Distribution of the SELMA translocon in secondary plastids of red algal origin and predicted ...

36 Pages·2012·2.43 MB·English

EC Accepts, published online ahead of print on 5 October 2012
Eukaryotic Cell doi:10.1128/EC.00183-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 Distribution of the SELMA translocon in secondary plastids of red algal origin
2 and predicted uncoupling of ubiquitin-dependent translocation from degradation
3 Simone Stork*1, Daniel Moog*1, Jude M. Przyborski2, Ilka Wilhelmi1’, Stefan Zauner1
4 and Uwe G. Maier#1,3
5 1Laboratory for Cell Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, D-
6 35032 Marburg, Germany. D
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7 2Laboratory for Parasitology, Philipps-University Marburg, Karl-von-Frisch Str. 8, D- lo
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8 35032 Marburg, Germany e
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9 3LOEWE-Zentrum für Synthetische Mikrobiologie (SynMikro), Hans-Meerwein-Straße, m

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10 D-35032 Marburg, Germany p
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11 *These authors contributed equally. a
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12 ‘Present address: Institute for Molecular Tumor Biology and Cancer Gene Therapy, rg
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13 Philipps-University Marburg, Emil-Mannkopff-Str. 2, D-35032 Marburg, Germany. n

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14 #E-mail: [email protected] il
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15 Running title: SELMA - uncoupling translocation from degradation 1
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16 Abstract
17 Protein import into complex plastids of red algal origin is a multistep process including
18 translocons of different evolutionary origin. The symbiont-derived ERAD-like machinery
19 (SELMA), shown to be of red algal origin, is proposed to be the transport system for
20 preprotein import across the periplastidal membrane of heterokontophytes,
21 haptophytes, cryptophytes and apicomplexans. In contrast to the canonical endoplasmic
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22 reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to w
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23 be uncoupled from proteasomal degradation. We investigated the distribution of known a
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24 and newly identified SELMA components in organisms with complex plastids of red d

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25 algal origin by intensive data mining, thereby defining a set of core components present m

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26 in all examined organisms. These include putative pore-forming components, a tp
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27 ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known c
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28 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was m
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29 expanded. These newly identified putative SELMA components as well as proteasomal g
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30 subunits were in vivo-localized as PPC proteins in the diatom Phaeodactylum
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31 tricornutum. The presented data allows us to speculate about the specific features of il
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32 SELMA translocation in contrast to the canonical ERAD system, especially the 2
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33 uncoupling of translocation from degradation.
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34 Introduction
35 Organelles like plastids, including those of secondary origin, almost completely rely on
36 protein import from the host cytosol (46, 65). The structure of complex plastids,
37 surrounded by three or four membranes required, in contrast to primary plastids, the
38 evolution of several additional protein transport mechanisms. Complex plastids arose
39 through secondary endosymbiosis, a process which describes the engulfment of a
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40 former free-living eukaryotic alga into a eukaryotic host cell (32-33). During evolution, w
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41 the symbiont was subsequently reduced in terms of compartmentalization and genome a
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42 size to an organelle strictly dependent on the host cell (16, 32). Different types of d

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43 secondary plastids exist in a very broad range of algae and protists, which can be m

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44 distinguished based on their evolutionary origin (e.g. a red or green alga derived tp
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45 symbiont), as well as on the amount of cellular reduction inside the host cell. Our c
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46 understanding of the evolution of organisms harboring a secondary plastid of red algal m
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47 origin has changed in the last few years. According to the chromalveolate hypothesis, g
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48 six major lineages were grouped together to be of monophyletic origin: cryptophytes,
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49 haptophytes, heterokontophytes, peridinin-containing dinoflagellates, apicomplexans il
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50 and the non-plastid containing ciliates, as well as several smaller lineages related to 2
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51 some chromalveolate members (15, 41). However, recent phylogenetic analyses have
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52 given rise to extended theories about the evolution of the lineages with a red algal g
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53 endosymbiont, including serial endosymbiotic events with secondary, as well as tertiary, t
54 endosymbioses (21-22, 26-27, 56, 61, 71, 75).
55 It has been shown that the lineages with an endosymbiont of red algal origin share
56 common plastid protein import mechanisms despite remarkable differences resulting
57 from specific features in plastid ultrastructure (10, 37, 65). Import into complex plastids
58 starts co-translationally at the endoplasmatic reticulum (ER) membrane where nascent
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59 precursor proteins are synthesized into the ER lumen. This transport step requires a
60 canonical N-terminal signal peptide (SP). In heterokontophytes, cryptophytes and
61 haptophytes, the outermost plastid membrane, termed chloroplast ER (cER) membrane,
62 is continuous with the endomembrane system of the host cell; therefore, the Sec61
63 mediated import already represents transport across the first membrane of complex
64 plastids. In contrast, the plastids of apicomplexans and peridinin-containing
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65 dinoflagellates are not connected to the endomembrane system. Thus, after import into o
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66 the ER lumen, proteins are likely to be transported to the plastid via vesicle transport lo
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67 mechanisms directly from the ER or via the Golgi apparatus (47, 57). e
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68 After the preprotein has entered the cER lumen, the SP is thought to be cleaved off and m

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69 a transit peptide-like sequence (TPL) is exposed at the new N-terminus. The TPL is tp
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70 required for further transport into the periplastidal compartment (PPC), which resembles c
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71 the naturally reduced cytoplasm of the endosymbiont, and further into the stroma of the m
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72 plastid. Such transit peptide-like sequences thereby fulfill an additional function in g
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73 contrast to transit peptides (TP) in primary plastids. Detailed characterization of the TPL
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74 revealed a difference between stroma- and PPC-localized proteins. Stromal proteins il
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75 possess an aromatic (Phe, Tyr, Trp) or bulky (Leu) amino acid at the +1 position of their 2
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76 TPL, in contrast to PPC proteins (4, 31, 34, 42). However, the observed AXA-FAP motif
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77 at the transition between SP and TPL of stromal proteins is not as well conserved in g
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78 haptophytes, apicomplexans and dinoflagellates as it is the case for heterokontophytes t
79 and cryptophytes (58). Furthermore, some membrane proteins of apicomplexan plastids
80 (apicoplasts) seem to carry intrinsic targeting signals instead of a bipartite targeting
81 signal (BTS) consisting of SP and TPL (1, 20).
82 For transport across the second outermost membrane, the periplastidal membrane
83 (PPM), a translocon model was proposed to consist of a recycled ER-associated
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84 degradation (ERAD) machinery of symbiont origin (67). Support for this model came
85 from the detection of symbiont-specific ERAD components encoded on the
86 nucleomorph of the cryptophyte Guillardia theta, this being the remnant nucleus of the
87 former red algal endosymbiont in the cryptophytes PPC (23). The canonical ERAD
88 removes aberrant or misfolded luminal (ERAD-L) and membrane (ERAD-M and ERAD-
89 C) ER proteins and tags them after retro-translocation in the cytosol with poly-ubiquitin
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90 moieties for subsequent proteasomal degradation (7, 40, 66). However, in the symbiont- o
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91 specific ERAD-like pathway (SELMA) the retro-translocation machinery of ERAD-L is lo
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92 postulated to be maintained, and possesses the capacity to transport proteins from an e
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93 ER luminal compartment into a cytoplasmic compartment, the PPC. This process is o
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94 supposed to be uncoupled from degradation. SELMA is conserved in all secondary t
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95 evolved organisms with a red algal endosymbiont, for which genomic data are available /e
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96 (26, 67-68). Proteins of the derlin family are still controversially discussed elements of s
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97 the ERAD-specific translocon. In the diatom Phaeodactylum tricornutum, two symbiont- o
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98 localized derlins (PtsDer1-1/PtsDer1-2) are expressed which form hetero- as well as o
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99 homo-oligomers and show interaction with transit peptide-like sequences of PPC- p
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100 localized proteins (38). These components are indeed involved in the transport of ,

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101 proteins into the plastid as indicated by a conditional knock-down mutant of the 19

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102 Toxoplasma gondii sDer1 protein which showed impairment in plastid protein import (2).
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103 The translocation process is predicted to be dependent on ubiquitylation, further s
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104 supported by the presence of a set of ubiquitylation enzymes (39, 67). Additional factors
105 proposed to be involved in SELMA are a symbiont-specific Cdc48 AAA-ATPase with its
106 co-factor Ufd1 and adaptor proteins (55, 67). Following translocation, the precursor
107 proteins are likely to undergo de-ubiquitylation and are either passed on to the
108 translocon in the third outermost membrane or folded in the PPC (13, 39, 55). Although
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109 a residual set of 20S proteasomal components was identified in the PPC of diatoms,
110 there is currently no link between SELMA and proteasomal degradation (55).
111 Having passed through the PPC, transport across the innermost plastid membranes
112 seems to be comparable to primary plastids with a translocon at the inner membrane of
113 chloroplasts (TIC) and a recently identified Omp85 protein which belongs to the family
114 of Toc75 proteins, the core components of the translocon at the outer membrane of
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115 chloroplasts (TOC) (1, 10, 13, 73). w
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116 Here, we present an update on the SELMA translocation model in organisms with a red d
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117 algal endosymbiont with focus on five heterokontophytes and apicomplexan parasites. fr
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118 In particular we mined the genomes of organisms that carry secondary plastids, h
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119 including recently published full genome sequences, for SELMA proteins. With this :/
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120 collected data set one would expect to define the degree of factor conservation and .a
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121 identify main components of the SELMA system which evolved to function in protein .o
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122 transport at a plastid membrane. Our results are compared to the respective host ERAD /
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123 system as well as to red algal ERAD components, from which SELMA originated. A
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124 Additionally, four new PPC-localized proteins similar to factors involved in ERAD could il 3
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125 be identified in the diatom P. tricornutum. We also extended the set of core proteasomal 01
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126 components in the PPC of heterokontophytes and discuss their putative function in y

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127 relation to SELMA. e
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129 Materials and Methods
130 Bioinformatic Analysis
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131 Protein sequences of ERAD and SELMA as well as proteasomal components were
132 collected from published data or retrieved via blastp and tblastn searches. As queries,
133 sequences from the Saccharomyces cerevisiae ERAD system and the P. tricornutum
134 SELMA system were used to search the genomic databases for Phaeodactylum
135 tricornutum v2.0 (12), Thalassiosira pseudonana (5), Fragilariopsis cylindrus
136 (http://genome.jgi-psf.org/Fracy1/Fracy1.home.html), Aureococcus anophagefferens
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137 (30), Emiliania huxleyi CCMP1516 main genome assembly v1.0 (http://genome.jgi- o
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138 psf.org/Emihu1/Emihu1.home.html) and Guillardia theta CCMP2712 v1.0 lo
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139 (http://genome.jgi-psf.org/Guith1/Guith1.home.html). Sequences from Ectocarpus e
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140 siliculosus (19) and Babesia bovis were searched at the National Center for o
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141 Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/guide/). t
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142 Apicomplexan sequences were retrieved from the Plasmodium Genomics Resource /e
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143 Version 9.0 (6) for Plasmodium, the Toxoplasma Genomics Resource v7.2 (29) for T. s
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144 gondi and Neospora caninum, TparvaDB Version 1.0 (74) and the National Center for o
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145 Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/guide/) for o
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146 Theileria parva and the Cryptosporidium Genomics Resource v4.6 (36) for p
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147 Cryptosporidium parvum. ERAD sequences for red algae were either retrieved from the ,

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148 genome projects of Cyanidioschyzon merolae (53) and Galdieria sulphuraria (Michigan 19

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149 State University Galdieria Database [http://genomics.msu.edu/galdieria/about.html]), or
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150 by local Blast (blast-2.2.10-ia32-win32) using expressed sequence tags (EST) of s
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151 Porphyridium cruentum and partial genome data from Calliarthron tuberculosum
152 (http://dbdata.rutgers.edu/data/plantae/) generated by Chan and colleagues (17).
153 In general a minimal e-value of 1e-04 was set as threshold for the identification of
154 ERAD/SELMA components on the protein level. However, in cases of weak query
155 sequence significance, also matches with a lower e-value were inspected. Additionally,
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156 criteria like domain structure and composition similarity (NCBI Conserved Domain
157 search) were applied for identification of relevant proteins (51). For proteasomal
158 components, all S. cerevisiae 20S protein sequences were used as queries to collect a
159 data set of putative proteasomal components which were then classified according to
160 the NCBI Conserved Domain Database (51) which differs from the S. cerevisiae
161 nomenclature (detailed information on different classifications can be found in (60)).
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162 All gene models of the identified proteins were aligned to genomic and EST sequences, w
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163 if available. Thereby, missing N- and C-termini were identified by searching for putative a
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164 start and stop codons in frame, respectively. If possible, intron borders of the gene d

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165 models were checked to be in agreement with EST data. The protein sequences were m

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166 additionally examined for N-terminal targeting sequences to discriminate symbiont tp
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167 proteins from host factors. PPC directed proteins are characterized by the presence of a c
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168 SP and a TPL. The SignalP 3.0 Server (24) was used for the prediction of a SP with a m
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169 cutoff of >0.5 by the HMM algorithm. Then, the sequences were analyzed with the g
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170 TargetP 1.1 Server (25) with default settings to define the SP as a secretory signal
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171 sequence and exclude mitochondrial targeting. In general, the TPL of PPC (symbiont) il
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172 proteins cannot be predicted accurately with available tools. For this reason, besides 2
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173 performing the prediction with the TargetP 1.1 Server (25) using signal peptide-
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174 truncated sequences in “plant” mode, the criteria defined in (55) were applied. In some g
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175 cases, a protein model was identified with high similarity to a known symbiont protein of t
176 the diatom P. tricornutum or the apicomplexan parasite P. falciparum but without SP
177 prediction. This can be caused by an incorrect gene model prediction due to the lack of
178 EST data or the presence of several putative start codons. Therefore, these proteins
179 were assigned as symbiont but marked to lack a signal peptide prediction.
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180 Analyses of transmembrane spanning regions were performed with TOPCONS (9),
181 domain and coiled-coil prediction was done using SMART (45). Protein sequence
182 alignments were performed with GENEDOC Software (version 2.6.002
183 [http://www.psc.edu/biomed/genedoc]).
184 Plasmid Construction and Transfection of P. tricornutum
185 The predicted PPC proteins were cloned and transfected into the diatom P. tricornutum. D
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186 The sequences of genes containing introns or without EST support were amplified from n
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187 cDNA, the rest from gDNA, cloned in front of egfp into P. tricornutum transfection d
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188 vectors. ptsubx, ptspng1, ptsubq were cloned into the nitrate-inducible pPha-NR vector fr
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189 (GenBank: JN180663), ptsnpl4, ptsβ1, ptsα3, pthβ7 and pthrpn10 into the light-inducible h
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190 pPha-T1 vector (GenBank: AF219942). For further information about sequences of in :/
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191 vivo-localized proteins as well as primer sequences see supplemental file 1. Biolistic .a
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192 transfection into P. tricornutum cells was performed as described previously (67, 77). .o
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193 Positive transformants were cultured under standard conditions as described before (3) /
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194 with 1.5 mM NH4+ in permanent cultivation. Protein expression under control of the Ap
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195 nitrate reductase promoter (pPha-NR vector) was induced by cultivation on 0.9 mM il 3
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196 NO3- for two days. 01
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197 Fluorescence Microscopy
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198 P. tricornutum transformants were fixed with 4 % paraformaldehyde/ 0,0075 % t
199 glutaraldehyde in 1x PBS buffer and analyzed with a confocal laser scanning
200 microscope Leica TCS SP2 using a HCX PL APO 40×/1.25 − 0.75 Oil CS objective.
201 Fluorescence of eGFP and chlorophyll was excited with an Argon laser at 488 nm and
202 detected with two photomultiplier tubes at a bandwidth of 500–520 nm and 625–720 nm
203 for eGFP and chlorophyll fluorescence, respectively.
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205 Results
206 1. Identification of ERAD and SELMA components in red algae and organisms
207 with a red algal endosymbiont
208 In order to identify new ERAD and SELMA components, all available genomic
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209 sequences of red algae and organisms with a red algal endosymbiont were screened o
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210 via BLAST search with queries from the best studied ERAD system of Saccharomyces lo
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211 cerevisiae (40, 66). The recently published genomes of heterokontophytes (the diatom e
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212 Fragilariopsis cylindrus, the brown alga Ectocarpus siliculosus, the harmful alga o
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213 Aureococcus anophagefferens), the nuclear genome of the cryptophyte Guillardia theta t
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214 and the apicomplexan Neospora caninum were included in these analyses. Because /e
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215 SELMA was shown to be phylogenetically derived from the ERAD system of the red s
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216 algal endosymbiont (26), we also included sequences from the red algae r
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217 Cyanidioschyzon merolae, Porphyridium cruentum, Calliarthron tuberculosum and n

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218 Galdieria sulphuraria in our analyses (see Material and Methods for detailed description r
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219 on used genome data). In contrast to the other chromalveolate groups, dinoflagellate ,
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220 plastids have only three surrounding membranes and very little is known about the 9

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221 mechanisms that transport proteins across these membranes (10, 65). Due to the
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222 paucity of genomic data for these organisms, we have not included peridinin-containing s
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223 dinoflagellates in this study.
224 We identified genes for conserved ERAD components in all investigated red algal
225 genomes (Table 1). However, due to incomplete data for Porphyridium cruentum (EST
226 data) and especially Calliarthron tuberculosum (partial genome data), only a subset of
227 ERAD factors could be identified. The collected data set for red algae implicates that the
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