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Research paper 1
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An unusual ERAD-like complex is targeted to the apicoplast of 3
Plasmodium falciparum. 4
Simone Spork1, Jan A. Hiss2, Katharina Mandel1, Maik Sommer3†, Taco W.A. 5
Kooij4, Trang Chu1, Gisbert Schneider2, Uwe. G. Maier3, and Jude M. 6
Przyborski1* 7
8
1Department of Parasitology, Faculty of Biology, Philipps University Marburg, 9
Marburg, Germany. 10
2Institute of Organic Chemistry & Chemical Biology, Johann Wolfgang 11
Goethe-University, Siesmayerstr. 70, 60323 Frankfurt, Germany. 12
3 Laboratory for Cell Biology, Philipps-University Marburg, Marburg, Germany. 13
4Department of Parasitology, Heidelberg University School of Medicine, Im 14
Neuenheimer Feld 324, 69120 Heidelberg, Germany. 15
†Current address: Molekulare Zellbiologie der Pflanzen, Biozentrum N200, 16
Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. 17
18
*Address correspondence to: 19
Jude M Przyborski 20
Department of Parasitology, Faculty of Biology, Karl von Frisch Strasse 8, 21
35043, Marburg, Germany. 22
Tel: +49-6421-2826596; Fax: +49-6421-2821531 23
E-mail: [email protected] 24
25
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00083-09 EC Accepts, published online ahead of print on 5 June 2009
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Abstract 26
Many apicomplexan parasites, including Plasmodium falciparum, harbour a 27
so-called apicoplast; a complex plastid of red algal origin which was gained by 28
a secondary endosymbiotic event. The exact molecular mechanisms directing 29
the transport of nuclear encoded proteins to the apicoplast of P. falciparum 30
are not well understood. Recently, in silico analyses revealed a second copy 31
of proteins homologuous to components of the ER-associated protein 32
degradation (ERAD) system in organisms with secondary plastids, including 33
the malaria parasite P. falciparum. These proteins are predicted to be 34
endowed with an apicoplast targeting signal, and are suggested to play a role 35
in the transport of nuclear encoded proteins to the apicoplast. Here we have 36
studied components of this ERAD-derived putative pre-protein translocon 37
complex in malaria parasites. Using transfection technology coupled with 38
fluorescence imaging techniques we can demonstrate that the n-terminus of 39
several ERAD-derived components targets GFP to the apicoplast. 40
Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues 41
of yeast ERAD components) localise to the apicoplast, where PfsDer1-1 42
tightly associates with membranes. Constantly, PfhDer1-1 localises to the 43
endoplasmic reticulum. Our data suggests that ERAD components have been 44
“re-wired” to provide a conduit for protein transport to the apicoplast. Our 45
results are discussed in relation to the nature of the apicoplast protein 46
transport machinery. 47
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Introduction 49
The apicomplexan parasite Plasmodium falciparum is the aetological agent of 50
malaria tropica, the most severe form of human malaria, responsible for over 51
250 million infections, and 1 million deaths annually (1). Many apicomplexan 52
parasites, including P. falciparum, harbour a so-called apicoplast; a complex 53
plastid of red algal origin which was gained by a secondary endosymbiotic 54
event (2, 3). Although, during the course of evolution, this plastid organelle 55
has lost the ability to carry out photosynthesis, it is still the site of several 56
important biochemical pathways, including isoprenoid and heme biosynthesis, 57
and as such is essential for parasite survival (4). As in other plastids, the vast 58
majority of genes originally encoded on the plastid genome have been 59
transferred to the nucleus of the host. As a result, their gene products 60
(predicted to constitute up to 10% of all nucleus-encoded proteins) must be 61
imported back into the apicoplast (5). The apicoplast is surrounded by four 62
membranes (6), and this protein import process thus represents a major cell 63
biological challenge, and has attracted much research interest, not least due 64
to the importance of P. falciparum as a human pathogen (7, 8). 65
The signals directing transport of nucleus-encoded proteins to complex 66
plastids, including the apicomplexan apicoplast, have been studied in great 67
detail in recent years, and reveal that such proteins are endowed with specific 68
N-terminal targeting sequences, referred to as a “bipartite topogenic signals” 69
(BTS), that directs their transport to this compartment (8). BTS are composed 70
of an N-terminal ER-type signal sequence, which initially allows proteins to 71
enter the secretory system via the Sec61 complex (9). Following this, proteins 72
are carried via an Golgi-independent transport step to the second outermost 73
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membrane, from where they are then translocated across the remaining three 74
apicoplast membranes, directed by the second part of the BTS, the transit 75
peptide (TP) (10). Based on evolutionary considerations, it has long been 76
suggested that transport across the inner two apicoplast membranes occurs 77
via a TOC/TIC- like protein translocase machinery, and this is supported by a 78
recent publication that provides evidence for an essential role of a 79
Toxoplasma gondii Tic20 homologue in this transport process (8, 11). Despite 80
this progress, it is still unclear how proteins travel across the second and third 81
outer apicoplast membranes. Several models have been discussed to 82
account for this transport step, including vesicular shuttle and translocon 83
based mechanisms (recently reviewed in: (12)), but until recently no actual 84
molecular equipment had been found which could account for these 85
membrane translocation events. To address this question, Sommer et al. 86
screened the nucleomorph genome of the chromalveolate cryptophyte 87
Guillardia theta (which, similar to P. falciparum contains a four membrane 88
bound plastid organelle) for genes encoding potential translocon related 89
proteins (13). Surprisingly, the authors identified genes encoding proteins 90
usually involved in the ER-associated protein degradation pathway (ERAD), 91
which recognises incorrectly folded protein substrates and retro-translocates 92
them to the cell cytosol for degradation by the ubiquitin-proteasome system 93
(14, 15). As such, the ERAD system functions as a translocation complex, 94
capable of transporting proteins across a biological membrane. Further 95
characterisation of one of these proteins (GtDer1-1, a homologue of yeast 96
Der1p, a component of the ERAD system) provided strong evidence for a 97
plastid localisation. These data suggested an attractive solution to the 98
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mechanistic problem of transport across the second and third outermost 99
membrane of complex plastids, by hypothesising a role for an ERAD-derived 100
protein translocon complex. Intriguingly, this study also identified several 101
members of this ERAD-derived translocon complex (apERAD) in the nuclear 102
genome of P. falciparum endowed with an N-terminal BTS (13). The BTS 103
derived from one of these proteins, PfsDer1-1, was sufficient to direct 104
transport of GFP to the apicoplast of P. falciparum, suggesting that this 105
ERAD-like machinery is ubiquitous among chromalveolates with four 106
membrane-bound plastids (13). In this current report we extend our study of 107
the P. falciparum apERAD complex. 108
109
Materials and methods 110
Bioinformatics 111
Homologues of the ERAD- pathway in apicomplexan parasites were identified 112
by Blast (16) search implemented in the Eukaryotic Pathogens Database 113
Resources (http://eupathdb.org/eupathdb/), and PlasmoDB (version 5.1 (17)). 114
Preliminary B. bovis sequence data was obtained from Washington State 115
University/USDA-ARS website 116
(http://www.vetmed.wsu.edu/research_vmp/program-in-genomics). 117
Sequences were analysed by SignalP 3.0 (18), PlasmoAP (5) and PATS (19) 118
for identification of N-terminal bipartite signals. Sequence alignments were 119
carried out using Clustal (20). Colour scheme used in figure 1 (Clustal 120
standard settings at: (http://www.ebi.ac.uk/Tools/clustalw2/index.html) ; 121
AVFPMILW, red Small (small+ hydrophobic (incl.aromatic -Y)); DE, blue 122
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(acidic); RK, magenta (basic); STYHCNGQ, green (Hydroxyl + Amine + 123
Basic); all others, grey. 124
For analysis of amino acids the the +1 positions following signal 125
peptide cleavage, predicted apicoplast and secreted non-apicoplast (signal 126
peptide containing) data sets were retrieved from PlasmoDB and subjected to 127
SignalP 3.0 analysis. Protein sequences and SignalP predictions were then 128
fed into a custom written Matlab script (available upon request to J.H.) which 129
performed in silico signal peptide cleavage, and sorting of the proteins 130
depending on the +1 amino acid (aromatic, non-aromatic). Alignments of the 131
20 amino acid sequences (FASTA format supplementary data, List S1) were 132
then prepared using Weblogo (21). 133
Trans-membrane domain prediction of all Plasmodium spp. PfDer1-1 134
sequences was carried out using PHOBIUS, TMHMM and MINNOU (22-24). 135
Amino acid sequences corresponding to predicted TM domains were 136
analysed for both length and hydrophobicity (scales: Woods (25) , Doolittle 137
(26)) and statistically analysed by Kolmogorov-Smirnov statistic (27). The KS 138
statistics regards the predicted length of the different tools for the TMDs as a 139
distribution in the host and the parasite respectively. If they differ on a 5% 140
niveau of the KS test, this means that the null hypothesis that both 141
distributions were draw from the same underlying distribution must be 142
rejected. A KS test was used because a standard distribution of the values 143
could not be assumed. 144
145
146
Expression constructs 147
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All primers used in generation of constructs are listed as supplementary data 148
in Table A2. Regions encoding the BTS of PfsUba1 (PF13_0182, bases 1 to 149
390), PfsCdc48 (PF07_0047, bases 1 to 420), PfsUb (PF08_0067 , bases 1 150
to 300) and PfDer1-2 (PFC0590c, bases 1-405) were PCR amplified from 151
genomic P. falciparum DNA, using specific oligonucleotides (xBTS_for, 152
xBTS_rev), introducing a 5´ XhoI and a 3´AvrII restriction site. The amplicons 153
were then digested with XhoI and AvrII and cloned into similarly digested 154
pARL_GFP_DHFR (28) in front of the GFPmut2 coding sequence. For 155
integration of the GFP coding sequence into the 3´ region of PF14_0498 156
(PfsDer1-1) 758 bp from the 3´ end of the gene (leaving out the stop codon) 157
were amplified from P. falciparum 3D7 gDNA using primers int_for/ int_rev, 158
introducing 5´ NotI and a 3´ KpnI restriction sites, and ligated into similarly 159
restricted pARL-GFP-DHFR (removing the CRT promotor region). For 160
integration of the GFP coding sequence into the 3’ end of PF13_0182 161
(PfsUba1), 968 bp from the 3’ end of the gene (leaving out the stop codon) 162
were amplified from P. falciparum 3D7 gDNA using primers int_for/ int_rev, 163
introducing a 5’ NotI and a 3’ KpnI restriction site, and ligated into similarly 164
restricted pARL-GFP-DHFR as above. For localisation of PfhDer1-1, the 165
entire cDNA sequence (missing stop codon) was amplified from 3D7 RNA 166
using the Superscript II 1-Step RT-PCR kit (Invitrogen) according to the 167
manufacturers protocol, using primers PfhDer1-1_F and PfhDer1-1_R. The 168
resulting PCR product was restricted with XhoI and KpnI and cloned into 169
similarly restricted pARL-GFP-DHFR. All constructs were verified by 170
automated sequencing. Plasmid DNA was isolated via the Qiagen Maxiprep 171
protocol. Regions encoding ACP_DsRed (ACP accession number: 172
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PFB0385w) and HSP_DsRed (Hsp60 accession number: PFL1545c) were 173
amplified from pSSPF2/PfACP-DsRed (kindly provided by S. Sato, (29)) using 174
ACP-F/HSP-F and DsRed-R, and cloned into the pARL-BSD vector, which 175
contains a blasticidin-s-deaminase selectable marker. 176
177
Cell culture, transfection and generation of GFP-integrant lines 178
The Plasmodium falciparum 3D7 line was cultured in human 0+ erythrocytes 179
according to standard protocols (30), except cultures were incubated in 180
gassed flasks. Transfection was carried out by electroporation of infected 181
human 0+ erythrocytes as previous described (31). GFP-transfectants were 182
selected with 5 nM WR99210 (kindly supplied by D. Jacobus) for human 183
DHFR- based vectors or 8,7 nM blasticidin for BSD based constructs. 184
Integrant parasites were selected by repeated drug cycling (3 weeks on, 3 185
weeks off), and integration checked via PCR. Positive parasite populations 186
were then cloned by limiting dilution. Integration was confirmed in each clone 187
by integration-specific PCR, (see text for details) followed by sequencing to 188
determine the exact integration site. 189
190
Immunofluorescence assays and live cell imaging 191
IFA assays were carried out following fixation using 4% Paraformaldehyde/ 192
0.00075% Glutaldehyde as previously described (32) except fixation was 193
carried out at 37°C for 30 minutes, and quenching was performed with 125 194
mM Glycine/PBS. Primary antibodies used: Rabbit anti-ACP (1:500, kindly 195
provided by Prof. Dr. GI McFadden), rabbit anti-BiP (1:2200, kindly provided 196
by Dr. T. Gilberger), chicken anti-GFP (1/1000, abcam), anti-chicken-Cy2, 197
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anti-rabbit-Cy3 (both 1/2000, DAKO) were all diluted in 3% BSA/PBS. 198
Hoechst 33258 (Molecular probes) was used in a concentration of 50 ng/ml 199
for fixed parasites or 10µg/ml for live parasites. MitoTrackerOrange (Molecular 200
Probes) was used at 20nM. All images were acquired at either 37°C (live 201
cells) or room temperature (fixed cells) on a Zeiss Cell Observer using 202
appropriate filter sets. Individual images were imported into Image J64 203
(version 1.39u, available at http://rsb.info.nih.gov/ij), converted to 8-bit 204
grayscale, subjected to background subtraction, and overlaid. Co-localisation 205
analysis was carried out using the ImageJ Plugin written by Pierre Bourdoncle 206
([email protected], available at 207
http://rsb.info.nih.gov/ij/plugins/colocalization.html). To create figures, TIF files 208
were imported into Powerpoint (Microsoft), assembled and slides exported as 209
TIFs. No gamma adjustments were applied to any images, and all data is 210
presented in accordance with the recommendations of Rossner and Yamada 211
(33). 212
213
Protein biochemistry and immunoblots 214
For membrane fractionation parasites were lysed in 50 mM Tris 2 mM EDTA 215
followed by a centrifugation step at 36000 g, 4°C for 30 min. The pellet was 216
re-suspended in 0.1M sodium carbonate buffer, 1 mM EDTA pH 11 and 217
incubated on ice for 30 min. The insoluble fraction was obtained by 218
centrifugation at 36000 g, 4°C for 30 min. Urea extraction was carried out as 219
previously described (34). High salt treatment was carried out by tris lysis (as 220
above) followed by incubation of membrane fractions in high salt buffer 221
(50mM Hepes, pH7.5, 0.6M KCl, 5mM DTT, 3mM MgCl2) on ice for 30 min 222
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after which pellet was separated from the supernatant and subjected to 223
Western Blot analysis. For triton-X-100 solubilisation, Tris lysed parasites 224
were re-suspended in different concentrations of triton-X-100/PBS. The 225
solution was incubated at 4°C overnight and centrifuged at 36000 g for 30 226
min. Protein samples were added to one volume of 2X Laemmli sample buffer 227
and boiled for 10 minutes. For Western blot analyses, protein samples were 228
prepared from saponin isolated parasites. 2x107 (4x107 for integrant lines) 229
parasite cell equivalents were separated by 10-15% SDS-PAGE and 230
transferred to nitrocellulose membrane. Primary monoclonal mouse anti-GFP 231
antibodies (Roche) were used in a concentration of 1/2000 for BTS fusion 232
proteins and 1/1000 for the integration line. Rabbit anti-Exp1 (1:500), and 233
monoclonal mouse anti-PfHsp70 (1/1000, a gift of Thierry Blisnick) have 234
previously been described (35). Anti-mouse HRP and anti-rabbit HRP (DAKO, 235
Hamburg) were used at 1/2000. Immunoblots were developed via 236
chemiluminescence using the ECL system. 237
238
Time course 239
Isolated parasites were collected from highly synchronised cultures every 6 240
hours over a period of 48 hours via saponin lysis, proteins separated via 10% 241
SDS-PAGE and transferred to a nitrocellulose membrane for subsequent 242
Western blot analysis, as detailed above. 243
244
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Results 245
246
Identification of apERAD proteins in apicomplexa 247
248
A previous bioinformatic analysis identified duplicated P. falciparum 249
homologues of ERAD components; the putative channel protein Der1p, the 250
AAA-type ATPase Cdc48, the Cdc48 co-factor Ufd1, and the E1 ubiquitin-251
activating enzymes Uba1 (13). In addition to these components, we are now 252
also able to identify further duplicated ERAD components; the E1 ubiquitin-253
activating enzyme Uba2, an E2 ubiquitin-conjugating enzyme Ubc, as well as 254
ubiquitin itself, Ub (Table 1). We hypothesised that, if an ERAD- like transport 255
system is essential for protein transport to the apicoplast, we should be able 256
to identify duplicated ERAD components in further members of plastid-bearing 257
apicomplexa. For this reason, we screened the genomic sequences of 258
Plasmodium spp., Toxoplasma gondii, Babesia bovis and Theileria parva for 259
homologues to ERAD components (36). We were able to identify duplicate 260
ERAD components in all these genomes (Table 1 and Table A3). Both ER 261
(hERAD) and apicoplast (apERAD) paralogues of Ubc4 and Ufd1 were 262
identified in all organisms, whilst other predicted proteins were less conserved 263
amongst species (Table 1 and Table A3). In Plasmodium spp. the genes 264
appear to be spread across all chromosomes, with no particular linkage 265
between the location of the ER and the respective apicoplast paralogues. All 266
genes seem to be located in perfectly syntenic regions, away from places of 267
large-scale chromosomal rearrangements or single gene insertions or 268
deletions (37). Based on regions of synteny, we are able to assign putative 269
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chromosome numbers to several of the “missing” components. Thus, a P. 270
knowlesi hUfd1 and a P. chabaudi sDer1-1 are both predicted to be encoded 271
on the respective chromosome 13 (Table 1). Ongoing assembly, gap closing 272
and analysis of these genomes is likely to reveal these genes. 273
In addition to the duplicated ERAD components mentioned, we were 274
also able to identify further components of the hERAD system (Hrd1, Hrd3, 275
Npl4, Table 1, Table A3). Npl4 is present in only a single copy, and is thus 276
unlikely to be involved in apERAD transport processes. Hrd3 was found to be 277
encoded twice in the genome, however neither copy is predicted to encode a 278
BTS, also negating a role in the apERAD system (Table 1 and Table A3). 279
Many, but not all, of the apERAD homologues encode a BTS, 280
suggesting that they are transported to the apicoplast. In several cases where 281
a BTS appears to be missing, closer inspection of the sequences reveals that 282
these protein sequences are incomplete, and do not include the entire N-283
terminal part of the protein (no initiation methionine, Table 1). In addition, 284
several proteins are predicted to possess an N-terminal TP, but are missing 285
an ER- type signal sequence, essential for entry into the secretory system 286
(Table 1). Prediction of intron/exon boundaries is notoriously challenging in 287
the P. falciparum system (38), and a plausible explanation for the lack of a 288
signal peptide suggests that the “missing” N-terminal signal can be found on a 289
5’ exon which has not been included in the gene model. Indeed, based on 290
cDNA sequences, we have previously re-annotated the gene model for 291
PFI0810c (encoding PfsUfd1), to include a signal peptide encoded on a 292
previously “missed” 5’ exon (13). Coding of BTS on “extra” exons is common 293
in many organisms, and may reflect their evolutionary history (39). An 294
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additional reason for failing to identify a recognisable BTS in homologues from 295
all organisms is that current software designed to predict apicoplast transit 296
peptides have been trained on P. falciparum proteins, and may therefore not 297
be able to consistently predict transit peptides of B. bovis or T. parva proteins 298
(19). Additionally, based on cDNA sequencing we have now re-annotated the 299
gene PF14_0653, encoding PfhDer1-1 (Genbank: FJ555561, Table 1). 300
Earlier studies have highlighted the importance of a DnaK (Hsp70) 301
binding site within P. falciparum transit peptides in high fidelity protein traffic to 302
the apicoplast (5), suggesting that binding of Hsp70 at the trans-side of the 303
target membrane plays a role in membrane translocation. Gould et al. and 304
Sommer at al. additionally identified putative periplastid resident Hsp70 305
proteins in Phaeodactylum tricornutum, respectively Guillardia theta (40). 306
During the course of our bioinformatic studies, we also identified a member of 307
the Hsp70 family predicted to have a BTS (encoded by Mal13P1.540). It 308
appears unlikely that this protein is a bona fide apicoplast protein as the 309
region predicted to be a transit peptide overlaps with the Hsp70 ATPase 310
domain, and the protein contains a C-terminal –KDEL ER retrieval sequence, 311
features not contained in the P. tricornutum or G. theta proteins. 312
In further support of our analyses, we also screened the genome of 313
Cryptosporidium parvum for ERAD components (36, 41). C. parvum, whilst 314
phylogenetically belonging to the phylum apicomplexa, no longer contains an 315
apicoplast, which appears to have been “lost” during the course of evolution 316
(42). Indeed, although we are able to identify many components of the 317
hERAD system (Table A3), we cannot find duplications of these genes, nor 318
predicted proteins with n-terminal extensions containing a BTS, further 319
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supporting a role for apERAD components exclusively in apicoplast related 320
processes. 321
322
PfsDer1-1, encoded by PF14_0498, is a structural orthologue of yeast Der1p 323
324
In yeast, Der1p is a central component of the ERAD system (43). This 325
approximately 25 kDa protein contains four membrane-crossing segments, 326
the first of which is predicted to act as a signal anchor, both recruiting the 327
protein to the ER and initiating its integration into the membrane (44). Der1p 328
integrates into membranes with both N- and C-termini exposed to the cytosol 329
(44). P. falciparum apicoplast Der1-1 (PfsDer1-1), shows 16% identity, 42% 330
similarity to yeast Der1p, and is predicted to contain a Der1- like domain 331
(PFAM: PF04511). Additionally, predicted membrane-crossing segments align 332
well between both sequences (Fig. 1A), and in all Plasmodium sDer1-1 and 333
hDer1-1 (Fig. 1A). These data support the hypothesis that PF14_0498 334
encodes a structural orthologue of yeast Der1p. 335
336
The N-terminus of apERAD homologues targets GFP to the apicoplast 337
338
We have previously demonstrated that the N-terminal region of PfsDer1-1 339
efficiently targets GFP to an intra-parasitic compartment suggested to 340
represent the apicoplast (13). We were interested in investigating whether 341
further predicted BTS derived from apERAD components are capable of 342
directing reporter protein transport to this organelle. We therefore generated 343
transfectant parasite lines expressing GFP fused C-terminally to predicted 344
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BTS derived from PfsCdc48, PfsDer1-2, PfsUba1, and PfsUb (referred to as 345
3D7PfsD1-2_BTS, 3D7PfsCcd48_BTS, 3D7PfsUba1_BTS and 3D7PfsUb_BTS respectively, 346
Fig. 2A). Live-cell imaging reveals that BTS derived from all four proteins are 347
sufficient to target GFP to a punctate structure within the parasite (Fig. 2B), 348
generally in apposition to both the mitochondrion and nucleus, suggestive of 349
an apicoplast localisation (45). To verify this, we carried out 350
immunofluoescence assays using antibodies against the acyl carrier protein 351
(ACP), an apicoplast marker (a kind gift of Geoff McFadden, University of 352
Melbourne). In all parasite lines, we observe co-localisation between the GFP 353
and ACP signals, confirming an apicoplast localisation (Fig. 2C). 354
Upon import to the apicoplast, transit peptides are generally cleaved by 355
a transit peptide pepidase (46). Western blot analysis of our transfectant lines 356
using anti-GFP antibodies reveals a multiple banded pattern. These bands 357
can, on the basis of their molecular weight, be assigned to either the full-358
length chimeric pre-protein with an uncleaved transit peptide (Fig. 3, asterix), 359
a lower molecular weight species probably representing the chimera after 360
transit peptide cleavage (Fig. 3, diamond), and a previously described 361
degradation product running at the size of GFP alone (Fig. 3, arrow) (9). The 362
relative size shift between pre-protein and mature protein varies between 363
BTS-GFP reporters. Transit peptides are known to vary greatly in length (20-364
100 amino acids (36)), and this result indicates that pre-PfsCdc48 and pre-365
PfsUba1 contain longer transit peptides than the other apERAD components 366
investigated here. 367
Taken together, these results show that predicted BTS derived from 368
PfsDer1-2, PfsCdc48, PfsUba1, and PfsUb are able to target GFP to the 369
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apicoplast, and suggest that their function is to mediate delivery of the full-370
length proteins to this compartment. 371
372
Full-length PfsDer1-1 and PfsUba1 localise to the apicoplast, whereas 373
PfhDer1-1 is an ER resident protein 374
375
Our preliminary results, based on bioinformatic sequence analyses and BTS-376
GFP reporters, strongly suggest that apERAD components localise to the 377
apicoplast, however we wished to verify that full-length gene products are also 378
present at the same location. Initially we attempted to fuse the full-length 379
gene encoding PfsDer1-1 to the GFP coding sequence, and express the 380
chimeric reporter from episomally maintained plasmids. Despite repeated 381
attempts at transfection, we were unable to obtain a drug-resistant population, 382
suggesting that expression of the reporter protein under these conditions is 383
toxic to the parasite, possibly due to incorrect levels/timing of expression. For 384
this reason, we decided to engineer the endogenous gene locus by single 385
crossover homologous recombination, incorporating the in-frame GFP coding 386
sequence into the 3’ end of the respective gene (shown schematically in Fig. 387
A4). After transfection, selection of drug-resistant parasites and drug cycling 388
to select for integration, we were able to isolate clonal parasite populations 389
that had integrated the transfection vector into their genome. To verify 390
integration into the correct gene loci, we carried out integration specific PCR 391
followed by sequencing of the PCR products and Southern Blot (S4B, data 392
not shown). In these parasites, expression of the PfsDer1-1-GFP or PfsUba1-393
GFP fusion protein is under control of the endogenous promoter. 394
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We analysed our clonal integrant parasite lines 3D7PfsD1-1 and 395
3D7PfsUba1 by Western blotting. In the case of 3D7PfsD1-1 , in addition to a band 396
migrating at the size of the mature, processed PfsDer1-1-GFP chimera (50 397
kDa), several higher molecular weight bands could be detected, probably 398
representing transit peptide cleavage intermediates (Fig. 4B). Analysis of 399
3D7PfsUba1 revealed a single >150kDa band, which was absent in the parental 400
3D7 strain, in agreement with the predicted molecular weight of the GFP-401
tagged chimeric protein. Cleavage intermediates could not be visualised, most 402
likely due to poor separation at this high molecular weight. The general 403
expression level of both proteins is low, and required us to load approximately 404
four times as many parasite cell equivalents (4x107 compared to 1x107) as is 405
usual in our laboratory, in order to obtain signals of sufficient strength for 406
analysis. 407
Epifluorescence microscopy of 3D7PfsD1-1 and 3D7PfsUba1 revealed only 408
a weak GFP signal, consistent with the low protein abundance noted above. 409
As sub-cellular localisation of the GFP-chimera by live-cell imaging was 410
limited by detection sensitivity, we carried out IFA using antibodies against 411
GFP and ACP. In both 3D7PfsD1-1 and 3D7PfsUba1 lines, GFP and ACP signals 412
co-localise (Fig. 4C), suggesting that both PfsDer1-1-GFP and PfsUba1-GFP 413
are resident apicoplast proteins. As a control, we expressed PfhDer1-1, fused 414
to GFP, from episomally maintained plasmids, generating 3D7PfsD1-1. 415
Fluorescence microscopy of this parasite line reveals GFP fluorecence in a 416
“perinuclear ring” structure, indicative of an ER localisation. 417
Immunofluorescent co-localisation using antibodies recognising the ER 418
marker protein BiP (a kind gift of Dr. T. Gilberger, Fig. 4C, bottom) 419
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substantiates this, with good co-localisation of GFP and BiP signals (Fig. 4C, 420
bottom). As an additional control, we co-transfected 3D7PfsD1-1 with a construct 421
encoding either the BTS of the apicoplast resident protein ACP or the 422
mitochondrial targeting sequence of mitochondrial PfHsp60, fused to the red 423
fluorescent protein DsRed. In fixed cells co-labelled with anti-GFP antibodies, 424
ACP-DsRed and GFP signals co-localise, whilst HSP-DsRed and GFP signal 425
do not (Fig. 4D). These data further support our hypothesis that the full-length 426
PfsDer1-1-GFP fusion protein is transported to the apicoplast, whereas 427
PfhDer1-1 is a bona fide ER resident protein.. 428
429
PfsDer1-1 tightly associates with apicoplast membranes 430
431
To function as part of a protein conducting channel for the translocation of 432
pre-proteins, PfsDer1-1 is expected to integrate into biological membranes. 433
We therefore investigated the membrane association of our PfsDer1-1-GFP 434
fusion construct. To this end, we carried out sequential membrane 435
fractionation and extraction on 3D7PfsD1-1 infected erythrocytes, followed by 436
Western blotting with anti-GFP antibodies. As a control for the fractionation 437
and extraction protocol, we also analysed the distribution of PfHsp70 (a 438
soluble protein of the parasite cytosol) and PfExp1 (a single spanning trans-439
membrane protein of the parasitophorous vacuolar membrane (35)). As 440
expected, PfHsp70 was found only in the soluble fraction following hypotonic 441
lysis with 50 mM Tris (pH 7.4) (Fig. 5A, STris), and PfExp1 largely in the 442
carbonate insoluble fraction (Fig. 5A, P) with a weak signal in the supernatant 443
following carbonate extraction, as previously described (47). GFP signals 444
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could be detected in only the final carbonate insoluble fraction, consistent with 445
a strong association of PfsDer1-1-GFP with a lipid bilayer (Fig. 5A, P). 446
Similarily, GFP signal could only be detected in the pellet fraction following 447
membrane extraction with urea (Fig. 5A., P), with control proteins PfHsp70 448
and PfExp1 present in the tris soluble fraction (Fig. 5A., STris), and final 449
membrane pellet fraction (Fig. 5A., P) respectively, as expected and 450
previously described (34). Upon treatment with increasing concentrations of 451
the detergent triton-X-100, the control protein PfExp1 could be easily 452
extracted from the membrane fraction (Fig. 5B, S), whereas PfsDer1-1-GFP 453
was highly resistant to solubilisation (Fig. 5B, P). Taken together, these data 454
verify that PfsDer1-1 is tightly associated with apicoplast membranes, and is 455
probably an integral membrane protein of this organelle. 456
457
PfsDer1-1 is expressed throughout the intra-erythrocytic life cycle 458
According to the study of Le Roch et al., mRNA abundance of genes 459
encoding PfapERAD components, including PfsDer1-1, increases 460
dramatically during the late trophozoite and early schizont stages of the 461
parasites intra-erythrocytic cycle (48). To investigate whether these mRNA 462
expression profiles correlated with protein abundance, we tightly synchronised 463
3D7PfsD1-1 parasite cultures using sorbitol (49), and removed samples for 464
analysis every six hours. Using anti-GFP antibodies, we could observe that 465
the protein appears to be present throughout the entire intra-erythrocytic 466
lifecycle (Fig. 5B), but that protein abundance increases throughout the ~48 467
hour cycle, with the highest levels of protein being found in late trophozoites 468
and early schizont stage parasites, consistent with the higher mRNA 469
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abundance during these stages, suggesting regulation at the transcriptional 470
level. It appears that highest expression of PfsDer1-1 takes place late in the 471
parasites developmental cycle, correlating well with the time period in which 472
both nuclear and apicoplast division is taking place (45). 473
474
Apicoplast targeting signals in P. falciparum proteins appear divergent from 475
those in most other chromalveolates 476
477
The study by Sommer et al. (13) leads us to predict that apERAD components 478
are situated either in the second outermost membrane itself (membrane 479
bound components such as PfsDer1-1), or in the space between the second 480
and third apicoplast membrane referred to as the periplastidic compartment 481
(PPC, soluble components such as PfsCdc48). This entails that, in contrast to 482
enzymes involved in biochemical pathways in the apicoplast stroma, apERAD 483
components only need to be targeted across two of the four membranes 484
surrounding the apicoplast. As the small size of the apicoplast, and diffraction 485
limits associated with light microscopy do not allow us to verify such a 486
localisation via light microscopy, we asked if bioinformatic analyses may 487
provide support for a PPC localisation. Previous studies on protein transport 488
to complex plastids of red algal origin have shown that differential sorting to 489
the plastid stroma (across four membranes), or the PPC (across only two 490
membranes) is directed by the amino acid present in the +1 position following 491
signal peptide cleavage. Proteins required to cross all four membranes 492
usually possess an aromatic amino acid, or leucine, at this position whereas 493
those localising to the PPC do not (12, 40, 50, 51). To investigate whether we 494
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could predict the localisation of PfapERAD components based on these 495
criteria, we analysed the amino acid residue at the +1 position following in 496
silico signal peptide cleavage. This analysis revealed that none of the proteins 497
studied is predicted to expose an aromatic amino acid at the N-terminus of the 498
transit peptide, and only one protein (PfsUba) exposes a leucine residue at 499
this point (Fig. A5). As a comparison, we also analysed the +1 amino acid of 500
sERAD components encoded by Phaeodactylum tricornutum, a diatom algae 501
that also contains a 4-membrane bound plastid. None of the P. tricornutum 502
sequences reveals an aromatic amino acid (or leucine) at the +1 position (Fig. 503
A5). In itself, this result is suggestive of a predicted PPC localisation for 504
PfapERAD proteins, however a larger bioinformatic analysis encompassing 505
the predicted +1 amino acids of 194 predicted apicoplast proteins reveals that 506
only 56 (29%) of these proteins do in actual fact obey the “+1 rule” (Fig. A5, 507
upper panel). As a negative bioinfomatic control, we analysed the +1 amino 508
acid of 277 P. falciparum proteins predicted to contain an N-terminal ER type 509
signal sequence, but no transit peptide. Of these 277 proteins, 67 (24%) 510
contain an aromatic amino acid, or leucine at the +1 site (Fig. A5, lower 511
panel). These data suggest that the “+1 rule” does not apply to P. falciparum 512
apicoplast targeted proteins, and as such, cannot be used as a tool to predict 513
a definitive localisation of P. falciparum proteins to either the PPC or 514
apicoplast stroma. 515
516
Predicted trans-membrane domains of sDer1-1 show unusual features 517
518
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To function as a protein conducting channel sDer1-1 must firstly co-519
translationally enter the secretory pathway before being carried to, and 520
inserting into, an internal apicoplast membrane. This is an unusual situation, 521
as it implies that a highly hydrophobic trans-membrane (TM) protein must 522
potentially cross several membranes before reaching its site of action. 523
Although transport of proteins destined to post-translationally insert into 524
membranes is poorly understood, several studies have revealed unusual 525
properties of predicted TM domains in proteins trafficked in this manner (52-526
54). For this reason, we performed a statistical comparison of the length 527
distribution of the predicted membrane spanning regions of all Plasmodium 528
Der1-1 orthologues (n=3sDer1-1 + 3hDer1-1). A Kolmogorov-Smirnov statistic 529
(27) reveals that the length of the first and the third TM domains differ 530
significantly (5% level) between ER and apicoplast copies (TM1: sDer1-1 19.3 531
+/- 0.7, hDer1-1 21.2 +/- 2.2; TM3: sDer1-1 19.8 +/- 5.4, hDer1-1 23.7 +/- 5.4. 532
Table A6). 533
534
535
Discussion 536
Here we report the identification and initial characterisation of an ERAD-537
derived potential pre-protein translocon complex within the apicoplast of the 538
human malaria parasite P. falciparum, referred to as apERAD. Utilising a 539
bioinformatic approach, we are able to show that all plastid-bearing 540
apicomplexan parasites so far studied encode an apERAD. Furthermore, 541
using transfection technology paired with protein biochemistry, we are able to 542
demonstrate that the predicted BTS derived from several apERAD 543
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homologues are sufficient to target GFP to the apicoplast. Additionally, as 544
proof-of-principle, we localised PfsDer1-1 and PfsUba1 (homologues of yeast 545
ERAD components) to the apicoplast. Taken together, these data are 546
consistent with a role for PfsDer1-1 in import of nucleus-encoded pre-proteins 547
to the apicoplast. 548
Although several models have been suggested to account for the 549
trafficking of nucleus-encoded pre-protein to the complex plastid of 550
chromalveolates containing four membrane bound plastids, until recently no 551
molecular machinery had been identified that could actually carry out such 552
transport processes (recently reviewed in (8)). Numerous early studies of 553
protein translocation across biological membranes revealed several criteria 554
which must be fulfilled by protein conducting channels (PCC) and their 555
associated factors, including the creation of a hydrophilic pore within the 556
target membrane, the means to distinguish substrate proteins, and the 557
necessity for a “driving force” to either pull, or push polypeptide chains 558
through the channel (55-57). The discovery of duplicated ERAD-derived 559
systems in chromalveolates thus provided an attractive solution to the 560
mechanistic problem, as an ERAD- based protein translocon would be able to 561
provide a hydrophilic pore across the membrane (based around sDer1-1, 562
possibly together with sDer1-2), and the necessary pulling force for passage 563
through the pore (provided by associated factors including sCdc48). 564
Integration of the GFP-coding sequence into the PfsDer1-1 and 565
PfsUba1 gene loci allowed us to localise these proteins by fluorescence 566
microscopy. Despite low endogenous expression levels of the reporter 567
protein, we were able to show that these proteins co-localise with the 568
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apicoplast marker ACP. Due to the small size of the apicoplast and diffraction 569
limits associated with light microscopy, we were not able to directly assign the 570
proteins to a particular sub-compartment of the apicoplast. Based on studies 571
of an ERAD-derived translocon in chromalveolate algae (13), and the diatom 572
algae P. tricornutum (58) we would suggest that PfsDer1-1 inserts into the 573
second outermost apicoplast membrane. Although we cannot formally 574
discount the possibility that PfapERAD components are transported to the 575
third outer, or indeed inner apicoplast membrane, this would appear unlikely 576
for several reasons. If PfsDer1-1 is involved in protein transport processes, 577
insertion in the third outer membrane would result in a topology incongruous 578
with transport into the apicoplast, requiring a reversal of the transport 579
direction, a situation for which no precedent exists (13). Additionally, a recent 580
study in T. gondii has convincingly demonstrated a role for TgTic20 in protein 581
transport to the apicomplexan apicoplast, a situation which is likely to also 582
hold true for P. falciparum, thus making a role for PfsDer1-1 in this process 583
unlikely. Future studies will aim to experimentally address the exact 584
localisation of this protein, possibly by using novel cell biological tools such as 585
self-assembling GFP (59). 586
PfsDer1-1 is extremely tightly associated with membranes, as 587
evidenced by membrane extraction experiments. Unexpectedly, we were 588
unable to solubilise PfsDer1-1 even under harsh (2% Triton-X-100) 589
conditions. Although insolubility in high Triton-X-100 concentrations may 590
seem indicative of an association with cytoskeletal components, there is no 591
evidence for such structures in the apicoplast. Analysis of further apicoplast 592
membrane proteins may shed light on this unusual solubility profile 593
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In yeast, Cdc48 mediated translocation through the ERAD translocon is 594
driven by ubiquitinylation of substrate proteins upon emergence from the 595
translocon (14, 60). In the course of this present study, we identified a 596
ubiquitin predicted to be endowed with a BTS for transport to the apicoplast 597
(PfsUb). The BTS derived from this protein was able to target GFP to the 598
apicoplast. PfsUb contains the essential K48 and K63 residues required for its 599
involvement in both mono-, and poly-ubiquitinylation of substrate proteins 600
(Fig. A7). We were not able to categorically identify high-molecular weight 601
ubiquitinylated forms of our GFP reporter proteins, suggesting that if 602
apicoplast targeted proteins are indeed ubiquitinylated during their trafficking, 603
either ubiquitin is subsequently cleaved from the proteins, or that the transit 604
peptide (a possible site of ubiquitinylation) is cleaved together with the 605
ubiquitin moiety from the mature protein. The latter situation would seem 606
unlikely, given that the transit peptide is required for further membrane 607
passage events. Whilst the exact molecular details remain to be dissected, 608
our data support a role for PfsUb in ubiquitinylation of apicoplast targeted pre-609
proteins. Possibly, ubiquitinylation of transit peptides emerging from the trans- 610
side of the translocon acts as a trigger for PfsCdc48 driven membrane 611
translocation. It is noteworthy that we failed to identify apERAD versions of 612
the ubiquitin ligases PfHrd1 or PfHrd3. Ubiquitin ligase-independent 613
monoubiquitinylation of substrate proteins has previously been described (61), 614
and the possibility exists that the reduced apERAD system instead relies on 615
ubiquitin conjugating (E2) enzymes to transfer ubiquitin to substrate proteins. 616
Alternatively, it is feasible that one of the two independent copies of PfHrd3 617
we identified is transported to the apicoplast, albeit in a manner which does 618
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not require a recognisable transit peptide. Such a mechanisms has been 619
demonstrated for the delivery of FtsH to membranes of the Toxoplasma gondii 620
apicoplast (62). 621
One observation which is of particular interest is that transit peptides 622
derived from apERAD components also appear to be cleaved upon organellar 623
import (Fig. 3). Transit peptide cleavage has previously been observed in pre-624
proteins transported to the lumen of the apicoplast, and a putative stromal-625
processing peptidase has been identified (46). Processing of PPC localised 626
plastid pre-proteins has previously been observed in Phaeodactylum 627
tricornutum, and was taken as evidence to suggest the existence of a 628
periplastid-processing peptidase (40) . Our results further support a model in 629
which pre-proteins destined for the PPC are processed by an as yet 630
unidentified protease. 631
In the course of our study, we also investigated whether it is possible to 632
distinguish between PPC and stromal apicoplast proteins, based on the 633
physiochemical properties of the amino acid residue exposed after signal 634
peptide cleavage. We find that, in contrast to several other systems studied, 635
there is no significant difference at the +1 position between proteins trafficked 636
across all four apicoplast membranes, and those that must only passage 637
across the outer two. Indeed, there is no significant difference between the +1 638
residue in apicoplast proteins and that of other secretory proteins. 639
Bioinformatic studies suggest that the phenyalanine motif is of ancestral origin 640
(shared in the common ancestor of the green and red lineages), and has 641
subsequently been lost in members of the green line. Additionally, transit 642
peptides derived from haptophyte algae (red lineage) also have a relaxed 643
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requirement for phenyalanine at this position (51). Our data suggest that, at 644
least at the level of the +1 position, Plasmodium falciparum transit peptides 645
appear to share some properties with those of haptophytes. As a logical 646
consequence of this, this result also suggests that P. falciparum apicoplast 647
proteins (which do not obey the +1 rule) are differentially sorted to either PPC 648
or plastid stroma based on sequence information in the downstream protein 649
sequence. Further studies will be required to experimentally verify this 650
hypothesis, as well as elucidate its significance for the nature of the apicoplast 651
protein import system. 652
In yeast, ERAD substrates are generally recognised on the basis of 653
distinct n-glycan modifications (63). Previous studies have determined that P. 654
falciparum has a low, if any capacity for n-glycosylation (64) suggesting that, 655
even for the hERAD system, the signal required for ERAD recognition could 656
differ to that common in yeast. As a result, how proteins are recognised by the 657
apERAD system must also remain a matter of speculation at this point. What 658
does appear clear is that, as all proteins trafficked to the apicoplast must first 659
enter the parasite’s ER, recognition of apERAD substrates must take place 660
via signals distinct from that for hERAD. Likewise, we failed, in all organisms 661
studied, to identify apERAD homologues of Npl4p, a protein which, in the 662
yeast system, usually interacts with Cdc48p and Ufd1p. This Cdc48p-Ufd1p-663
Nlp4p is involved in recognition of polyubiquitinylated ERAD substrate 664
proteins. A recent study of sERAD in the diatom P. tricornutum suggests that 665
sDer1 itself is able to distinguish substrates (58). Thus PfsDer1 itself may play 666
a role in substrate recognition. It is clear that further studies will be required to 667
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dissect the exact sequence and/or substrate recognition requirements for this 668
translocation process. 669
To conclude, our data provide strong evidence for the presence, in the 670
apicoplast of the malaria parasite P. falciparum and of other apicomplexans, 671
of components of an ERAD-derived translocon complex. Specifically, we can 672
show that PfsDer1-1 and PfsUba1 are localised to the apicoplast, and that the 673
BTS derived from further sERAD components are also capable of targeting 674
reporters to the apicoplast, suggesting that these proteins also fulfil their 675
biological function in this compartment. This translocon complex is likely to be 676
required for the import of nucleus-encoded pre-proteins to the organelle. 677
Exactly how these components function in a co-ordinated fashion to allow 678
passage of proteins across the multiple membranes of the apicoplast remains 679
to be studied in detail, but will provide the basis for future research efforts. 680
The unusual intra-cellular lifecycle of the malaria parasite has already 681
revealed several novel cell biological phenomena, and our study suggests 682
that P. falciparum still has many tricks up its sleeve. 683
684
Acknowledgements 685
This work was supported by a PhD fellowship of the Philipps University 686
Marburg (S.S.), the International Max-Planck Research School (C.T.), DFG 687
grant PR1099/2-1 (J.M.P), and a long-term fellowship of the European 688
Molecular Biology Organization (T.W.A.K., ALTF-763-2006). We wish to 689
especially thank Thierry Blisnick, Lars Bullmann, Franziska Hempel, Ming 690
Kalanon, Simone Külzer, Christof Taxis, Klaus Lingelbach, Geoff McFadden, 691
and our co-workers at the Marburg University Hospital blood bank for 692
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essential reagents and fruitful discussions. We would like to especially 693
acknowledge the help and advice of the entire PlasmoDB team. 694
695
696
697
698
699
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P. falciparum P. vivax P. yoelii P. knowlesi P. berghei P. chabaudi
ER Apicoplast ER Apicoplast ER Apicoplast ER Apicoplast ER Apicoplast ER Apicoplast
Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch. Acc. No. Ch.
Cdc48 PFF0940c/ Mal8P1.92
6 PF07_0047 7 PVX_114095 11 PVX_0880852 1 PY03639 11 PY057872, 3 12 PKH_113000 11 PKH_010920 1 PB000171.02.0
11 PB000404.03.03
12 PC000445.00.0
11 PC000038.00.01, 2, 3
12
Der1-1 PF14_0653§ 14 PF14_0498 14 PVX_117040 12 PVX_117865 12 PY028701 13 PY06810 13 PKH_123690 12 PKH_125400 12 PB000599.01.01
13 PB000620.00.01, 2
13 PC301995.00.01
13 - 13†
Der1-2 PF10_0317 10 PFC0590c1 3 PVX_111100 6 PVX_1198101 8 PY03142 5 PY022832 6 PKH_061690 6 PKH_0825801 8 PB001618.02.0
5 PB301046.00.01, 2
4 PC000384.05.0
5 PC300480.00.01, 2, 3
4
Hrd1* PF14_0215 14 - - PVX_085355 13 - - PY000251, 2 10 - - PKH_132660 13 - - PB001204.02.0
10 - -
PC000714.01.01, 2 / PC000098.01..01
10 - -
Hrd3$ PFC0550w/ PF14_0462
3/14 - - PVX_118065/PVX_119750
12/8 - - PY04510/PYO51121
4/13 - - PKH_125790/PKH_082700
12/8 - - PB000257.00.01/PB000807.02.0
4/13 - - PC300752.00.01/PC000360.02.01
4/13 - -
Npl4 PFE0380c 5 - - PVX_097945 10 - - PY05126 11 - - PKH_102520 10 - - PB001631.02.0
11 - - PC000343.04.0
11 - -
Ub PFL0585w 12 PF08_0067 8 PVX_084620 13 PVX_0896203 5 PY039711 6 PY005392 7 PKH_131070 13 PKH_051680 5 PB000763.03.0
6 PB000118.00.01
7 PC000735.00.0
6 PC300085.00.0
7
Uba1* PFL1245w 12 PF13_0182 13 PVX_123920 14 PVX_082590 12 PY01879 14 PY01851 / PY064131, 2
13 PKH_144260 14 PKH_121970 12 PB000204.03.01
14 PB000355.02.01, 2
13 PC001230.02.01
14 PC000262.00.0
13
Uba2 PFL1790w 12 PF13_0344 13 PVX_100800 14 PVX_115230 11 PY05539 14 PY028462, 3 11 PKH_145560 14 PKH_110530 11 PB000980.01.0
14 PB301007.00.0
11 PC000817.00.0
14 PC000463.02.02, 3
11
Ubc PFL0190w 12 Mal13P1.227 13 PVX_084235 13 Pv0831752 12 PY030251 4/ 6 PY00590 13 PKH_130250 13 PKH_120890 12 PB000336.03.01
6 PB000746.02.01, 2
13 PC000554.00.0
6 PC000271.02.01, 2
13
Ufd1* PF14_0178 14 PFI0810c1 9 PVX_085555 13 PVX_099250 7 PY016401/PY016411/PY016421
10 PY045761, 2 8 - 13† PKH_071380 7 PB000251.00.0
10 PB001013.03.01, 2, 3
8
PC105816.00.01/ PC000154.03.01
10 PC000751.03.01, 2, 3
8
§Revised. Genbank: FJ555561,
1Gene model incomplete/ lacking initiation methionine,
2 No signal peptide,
3No transit peptide,
† Gene not detected, chromosomal location inferred from synteny, - Not detected,
$ Gene duplication, * Multiple PlasmoDB entries refer to only one actual gene.
Spork et al. Table 1.
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