Biochimica et Biophysica Acta, 1071 ( 1991 ) 221 - 253

221

© 1991 Elsevier Science Publishers B.V. All rights reserved 0304-4157/91/$03.50

BBAREV 85384

Review

Chloroplast protein topogenesis" import, sorting and assembly A. Douwe de Boer and Peter J. Weisbeek Department of Molecular Cell Biology and Institute of Molecular Biology, Unicersityof Utrecht, Utrecht (The Netherlands) (Received 20 December 1990)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

221

II.

Proteins of the chloroplast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chloroplast envelope and intermembrane space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Stioma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Thylakoid membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Thylakoid lumen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

223 223 227 227 227

III.

Organization of chloroplast targeting signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Transit peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Thylakoid transfer domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

280 280 229

IV.

Translocation across the envelope membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cytosolic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Import receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Energetics of chloroplast import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The stromal processing peptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

232 232 234 236 237

V.

Routing within the chloroplast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Insertion into envelope membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Translocation across the thylakoid membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Insertion into the thylakoid membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Stromal factors and energetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The thylakoidal processing peptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

238 238 240 241 243 244

Vl.

Modification and assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

244

VII. Protein import into other plastids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

246

VIII. Protein sorting in plant cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

246

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

247

I. Introduction Abbreviations: DHFR, dihydrofolate reductase; LHCP-II, light harvesting complex protein of photosystem II; pmf, proton-motive force; PSI, photosystem I; PSII, photosystem If; rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; SPP, stromal processing peptidase; TPP, thylakoidal processing peptidase. Correspondence: A.D. de Boer, Agrotechnical Research Institute (ATO-DLO), P.O. Box 17, 6700 AA Wageningen, The Netherlands.

L i k e all o t h e r e u k a r y o t i c cells, p l a n t cells ar,; subdiv i d e d by m e m b r a n e s i n t o d i f f e r e n t c o m p a r t m e n t s c a l l e d o r g a n e l l e s [1]. T h e s e o r g a n e l l e s s e p a r a t e t h e d i f f e r e n t b i o c h e m i c a l p r o c e s s e s t h a t t a k e p l a c e in t h e e u k a r y o t i c cell. A p a r t f r o m t h e t y p e s o f o r g a n e l l e s t h a t a r e s h a r e d

222 with other eukaryotes, such as the nucleus, mitochondria, the endoplasmic reticulum and microbodies, plant cells are characterized by plastids [2] and vacuoles [3]. Many different types of plastids are known, all performing separate functions in different organs of the plant, but originating in development from one form, the proplastid [2,4]. The most well known plastid, the chloroplast that resides in green tissue, is the actual site of photosynthesis [5]. Many proteins are needed to regulate and catalyze the various processes occurring in all of these organelles. Most of the organellar proteins are coded for by the nucleus apart from a small subset that is encoded for by the limited amount of DNA present in two types of organeUes, the mitochondria [6,7] and the plastids [8,9]. Both mitochondria and plastids are thought to have arisen from two independent endosymbiotic events, in which a orimitive eukaryotic cell took up a prokaryote resulting in the gain of specific functions [10,11]. Mitochondria have similarities with certain types of purple bacteria [12,13], whereas plastids have similarities with certain cyanobacteria especially Prochloron [11]. In the course of evolution most of the original prokaryotic genome was either lost or transferred to the nucleus [11,14,15]. This confronted the cell with the problem of protein transport of nuclear encoded proteins from the cytoplasm into these new organelles and the problem of sorting between proteins destined for either of these two organelles and pre-existing organelles. The mechanism of protein translocation into organeiles in a cell has been a major topic during the last two decades [16]. The best studied organelles in this respect are the endoplasmic reticulum, especially the secretory pathway of protein transport (for reviews see Refs. 17 and 18), the mitochondria (for reviews see Refs. 19 and 20) and to a lesser extent the plastids, especially the chloroplast that is the subject of this review (for other recent reviews see Refs. 21-23). Considerable knowledge on mitochondrial protein import has been obtained from studies with Saccharomyces cerevisiae and Neurospora crassa, but recently import into plant mitochondria has also gained some attention [24-27]. Far less is known about import into the nucleus (for a review see Ref. 28), import into microbodies (for reviews see Refs. 29-31) and protein transport through the endoplasmic reticulum other than secretion, e.g., routing to the Golgi apparatus or to the lysosome (for reviews see Refs. 32 and 33). This review will evaluate and discuss what is known about protein transport into and within chloroplasts. This will be discussed in relation to what is known from other systems, especially protein import into mitochondria since this organelle is also supposed to have entered the eukaryotic cell as an endosymbiont. Both organelles had to evolve a protein translocation

Thechloroplast

j ~ t h y l a k o i d S /'/'

, ,, {--- -~ ~

membrane

~-..C-- \x"~-thylakoidlu,~len .-', \\

~envelope innermembrane_~~/~ intermembranespace~ ~ X~ outermembrane---1 contactsite Fig. ]. Schematic representation of a chloroplast. The six diffe~cnt locations for proteins to reside arc indicated together with a contact site in the envelope. The contact site is drawn as a fusion between inner and outer membrane, it is, however, not excluded that both membranes are just held in close proximity.

device to import proteins from the surrounding cytoplasm, but differences between the two devices were necessary to be able to discriminate between proteins destined for either organelle. Chloroplasts are in themselves also subdivided by membranes into three separate compartments, the inter-membrane space, the stroma and the thylakoid lumen (Fig. 1). Consequently there are, with the two envelope membranes and the thylakoid membrane, six different locations for proteins to function (see Section If). Therefore, a large part of this review deals with protein routing inside chloroplasts (see Section V). Routing inside the chloroplast will be compared with both mitochondrial routing and protein transport in bacterial cells (for reviews see Refs. 34 and 35). Unfortunately there is not much known about protein transport in cyanobacteria because these prokaryotes would provide a good model system for routing inside the chloroplast. It would especially be useful to specify the signals that are responsible for discrimination between excretion into the periplasmic space and translocation across the thylakoid membrane or the signals specifying whether a membrane protein is targeted into the thylakoid membrane or into the inner membrane (see Section V). Protein transport and routing inside chloroplasts are outlined by the following events (see below), that will be dealt with in more detail in Sections III-IV. Nuclear encoded chloroplast proteins are synthesized in the cytoplasm on free polysomes as higher molecular weight precursor proteins with N-terminal transit peptides, that are needed for chloroplast targeting. The transit peptides are removed after or during translocation by a stromal processing peptidase. Import into chloroplasts usually requires proteinaceous receptors in the envelope membrane, and ATP i~ ~i¢edcd both for binding

223 and translocation. Sometimes cytosolic factors are required for keeping the precursor soluble and in the correct conformation. Besides the transit peptide there is additional information present in the protein for proper routing inside the chloroplast. Proteins that have to cross the thylakoid membrane contain a second N-terminal extension behind the transit peptide that is removed by a thylakoidal processing peptidase. Stromal factors and ATP are often needed for correct integration into membranes, translocation across the thylakoid membrane and, assembly into protein complexes. Sometimes proteins are modified during or after routing inside the chloroplast (see Section VI). Protein transport into pla~tids other than chloroplasts will be discussed briefly in Section VII. Very little is known about the signals required for proper targeting to the correct organelle, a problem that is even more complex in plant cells, due to the presence of additional organelles. We will review what is currently known about this in Section VIII. Most experiments on protein transport into chloroplasts are performed in vivo with isolated intact chloroplasts (referred to by some authors as 'in organello', because the experiments are performed with intact organelles). Different protocols based on density centrifugation in silica sols, from which percoll is most often used, have been described to isolate intact chloroplasts [36-40]. Initially radiolabelled precursor proteins were obtained by translation of total poly-A RNA in a wheat ~e"m iysate and the precursor of interest was detected by immune precipitation with antibodies [41]. When cloned genes were obtained, a specific RNA species was separated from the pool of poly-A RNA by hybridization to a DNA template, before translation [42]. Mutagenesis experiments to alter the transit peptide or the mature protein, and the use of fusion proteins were not possible until in vitro transcription plasmids became available [43,44]. All these procedures however did have the disadvantage, that an in vitro translation system had to be used by which only low amounts of radiolabelled precursor protein, contaminated by a large amount of nonlabelled proteins, could be made. Presently this problem is overcome by the expression of precursor proteins in Escherichia co!i and subsequent purification [45-47]. Several transport studies have been performed in vivo in transgenic plants using either marker proteins or antibodies to monitor the location of the proteins in the cell (for example see Refs. 48-50). II. Proteins of the chloroplast More than 250 different spots are found after two dimensional gelelectrophoretic analysis of chloroplast proteins, a figure almost certainly underestimating the

true number of proteins present [8,51]. These proteins perform diverse functions such as photosynthesis and CO 2 fixation, biosynthesis of many organic molecules, regulation and transport [2,9,52]. Between 30-40% of these chloroplast proteins are encoded for by the chloroplast genome, about 140 different proteins were found to be imported into chloroplast from the cytoplasm [53,54]. Irrespective of their site of synthesis all these proteins have to be routed to their specific location and therefore should contain routing information coded in the protein. An exception are those proteins that are synthesized in the stroma and also have their function in this compartment. The nucleotide sequences for three different chloroplast genomes, liverwort [55], tobacco [56], and rice [57] have been determined. There are about 150 structural genes in the tobacco chloroplast genome, coding for about 90 different proteins of which more them 50 have been assigned to known chloroplast proteins [58,59]. More and more genes for nuclear encoded chloroplast proteins are isolated and sequenced (Table I), but this is still a very small fraction of the actual number of proteins that are present.

II-A. Chloroplast envelope and intermembrane space The chloroplast is surrounded by a two-membrane envelope. The two membranes are separated from each other by an intermembrane space. There are specific points, called contact sites, at which the two membranes are fused or held in close proximity [60-62]. These contact sites are considered to be important with respect to protein transport (see Section IV-A). Over the years various protocols for envelope isolation have been developed, and several methods have been described for the separation of inner and outer membranes [63-66]. These methods make use of the differences in buoyant density of the two membranes in a sucrose gradient. This difference is due to the dissimilar protein content of the two membranes, the inner membrane has a ratio of lipid to protein of 0.8 (w/w), whereas the outer membrane has a ratio of 3.0 (w/w), resulting in a density of 1.13 g / m l for the inner and 1.08 g/ml for the outer membranes respectively [67]. Contact sites co-purify with the inner membrane fraction [61]. Both envelope membranes are rich in galactolipids, a feature unique with respect to other organelles and they do not contain any phosphatidylethanolamine or cardiolipin, as do mitochondrial membranes. The lipid composition of the two membranes is quite different, the outer membrane contains 32% phosphatidylcholine and 17% monogalactosyldiacylglycerol against 6% and 49%, respectively, for the inner membrane [67]. Chloroplasts are strongly negatively charged, probably due to both lipid and protein composition [68,69].

224 TABLE I Cloned nuclear encoded chloroplast proteins

Nuclear encoded chloroplast proteins that are cloned and sequenced are listed. They are ordered with respect to their final location in the chloroplast. Peripheral thylakoid membrane proteins are considered as membrane proteins here. The length of the transit peptide, mature and precursor protein is given as the number of amino acid residues. I!! defined numbers are put between brackets. The > stands for more than, to indicate that the data are not derived of a full length clone. Abbreviations: ELIP, early light induced protein; EPSP, 5-enolpyruvylshikimate-3phosphate synthase; FNR, ferredoxin-NADP + oxidorcductase; Hsp, heat shock protein; rid, not determined. Protein

Organism

Number of amino acid residues transit peptide

Envelope proteins 10 kDa envelope protein 30 kDa envelope protein 30 kDa envelope protein Stromal proteins acetolactate synthase acetolactate synthase ac~, carrier protein acyl corner protein acyl corner protein acyl corner protein I acyl corner protein 1 acyl comer protein ADP-81ucose pyrophosphorylase carbonic anhydrase carbonic anhydrase EPSP synthase EPSP synthase EPSP synthase ferredoxin ferredoxin ferredoxin ferredoxin fructose 1,6 bisphosphatase glutamine synthetase glutamine synthetase glutamine synthetase glutamine synthetase glyceraldehyde 3 phosphate dehydrogenase subunit A glyceraldehyde 3 phosphate dehydrogenase subunit A glyeeraldehyde 3 phosphate dehydrogenase subunit A glyceraldehyde 3 phosphate dehydrogenase subunit B glyceraldehyde 3 phosphate dehydrogenase subunit B glyceraldehyde 3 phosphate dehydrogenase subunit B glycerol 3 phosphate acyltransferase Hsp21 Hsp22 hydroxy methylbilane synthase NADP-malate dehydrogenase NADPH-protochlorophyllide oxidoreductase nitrite reductase phosphoglycerate kinase phosphoribu!okinase phosphoribulokinase pyruvate, orthophosphate dikinase ribosomal protein L12

spinach pea spinach

72 (83-89)

Refs.

mature protein

precursor

62 330 (318-321)

402 404

101 170 171

nd nd 84 83 83 81 83 83 456 224 224 444 445 445 98 96 97 08 (358) 381 371 371 372

667 670 135 134 134 137 142 137 483 328? 254 520 516 521 146 148 149 148 409 430 427 429 428

315 315, 316 303 304 305 317 318 319 306 320 321 322 323 323 324 325 326 327 328 329 330, 331 332, 333 334

tobacco Arabidopsis turnip rape rape spinach barley Arabidopsis rice pea spinach Arabidopsis petunia tomato Silene Arabidopsis pea spinach wheat pea barley bean rice

nd nd

tobacco

> 58

336

nd

335

maize

66

338

404

336

pea

68

337

405

337

> 53

386

nd

335

80

367

447

337

368 368 ( 185-186) 157 341 375

451 396 232 480 432

337 338 339 1O0 109 340

314 562 408 351 351 876 133

388 594 480 402 404 947 189

341 342 343 344 345 346 347

tobacco pea spinach squash pea Chlamydomonas Euglena maize barley spinach wheat spinach wheat maize spinach

51 51 51 56 59 54 28 104? 33 76 72 76 48 52 52 50 (51) 49 46 48 46

83 28 (46-47) 139 57 74 32 72 51 53 71 56

Note

tl a.b a,c

il

d

c

225 TABLE I (continued) Protein

ribosomal protein Li3 ribosomal protein LI8 ribosomal protein L24 ribosomal protein L25 ribosomal protein L9 ribosomal protein PSrp-1 rubisco activase rubisco activase rubisco binding protein small subunit of rubisco small subunit of rubisco UDP glucose: starch glucosyl transferase (waxy) UDP glucose: starch glucosyl transferase (waxy) superoxide dismutase superoxide dismutase superoxide dismutase thioredoxin f tryptophane synthase TufA unknown (cs gene, pale mutation) unknown (ELIP) a-glucan phosphorylase Thylakoid membrane proteins FNR FNR FNR LHCP-I type I LHCP-I type It LHCP-I type It subunit It PSI (psaD) subunit II PSI (psaD) subunit IV PSI (psaF) subunit IV PSI (psaF) subunit IV PSI (psaF) subunit V PSI (psaG) subunit V PSI (psaG) subunit VI PSI (psaH) subunit VI PSI (psaH) subunit VI PSI (psaH) subunit VIII PSI (psaK) ELIP (HV60) ELIP (HV90) ELIP (HV58) 10 kDa subunit PSII (oecF) l0 kDa subunit PSII (oecF) CP24 PSI LHCP-II LHCP-II type I LHCP-II type II LHCP-II type II LHCP-II type Ii LHCP-II type II LHCP-II type II gamma subunit ATPsynthase (atpC)

Organism

Refs.

transit peptide

mature

precursor

(60) 49 39 30 34 66 58 58 nd 57 45

(190) !!! 155 87 160 351 414 415 541 nd 140

250 ]60 194 117 194 404 472 473 nd nd 185

348 349 349 349 349 350 35! 352,353 271 23 354

maize

72

533

605

307

barley pea tomato petunia spinach

nd 48 63 65 77 (79) 67 nd 47 50

nd 154 155 154 113 (391) 409 nd 149 916

nd 202 218 219 190 470 476 430 196 966

308 355 356 357 358 359 15 360 361 107,309

53 52 52 nd nd nd 50 50 34 46 24 69 30 49 48 30 26 (38) (38) (30) 41 39 51 nd 35 35 nd 36 36 36 41 35 70 75 68

314 308 313 nd nd nd 162 158 91 101 73 98 96 95 95 100 87 (129) (134) (199) 99 99 210 nd nd 229 229 229 nd 229 323 288 187 147 179

369 360 365 246 270 270 221 208 125 147 97 167 126 144 143 130 113 167 172 231 140 138 261 257 nd 264 nd 265 nd 265 364 323 257 222 247

362 363 364 365, 366 367 368 369, 370 371 370 372 223 90 240 241 373 240 240 374 374 374 375 376, 377 88 378 23 251 379 380 381 382 297 383 298 88 236

spinach pea pea pea pea spinach spinach

Arabidopsis wheat consensus

Chlamydomonas

Arabidopsis Arabidopsis Arabidopsis pea potato spinach pea

Mesenbryanthamtm tomato tomato petunia spinach tomato spinach barley

Chlamydomonas spinach

Chlamydomonas spinach barley

Chlamydomonas Chlamydomonas barley barley barley spinach potato spinach

Chlamydomonas consensus

Lanna Pinus petunia

Silene tomato spinat h

gamma subunit ATPsynthase (atpC) Chlamydomonas delta subunit ATPsynthase (atpD) spinach subunit II ATPsynthase (atpE) Rieskc FeS protein (petC)

Number of amino acid residues

spinach spinach

Note

226 TABLE I (continued) Protein

Thylakoid lumen proteins plastocyanin plastocyanin plastocyanin plastocyanin plastocyanin plastocyanin cytochromc C552 33 kDa subunit (oecA) 33 kDa subunit (oecA) 33 kDa subunit (oecA) 33 kDa subunit (oecA) 23 kDa subunit (oecB) 23 kDa subunit (oecB) 23 kDa subunit (oecB) 23 kDa subunit (oecB) 16 kDa subunit (oecC) 16 kDa subunit (oecC) subunit ! !I PSI (psaE) subunit ill PSI (psaE)

Organism

Arabidopsis Silene tomato pea spinach

Chlamydomonas Chlamydomonas pea spinach

Arabidopsis Chlamydomonas spinach pea mustard

Chlamydomonas spinach

Chlamydomonas spinach

Chlamydomonas

Number of amino acid residues

Refs.

transit peptide

mature protein

precursor

72 66 71 69 69 47 58 81 84 85 52 81 73 81 57 83 51 77 62

09 99 99 99 99 99 90 248 247 247 241 186 nd 186 188 149 149 154 181

17 i 165 170 168 168 146 148 329 331 332 293 267 nd 267 245 232 200 231 243

384 385 386 387 388 389 390 391 392 393 394 395 23 396 397 395 394 90 223

Note

P P.q

a b c d

Non-chloroplast protein. The consensus of 5 different clones with the same transit and mature length is given. The consensus of 2 different clones with the same transit and mature length is given. The transit peptide is unusually long with respect to other transit peptides and the other known carbonic anhydrase transit peptide (see Ref. 321) and the transit peptide given is only based on DNA sequence data. c The sequence given in Ref. 333 possibly contains a mistake in transit peptide coding region. f No import studies have yet been performed with this precursor protein, therefore it is not absolutely sure that a transit peptide is not needed for chloroplast import in this case. g The cloned sequence only contains a partial transit peptide. h The number of amino acid residues for the transit peptide is derived from the consensus sequence. The number of amino acid residues for the mature and the precursor is not given (for details see Ref. 23). i This subunit was originally called subunit ill but later renamed (see Ref. 241). k This protein is also called P(30). i This protein is also called P(35). m This protein is also called P(28). n This protein is also called P(37). o The protein was originally published as ST-LS1. ~' This subunit was originally called subunit IV but later renamed (see Ref. 241). '~ This protein is also called P(21).

Up till now it has not been possible to disrupt the outer membrane of intact chloroplasts witheut lysis of the whole chloroplast, despite the differences between the two membranes. This is in contrast with the situation in mitochondria where treatment with low concentrations of digitonin results in release of the intermembrahe space contents while leaving the inner membrane intact [70]. The chloroplast outer membrane is permeable to molecules of molecular mass up to 7-13 kDa, due to the presence of porin-like proteins [71]. Therefore, small proteins including some proteases are also able to penetrate the intermembrane space. Transport of small molecules over the relatively impermeable inner envelope membrane is mediated by various carrier

proteins [72,73]. Several of these translocators have been studied in more detail; the phosphate translocator is a 30 kDa inner membrane protein [74-76] and the dicarboxylic acid translocator in Arabidopsis is probably a 42 kDa protein [77]. About 75 different proteins have been identified in envelope membranes [78]. Besides a function in metabolite transport these proteins are involved in protein translocation, different biosynthetic processes like synthesis of lipids, pigments, terpenes and flavonoids, and several other functions. Most of these proteins are nuclear encoded (for a review see Ref. 67). The genes for only two envelope proteins have been cloned and sequenced (Table I). Although it has been reported that several envelope proteins are coded for by the chloroplast genome, no

227 chloroplast encoded membrane protein is actually identified [67,58]. Due to the inability to isolate the intermembrane space contents, there is very little known about proteins present in this compartment. Up till now only one protein with a molecular weight of 64 kDa, that can be phosphorylated by 32p-ATP, has been assigned to this compartment. The protein fractionates with the soluble stromal proteins, whereas the kinetics of the labeling reaction show that it is phosphorylated at the same time as some envelope proteins, but before phosphorylation of certain stromal proteins [79]. Recently however, the 64 kDa protein has been identified as phosphoglucomutase, a protein that is believed to be functional in the stromal space [79a].

II-B. Stroma Two-dimensional gel analysis of stromal proteins reveals about 140 spots, again probably underestimating the number of different proteins present [8]. These proteins participate in DNA replication, transcription and translation [9], most of the biosynthetic pathways that occur in the chloroplast and several other metabolic reactions [52]. The stroma is also the site for C O 2 fixation by rubisco [80], e protein complex that is considered to be the most abundant in the world [81]. Few stromal proteins are chloroplast encoded, including the large subunit of rubisco, some ribosomal proteins, a translation initiation factor and several subunits of the RNA polymerase [58]. The remaining stromal proteins are nuclear encoded and have to be imported from the cytoplasm. Several nuclear genes for stromal proteins have been cloned and sequenced (Table I). The stromal contents can be obtained after lysis of intact chloroplasts, removal of the thylakoids by lowspeed centrifugation, and removal of the envelope membranes by high speed centrifugation [82]. The stromal fraction is expected to be contaminated by proteins from the intermembranc space, although the proteins of this latter compartment will only constitute a very minor amount of the total protein.

II-C. ThylakoM membrane Thylakoid membranes have been studied extensively as the site for photosynthetic electron transfer and phosphorylation [5]. The thylakoid membranes are arranged in stacked (grana lamellae) and unstacked (stroma lameUae), regions and four major protein complexes, that are unevenly distributed over the two regions, have been identified [83]. Photosystem I, its light harvesting complex and the F0/Ft-ATPsynthase complex are mainly found in the stroma lamellae, whereas photosystem II and light harvesting complex II are

mainly situated in the grana stacks. The cytochrome b 6 / f complex is evenly distributed over the two regions. A few proteins, such as the thylakoidal targeting peptidase (see Section V-E), are present in the membrane not associated witF these complexes. The thylakoids can be isolated after lysis of intact chloroplasts by low speed centrifugation, and it is possible to separate grana lamellae from stroma lamellae by digitonin treatment [84,85]. Thylakoid membranes, free of luminal proteins and some loosely bound thylakoid membrane proteins, like the a- and/3-subunit of the FrATPsynthase, can be obtained after sonication of the thylakoids, by high speed centrifugation [50]. The protein content of the thylakoid membrane is rather high (the lipid to protein ratio is 0.4 (w/w)), but the lipid composition is quite similar to the inner membrane [67]. The subunits of these protein complexes are both encoded for by the chloroplast and by the nucleus [86,54], and models for the arrangement ot these subunits in the different complexes have been devised [54,87,88]. The genes for several nuclear encoded thylakoid membrane proteins have been cloned and sequenced (see Table I). All subunits are synthesized as higher molecular weight precursors with a cleavable transit peptide and non-cleavable routing signals (see Section V-C). The chloroplast encoded thylakoid membrane proteins also contain non-cleavable sequences for routing. The only exception is cytochrome f, which has its main function at the luminal side of the thyiakoid membrane and therefore most of the protein has to cross the membrane (see Section V-C).

II-D. Thylakoid htmen The thylakoid lumen is believed to be a space completely enclosed by the thylakoid membrane system without any contact with the stromal compartment. The lumen is very acidic since protons are continuously pumped into this space by the photosynthetic activity of the membrane. The resulting pmf drives the synthesis of ATP by the Fi/F0-ATPsynthase [5]. Water splitting and the resulting oxygen evolution also takes place in this compartment [89]. Several luminal proteins have been identified, being three proteins of the water splitting complex, the electron carrier plastocyanin and the plastocyanin binding protein, subunit III of PSI. The C-terminal portion of subunit III however contains two hydrophobic segments, that might act as membrane anchors although their hydrophobic moments are rather low [90]. All of the above five proteins are nuclear encoded and their genes have been cloned from several organisms (Table I). The precursor contains in all cases an N-terminal extension that is composed of two domains. The first domain is the equivalent of a real transit peptide and is

228 needed for targeting into the chloroplast, whereas the second domain contains routing information for thylakoid transfer (see Sections III-B and V-B) [91,82].

I11. Organization of chloroplast targeting signals Nuclear encoded chloroplast proteins are synthesized in the cytoplasm as higher molecular weight precursor proteins as first reported by Dobberstein et al. [92]. They showed that the small subunit of rubisco from Chlamydomonas is synthesized in a wheat germ system as a 20 kDa precursor with a 3.5 kDa extension that is removed upon incubation with post-ribosomal supernatant. The processing activity present in this supernatant originated from lysed chloroplasts (see Section IV-D). Soon thereafter more proteins were found to be synthesized as precursor proteins, e.g., higher plant small subunit of rubisco, LHCP-II and ferredoxin [93-95]. The extension was found to be N-terminal [96], and is called a transit peptide to distinguish it from the structurally and functionally different signal sequence required for co-translational transport [97]. Nuclear encoded chloroplast proteins are synthesized on free polysomes instead of membrane bound ones [92] and enter the chloroplast post translationally [98,99]. Up till now all known proteins that are actually imported into chloroplasts are found to be synthesized as precursor proteins with an N-terminal targeting signal. One nuclear encoded heat shock protein from Chlamydomonas, that normally functions inside the chloroplast, does not reveal the presence of a N-terminal extension, as deduced from its nucleotide sequence [I00]. However, no in vitro import studies have yet been undertaken with this protein. Only one other nuclear encoded chloroplast protein, that functions in the outer envelope membrane is found to be synthesized as a native protein without a cleavable transit peptide. This protein, with unknown function, enters the membrane without being processed (see Section V-A and Ref. 101). When increasing numbers of genes for nuclear encoded chloroplast proteins were cloned and sequenced it became apparent that the conformity between the N-terminal extensions was not just homology blocks of identical amino acids, as was originally thought [102], but more of a conformity in charge distribution and

.....

+

....

+

.

+

..

secondary structure with some preferential positions for certain amino acids [103]. The structural features of these transit peptides will be discussed below. Proteins destined for the thylakoid lumen are synthesized as precursors with an elongated N-terminal targeting signal, that is composed of two functionally distinct domains [82,91,104,105]. The first domain is analogous to the transit peptide of stromal proteins and it can be exchanged by a stromal transit peptide without any effect on import and routing [104,91]. The second domain, the thylakoid transfer domain, is comparable to a standard signal sequence as can be found in precursor proteins destined for the endoplasmic reticulum in eukaryotic cells or in the precursors of proteins that are excreted in the periplasmic space in prokaryotes [103,106]. The features of this second domain will be discussed in Section II-B. Thylakoid membraae proteins that normally have to cross the membrane partially do not contain cleavable extensions comparable to the thylakoid transfer domain, but seem to have internal domains in the mature protein. An exception is the chloroplast encoded protein cytochrome f, and subunit III of PSI if this protein is indeed a membrane protein (see Section II-D).

IliA. Transit peptides Statistical analysis of 18 chloroplast transit peptides with known cleavage sites show practically no homology blocks, apart from the presence of the N-terminal dipeptide methionine-alanine. This dipeptide probably signals the removal of the N-terminal methionine from the precursor after translation [103]. The size of the stromal target~,~i domain in higher plants ranges from about 28 to ~"!~ amino acids (see Table I, stromal and thylakoid membrane proteins). Although the reason for these differences in length is not known, similar proteins in different organisms have comparable transit peptide lengths. Therefore, it is conceivable that the transit peptide is adjusted to the mature protein in the course of evolution. For example the transit peptide of the enzyme a-glucan phosphorylase is extremely basic, and it might have a function in reducing the acidic nature of the mature protein during transport [107]. Transit peptides of the alga Chlamydomonas are usually smaller, ranging from 24 to 45 amino acids. These transit peptides are also unusual in that they show intermediate characteristics

-

+..

.

~...+++.

.

MASLSATTTVRVQPSSSSLHKLSQGNGRCSSIVCLDWGKSSFPTLRTSRRRSFISA,A K

I

Fig. 2. The transit peptide of the spinachacyl carrier protein is shown as an exampleof a typicaltransit peptide [317]. The C- and N-terminal part of the transit peptide is undtrlined. Aminoacids that match the consensuscleavagesite are shown in bold face. The actual cleavagesite is markedwith an asterisk.The chargesof the aminoacidsare givenabove the sequence.Hydroxyamino acidsare markedabovewith dots.

229 between mitochondrial and higher plant chloroplast targeting peptides [108]. A very large transit peptide, of 139 amino acids, is present in the precursor for the enzyme hydroxy methylbilane synthase of the alga Euglena. This alga, however, contains an additional third membrane around the chloroplast that is probably derived from the endoplasmic reticulum. Therefore, this transit peptide might turn out to be a composite targeting sequence [109]. Transit peptides (Fig. 2) are characterized by high amounts of the hydroxy amino acids serine (about 20%) and threonine and the lack of tyrosine and negatively charged amino acids [21,23,102,103]. Statistical analysis reveals the presence of three regions in transit peptides [103,110]. Firstly, an amino terminal domain of 10 amino acids with almost no proline, glycine and charged amino acids, and normal levels of serine. Secondly, a central region variable in length with high amounts of hydroxy amino acids and containing positively charged amino acids. The third region is composed of the C-terminal 10 amino acids before the processing site. This region has, theoretically, a high potential for forming amphiphilic g-strands. Helix forming amino acids like leucine and lysine are excluded from the region and arginine residues are often present in the region - 6 to - 1 0 and at position - 2 . The change between a/J-sheet and a more helical two dimensional structure is thought to mark the cleavage site. The consensus cleavage site, (lle/Vai)-X(Ala/Cys)$ Ala, has been derived [110]. There is, however, a lot of deviation from the consensus cleavage site as might be expected when apart from primary also secondary structure is important for signalling the cleavage site. Several groups have tried to dissect the transit peptide into functional domains, such as binding domains and those domains participating in processing or translocation. However, the results obtained by mutagenesis experiments on the transit peptide do not provide a clear picture. Deletions in the C-terminus of the transit of the small subunit of rubisco [111,112], ferredoxin [113] and plastocyanin [91] show a strong reduction in both binding efficiency and import. Deletions of two amino acids in the C-terminus of the transit do not affect binding [113,91]. Binding and import is also severely reduced in N-terminal deletion mutants [91,111,112]. Deletions in the central region result in mutants that are sometimes still imported with reduced efficiency, although no binding is observed [112]. This is also observed for a 9 amino acid deletion in the C-terminus of the transit peptide of the small subunit. However, the processing of this mutant to its mature size is aberrant [114,115]. Deletion of an additional three amino acids reduces the efficiency of import even more and no mature sized protein is found inside the chloroplast ar,.~___ore, although the aberrantly pro-

cessed protein is still incorporated into the rubisco holoenzyme [115]. It is not known whether these binding mutants bypass the receptor and enter the chloroplast through a different pathv,ay that is less efficient and specific as has been observed in the mitochondrial system [116]. In summary, the transit pepfide as a whole seems to be important for binding and translocation, whereas the C-terminal region is presumably more specifically involved in defining the processing site. Besides the transit peptide, regions in the mature protein have been reported to be important for efficient import into chloroplasts [117,118]. Results obtained with the bacterial protein neomycin phosphotransferase II fused to the transit peptide of the small subunit of rubisco show that the import efficiency is increased when the 23 N-terminal amino acids of the mature small subunit protein are included in the construct [117,118]. A cytosolic heatshock protein without these 23 N-terminal amino acids of the small subunit mature protein translocates less efficiently into the chloroplast than the wildtype small subunit protein; however, it was not tested whether the addition of this sequence would improve the efficiency [119]. No improvement of the efficiency was observed when a brome mosaic virus coat protein was used as a passenger, not even the inclusion of the complete small subunit mature sequence resulted in efficient translocation [120]. In almost all cases passenger protein¢, were imported into chloroplasts both in vitro and ,n vivo when a transit peptide was present (see Table I1). Although the efficiency of import was generally not determined, these data show that the transit peptide itself is sufficient for targeting proteins into the chloroplast. Only binding but no import is observed when basic passenger proteins are used, probably due to the negative charge of the chloroplast (Ref. 113 and Van der Pias and Weisbeek unpublished results). Since a transit peptide co-evolved with its mature protein (see above), the presence of amino acids of the mature in addition to the transit peptide in front of the passenger protein, probably only has a function in presenting the transit peptide in a more natural way. In conclusion we consider it unlikely that the mature part of a chloroplast precursor protein itself contains relevant information for translocation into the chloroplast.

III-B. Thylakoid transfer domains Precursor proteins destined for the thylakoid lumen contain an amino temfinal extension, which we will call a chloroplast targeting signal, that is composed of a transit peptide and an additional sequence necessary for thylakoid transfer [91]. This additional sequence, the thylakoid transfer domain, is rather short and has all the characteristics of signal sequences [23,103,121,122]. Thylakoid transfer domains like signal

230 charged amino acids in position - 3 and - 1 [121,106]. The consensus cleavage site is AIa-X-AIa$[103]. A similar arrangement is present in the thylakoid transfer domains of cyanobacterialthylakoid lumen proteins and

sequences contain a short positively charged N-terminal domain (n-region), a central hydrophobic domain of 14-22 amino acids (h-region) and a carboxy terminal domain of 5-6 residues (c-region) with small unTABLE II

Fusion proteins used for import studies into chloroplasts Fusion proteins are listed with their targeting domain and mature parts specified. Localization inside the chloroplast is given, but only for the fraction(s) that contained most of the imported protein. It is specified whether the experiments were performed in transgenic plants (in vivo) or with isolated chloroplasts (in vitro). When next to the transit peptide almost the complete mature is also present in front of the passenger, both mature proteins are given separated b y / . When the transit peptide and the thylakoid transfer domain are derived from different sources both domains are given separated by / . Abbreviations: ATP-B, subunit /3 of the FI-ATPsynthase; BLA, Escherichia coli /3-1actamase; CAT, chloramphenicol acetyltransferase; CP, brome mosaic virus coat protein; E, E. coli; El3, possitively charged nonsense protein; FD, ferredoxin; GO, glycolate oxidase; GUS,/3-glucuronidase; HSP, heatshock protein; LSU, large subunit of rubisco; MIgM, mouse immunoglobin M; nd, not determined; NPTII, neomycin phosphotransferase II: OECI, 33 kDa subunit of the water splitting complex; P, petunia; pBLA, precursor of E. coil/3-1actamase: PC, plastocyanin: PSB-A, 32 kDa DI protein of PSII; S soybean; SOD, superoxide dismutase; SSU, small subunit of rubisco; St, Sahnonella thyphbnurium; VSVG, vesicular stomatitus virus glycoprotein; WAXY, UDP-glucose: starch glucosyl transferase. Targeting signal

Mature protein (passenger)

Import into the chloroplast

Localization inside the chloroplast

in vitro/ in vivo

Refs.

Note

SSU SSU (S) SSU SSU SSU SSU SSU SSU SSU SSU SSU SSU SSU SSU SSU SSU SSU FD FD FD FD FD LHCP-II LHCP-ll LHCP-ll EPSP-synthase (P) WAXY SSU/OECI FD/PC OECI OECI OECI PC PC PC PC PC PC PC

NPTll SSU (pea) 17.5 kDa HSP OECI CP LSU ATP-B pBLA LHCP-II glutamine synthetase CAT SSU/VSVG SSU/MIgM SSU/CP EPSP-synthase (St) PSB A NPTII PC SOD BLA El3 PC SSU NPTll GUS EPSP-synthase (E) GUS OECI PC SSU GO DHFR DHFR SOD FD BLA PC/BLA BLA PC/BLA

yes yes yes yes yes yes yes yes yes yes yes yes yes no yes yes yes yes yes yes no yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes

stroma stroma stroma stroma stroma (inefficiently) stroma stroma stroma thylakoid membrane stroma stroma stroma stroma stroma thylakoid membrane stroma stroma stroma stroma stroma thylakoid lumen stroma stroma stroma stroma nd thylakoid lumen thylakoid lumen stroma/thylakoid lumen stroma thylakoid lumen stroma stroma stroma stroma thylakoid membrane thylakoid lumen thylakoid lumen/ membrane

in vitro in vitro in vitro in vitro in vitro in vitro m vitro m vitro m vitro m vitro m vitro m vitro m vitro m vitro both in vivo in vivo in vitro in vitro both in vitro in vivo in vitro in vivo in vivo both m vitro m vitro m vitro m vitro m vitro m vitro m vitro m vitro m vitro m vitro m vitro m vivo m vivo

48, 117 119 119 104 120 214, 256 214, 256 256 208, 210, 256 256 256 221 221 120 398 212 48, 118, 399, 400 123 123 see note ll3 50 2O8 209 49 401 173 104 91 104 104 104, 124 91 123 123 see note see note see note see note

c

a b c d

This passenger protein contains a stop-transfer sequence. Probably the structure of this fusion protein interferes with import. De Boer et al.,, EMBO J. in press. Import into the thylakoid lumen was rather inefficient.

d

231 TABLE III

Tt~ylakoid transfer domains Thylakoid transfer domains are listed with their processing sites aligned to the right. When the processing site has not been determined a question mark is given. Three amino acid residues in front of the processing site are bold faced to indicate the consensus processing site. Or~lya~ amino acids N-terminal of the processing site of nuclear encoded eukaryotic proteins are given. A < is put in front of these sequences to indicate that the targeting sequence is cut off at the N-terminus. Positive and negative charges are listed above the sequence and the hydrophobic core is underlined. Protein

Organism

Refs.

Arabidopsis

384

Thylakoid transfer domain

Eukaryotic +

Plastocyamn

÷

+

+

Plastocyanm

Silene

385

÷

÷-