciiiu.s co/i. Colons indicate identities and dots indicate conservative replacements according to Schwartz and Dayhoff [381.

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REFERENCES [I] Bieker, K.L., Phillips, G.J. and Silhavy. T.J. (1990) J. Bioenerg. Biomemb. 22. 29 I-3 IO.

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Fig. 4. Hydropathy plots of SLXY protcins from C~_t~‘p~o~~~o~~~~.s @. Bucilitrs srrbrilisand E,sclwrichiu cd’. PIOISwere obtained using the Kytc-Doolittle option of DNA Strider 1351.The positions of putative transmembrane segments are indicated by roman numerals I-X.

[2] Hartl, F.-U.. Lecker. S.. Schiebrl, E., Hendrick, J.P. and Wickner, W. (1990) Cell 63. 269-279. [3] Lill. R., Dowham, W. and Wickncr, W. (1990) Ccl! 60,271-280. [4] Brundagc, L.. Hendrick, J,P.. Schiebel, E., Dreissen. A.J.M. and Wickner. W. (1990) Cell 62, 649-657. [S] Gardel, C., Johnson, K.. Jacq, A. and Reckwith. J. (1990) EMBO J. 9. 3209-3216, [6] Nakamurs, K., Nakamura. A.. Takamatsu. H., Yoshikawa, H. and Yamanc, K. (1990) J. Biochem, 107,603-607. [7] Sub. J.W,, Boylan, S.A.. Thomas. SM., Dolan, KM.. Oliver, D.B. and Price, CW. (1990) Mol. Microbial. 4, 305-314. [S] Overhotf, G.. Klein, M.. Spcia, M. and Frcudl, R, (1991) Mol. Gcn. Gcner. 228,417-423. [9] Schmidt, G.W. and Misbkind, ML. (1986) Annu. Rev. Biochem. 55, 879-912. [IO] Kecgstra, K. and Olsen, L.J. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40. 471-501.

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Schncll. D.J,. Blob& Ct. and Pain, D. (1990) J. Cell Biol. I I I, 1825-1838. Franzen. L.-G., Rochaix, J.-D. and von Heijnc. G. (1990) FEBS Lett. 260, 165-168. von Hcijnc. G. and Nishikswa, K. (1991) FEBS Lett. 278, l-3. Li, H.-M., Moore, T. and Keegstra, K. (1991) Plant Cell 3, 709717. Gavel. Y. and von Hcijnc. G. (1990) FEUS Lctt. 261, 455-458. Last, D.I. and Gruy. J.C. (1989) Plant Mol. Biol, 12, 655-666. Howr.C,J, and Wallace, T.P.(1990) Nucleic Acids Res. IX, 3417, Gibbs, SP, (1981) Int. Rev. Cy~ol. 72.4999. Gibbs, S.P. (1981) Annu. N.Y. Acad. Sci. 361. 193-208. Douglas, SE.. Murphy. CA., Spencer, D.F. tind Gray, M.W. (1991) Nature 350, 148-151. Greenwood, A.D., GritTiths, H.B. and Santore. U.J. (1977) Brit. Phycol. J. 12, 119. Roith. M. and Douglas. S.E. (1990) Plant Mol. Biol. l&585-592. Jenkins. J.. Hillcr. R-G., Spcirs, J. and GodovacZimmermann, 5. (1990) FEBS Lett. 273. 191-194. Gibbs, S.P. (1979) J. Cell Sci. 35, 253-266, Ohyama, K., Fukuzawa, H., Kohchi. T., Shirai, H., Sano. T.. Sano. S., Umcsono, K., Shiki, Y., Takcuchi, M.,ChanS, Z., Ao~a, S., inokuchi. H. and Ozeki. H. (1986) Nature 322, 572-574. Shinozaki, K., Ohmc. M., Tanaka, M.. Wakasugi.T.. Hayashida. N,. Matsubayashi, T.. Zaita, N., Chunwongse, J., Obokata. J.. Yamaguchi$%inoi?irki. K.,Ghto. C,.Torazawa, K., Meng. B.-Y.. Sugita, M.. Deno, H.. Kamogashira. T., Yamada, K., Kusuda. J., Takaiwa, F.. Kate, A.. Tohdoh. N., Shimada, H. and Sugiura. M. (I 98G) EM130 J. 5, 2043-2049, Hiratsuka, 1.. Shimada. H,, Whittier, R.. lshibashi. T.. Sakamoto. M., Mori, M.. Kondo. C., Honji. Y., Sun. C-R.. Men& B-Y.. Li, Y.-Q,, Kunno. A.. Nishizawa, Y., Hirai, A.. Shinozaki. K. und Sugiura. M. (1989) Mol. Gen. Genct. 217, 185-194, WI Baldauf, S.L. and Palmer. J.D. (1990) Nature 344, 262-265. [29] Gantt, J.S.. Bdldauf, S.L.. Colic. P.J.. Wcedcn, N.F. and Palmer, J.D. 119911 EMBO J. IO. 3073-3078. [301 Douglas. S.E. and Durnford, D,G. (1989) Plant Mol. Biol. 13, 13-20, [3ll Wang, S. and Liu, X.-Q. (1992) Proc. Natl. Acad. Sci. USA (in press). DouBlas. SE. (1991) Curr. Gcnct, 19, 289-294. 1::; Douglas. SE. (1988) Curr. Genct. 14, 591-595. [341 Sangcr, F., Nicklen, S. and Coulscn, A.R. (1977) Proc. Natl. Acud. Sci. USA 74. 5463-5467. Marck, C. (19SR) Nucleic Acids Res. 16. 1829-1836. :;z,’ Pearson, W.R. and Lipn-.an. D,J. (1998) Proc. Natl. Acad. Sci. USA 85.2444-2448. 1371 Akiyama, Y. and Ito. K. (1987) EMBO J. 6, 3465-3470. [3Sl Schwartz, R.M. and Dayhoff, M.O. (1978) in: A;ias of Protein Sequence and StruciIa--, x A. 5, suppl. 3 (M.O. Dayhoff, cd.) pp, 353-358, Natio%, Biomedical Research Foundation, Washington.

A secY homologue is found in the plastid genome of Cryptomonas phi.

An open reading frame with significant similarity to the secY gene of Escherichia coli has been found within a ribosomal protein operon on the plastid...
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