Protein Targeting into Diatom Plastids
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz, Fachbereich Biologie
vorgelegt von Sascha Vugrinec
Tag der mündlichen Prüfung: 24. März 2011
Referent: Prof. Dr. Peter G. Kroth
Referentin: Prof. Dr. Iwona Adamska
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-133680
Kieselalgen sind wie eine Reihe weiterer Algengruppen durch sekundäre Endocytobio- se entstanden, bei der eukaryotische Algen von eukaryotischen Wirtszellen aufgenom- men und anschließend zu „komplexen“ Plastiden reduziert und spezialisiert wurden.
Vergleichbar mit der Entstehung primärer Plastiden mussten dabei Transportmecha- nismen entwickelt werden, um Präproteine zurück in die Plastide zu transportieren.
Sieben putative Untereinheiten der Translokatoren der inneren Chloroplastenmem- bran (Tic) sind in den Genomen der KieselalgenPhaeodactylum tricornutum undTha- lassiosira pseudonanakodiert. Fusionsproteine mit Tic-Präsequenzen oder Volllängen- Fusionsproteine mit GFP zeigen, dass die untersuchten Tic-Komponenten plastiden- assoziiert sind. Mit Hilfe weitererin vivo-,in vitro- undin silico-Studien, konnten wir die einzelnen Tic-Komponenten ausP. tricornutum genauer charakterisieren.
Wir konnten weiterhin zeigen, dass zweigeteilte Präsequenzen aus unterschiedlichen Algengruppen in der Kieselalge P. tricornutum funktionell sind, wenn sie heterolog als GFP Fusionsproteine exprimiert werden. Interessanterweise ermöglichte auch ein modifiziertes Signal-Peptid einer Carboanhydrase aus Arabidopsis thaliana, welche vermutlich über das Endoplasmatische-Reticulum in die Chloroplasten vonA. thaliana transportiert wird, den GFP-Import inP. tricornutum-Plastiden. Das deutet auf einen konservierten Weg für den Proteinimport in sekundäre Plastiden, der mit dem Signal- Peptid-abhängigen Importweg in die Chloroplasten höherer Pflanzen verwandt ist.
Colokalisations-Analysen zeigen, dass natürliche und künstliche Kieselalgen Präse- quenzen zur Akkumulation von GFP in einer als „blob“-artig bezeichneten Struktur führen, und dass diese Struktur in beiden Fällen identisch ist.
Im Moment stehen mehrere Modelle zum Proteinimport in die „komplexen“ Plasti- den von Kieselalgen zur Diskussion, welche sich in der Art und Weise unterscheiden, wie sie den Transport über das CER-Lumen in den Zwischenmembranraum beschrei- ben. Das Poren Model versucht dies über den Transport durch eine Verbindung zwi- schen dem CER und dem Zwischenmembranraum zu erklären. Mit Hilfe des selbst- assemblierenden GFP Systems konnten wir zeigen, dass Proteine offenbar nicht auf direktem Wege vom CER in den Zwischenmembranraum gelangen können.
Weiterhin kann man aus diesen Ergebnissen schlussfolgern, dass kernkodierte Plas- tiden-Proteine aus Kieselalgen, mit Hilfe von Translokatoren über die vier Plastiden- Hüllmembranen transportiert werden und nicht über die ebenfalls diskutierten Vesikel oder Poren.
Diatoms, like many other algal groups, evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent reduction and specialisation to a “complex” plastid. Comparable to the evolution of primary plastids, targeting mechanisms had to be developed to reimport preproteins into the plastid.
Seven putative subunits of the translocons at the inner envelope membrane of chloro- plasts (Tic) are encoded in the genome of the diatomsPhaeodactylum tricornutumand Thalassiosira pseudonana. Fusion proteins of Tic presequences or full length fusions to GFP show that the investigated Tics are plastid associated.
Fusion proteins consisting of bipartite plastid targeting presequences from various algal groups show that they are also functional when heterologously expressed as GFP fusion proteins in the diatom P. tricornutum. Interestingly, also the modified signal peptide of a carbonic anhydrase fromArabidopsis thaliana, which apparently is targeted toA. thaliana plastids via the endoplasmic reticulum, is able to direct GFP intoP. tricornutum plastids. This indicates that a conserved transport route is used for protein import into all secondary plastids, and that this route might be related to the signal peptide dependent route to plastids of higher plants.
Colocalisation analyses suggest that native and artificial presequences from diatoms lead to an accumulation of GFP in a “blob”-like structure and that this structure is identical in both cases.
At the moment, several models for the import of proteins into the “complex” plastids of diatoms are discussed. The models differ in the way they explain transport from the CER into the interenvelope space (ies). In the “pore model” it is proposed that a connection between the CER lumen and the ies might route nucleus encoded proteins across the periplastidic space. With the aid of a self-assembling GFP system we could show that proteins cannot reach the ies from the CER on a direct way.
Furthermore from these results it can be concluded that nucleus-encoded plastid proteins from diatoms pass the four plastid envelope membranes via translocators and not via pores or vesicles which are also proposed.
Zusammenfassung/Abstract iii
Proteinimport in Plastiden von Kieselalgen . . . iii
Protein Targeting into Diatom Plastids . . . iv
List of Figures ix List of Tables xi 1. General Introduction 1 2. Characterisation of the Tic-complex in the diatom P. tricornutum 7 2.1. Abstract . . . 8
2.2. Introduction . . . 9
2.3. Materials and Methods . . . 12
2.3.1. Culture conditions . . . 12
2.3.2. Sequence analysis and prediction programs . . . 12
2.3.3. PCR and construction of the plasmids . . . 13
2.3.4. Phylogenetic analyses . . . 15
2.3.5. SDS-PAGE and western blots . . . 15
2.3.6. Carbonate extraction . . . 15
2.3.7. Nuclear transformation . . . 16
2.3.8. Microscopy . . . 16
2.4. Results . . . 18
2.4.1. Localisation . . . 18
2.4.2. Interaction experiments using the split-GFP system . . . 30
2.4.3. Phylogeny of Tic components . . . 33
2.5. Discussion . . . 35
2.5.1. Putative channel-forming components . . . 36
2.5.2. Predicted regulatory subunits . . . 40
2.5.3. Tic component of the interenvelope space . . . 41
3. Cross-phyla functionality of plastid targeting presequences in chromists 53
3.1. Abstract . . . 54
3.2. Introduction . . . 55
3.3. Materials and Methods . . . 59
3.3.1. Culture conditions . . . 59
3.3.2. Sequence analysis and prediction programs . . . 59
3.3.3. Isolation of RNA and cDNA synthesis . . . 59
3.3.4. Construction of the plasmids . . . 60
3.3.5. Nuclear transformation . . . 61
3.3.6. Deglycosylation . . . 61
3.3.7. SDS-PAGE and western blots . . . 62
3.3.8. Microscopy . . . 62
3.4. Results . . . 63
3.4.1. Localisation experiments using heterologous-presequence:GFP fusion proteins . . . 63
3.4.2. Processing analysis of AtCAH1 . . . 68
3.4.3. Deglycosylation of PRK . . . 68
3.5. Discussion . . . 70
3.5.1. Conserved targeting signals from various organisms with pri- mary and secondary plastids . . . 70
3.5.2. A putative ancient pathway in P. tricornutum . . . 71
4. Comparison between native and artificial diatom sequences that direct GFP into BLSs 75 4.1. Abstract . . . 76
4.2. Introduction . . . 77
4.3. Materials and Methods . . . 79
4.3.1. Culture conditions . . . 79
4.3.2. Sequence analysis and prediction programs . . . 79
4.3.3. PCR and construction of the plasmids . . . 80
4.3.4. SDS-PAGE and western blots . . . 81
4.3.5. Nuclear transformation . . . 81
4.3.6. Microscopy . . . 82
4.4. Results and Discussion . . . 83
4.4.1. Colocalisation experiments in Phaeodactylum tricornutum . . . . 83
4.4.2. Processing of preproteins . . . 88
5. Is there a connection between the CER-lumen and the interenvelope space? 93 5.1. Abstract . . . 94
5.2. Introduction . . . 95
5.3. Materials and Methods . . . 98
5.3.1. Culture conditions . . . 98
5.3.2. PCR and construction of the plasmids . . . 98
5.3.3. Nuclear transformation . . . 99
5.3.4. Microscopy . . . 99
5.4. Results and Discussion . . . 100
6. General Discussion 105 A. Supplementary Data 115 A.1. Supplementary Material, Chapter 2 . . . 116
A.2. Supplementary Material, Chapter 4 . . . 133
B. Author Contributions 135
C. List of Publications 137
D. Acknowledgements 139
Bibliography 141
1.1. Plastid evolution by endocytobiosis . . . 4
1.2. Diatom model organisms . . . 5
2.1. Tic complex in higher plants . . . 11
2.2. Principle of the self-assembling-GFP system . . . 21
2.3. Control constructs for self-assembling GFP system . . . 22
2.4. Localisation of PtTic22 using a self-assembling-GFP system . . . 23
2.5. Orientation of PtTic20 . . . 25
2.6. Orientation of PtTic21 . . . 26
2.7. Carbonate extraction efficiency test . . . 28
2.8. Western blot analyses from Tic proteins using carbonate extraction . . 29
2.9. Control western blot from carbonate extraction . . . 30
2.10. Control constructs for split-GFP system . . . 31
2.11. Interaction assay of PtTic21 with PtTic22 . . . 32
2.12. Phylogenetic tree of Tic20, Tic22, Tic21 and Tic110 . . . 34
2.13. Putative Tic complex in Phaeodactylum tricornutum . . . 35
2.14. Schematic representation of all identified Tics in P. tricornutum . . . . 44
2.15. Localisation of PtTic20 . . . 46
2.16. Localisation of PtTic21 . . . 47
2.17. Localisation of PtTic22 . . . 48
2.18. Localisation of PtTic32 . . . 49
2.19. Localisation of PtTic55 . . . 50
2.20. Localisation of PtTic62 . . . 51
2.21. Localisation of PtTic110 . . . 52
3.1. Evolution of algae by primary and secondary endocytobiosis . . . 56
3.2. Heterologous fusion proteins . . . 64
3.3. Localisation of heterologous presequence:GFP fusion proteins in P. tri- cornutum . . . 65
3.4. Localisation of heterologous presequence:GFP fusion proteins inP. tri-
cornutum . . . 66
3.5. Localisation of heterologous presequence:GFP fusion proteins inP. tri- cornutum . . . 67
3.6. Processing analyses of AtCAH1 . . . 68
3.7. Deglycosylation of PRK . . . 69
4.1. Co-localisation control constructs . . . 84
4.2. Co-localisation experiment in Phaeodactylum tricornutum . . . 86
4.3. Co-localisation inThalassiosira pseudonana . . . 87
4.4. Processing analyses of nBLS and aBLS . . . 89
5.1. Import pathways . . . 96
5.2. Pore-test using self-assembling GFP system . . . 101
6.1. Schematic illustration of possible import pathways in P. tricornutum plastids. . . 107
6.2. Schematic illustration of the assumed import pathway in P. tricornu- tum plastids. . . 114
A.1. Alignment of Tic22 proteins . . . 119
A.2. Alignment of Tic32 proteins . . . 120
A.3. Alignment of Tic55 proteins . . . 121
A.4. Alignment of Tic62 proteins . . . 122
A.5. Signal peptide prediction for P. tricornutum Tics . . . 123
A.6. Signal peptide prediction for P. tricornutum Tics . . . 124
A.7. Signal peptide prediction for T. pseudonana Tics . . . 125
A.8. Signal peptide prediction for T. pseudonana Tics . . . 126
A.9. Transmembrane helix prediction P. tricornutum using TMHMM . . . . 127
A.10.Transmembrane helix prediction P. tricornutum using TMMOD . . . . 128
A.11.Transmembrane helix prediction in P. tricornutum using SPLIT . . . . 129
A.12.Transmembrane helix prediction in T. pseudonana using TMHMM . . 130
A.13.Transmembrane helix prediction in T. pseudonana using TMMOD . . . 131
A.14.Transmembrane helix prediction in T. pseudonana using SPLIT . . . . 132
2.1. Overview from identified Tics . . . 37
6.1. Support of different import models . . . 113
A.1. Presequence prediction for diatom Tic proteins . . . 117
A.2. Transmembrane domain prediction from diatom Tic proteins . . . 118
A.3. Presequence prediction from P. tricornutum and T. pseudonana . . . . 134
General Introduction
Diatoms are one of the most common types of phytoplankton with an estimated number of 200,000 species worldwide [97]. This group of eukaryotic algae can be found in the upper zone of the ocean and other water bodies and in some terrestrial habitats, in principle almost in all areas with sufficient light and nutrients are available [151].
Diatoms are photoautotrophic organisms meaning that they can use the light energy to convert carbon dioxide (CO2) into organic compounds. The contribution of diatom photosynthesis to marine primary productivity has been estimated to be around 40 % [37, 116]. On a global scale it can be estimated that diatoms contribute at least 20 % of the annual primary productivity. That means that diatom photosynthesis in the sea generates about as much organic carbon as all the terrestrial rainforests combined [44]. Therefore, diatoms are an important food source for the zooplankton, as well as suspension and filter feeders. In the open ocean, a relatively large amount of diatom biomass sinks from the surface into deeper water layers and becomes food for different organisms [134]. A small fraction of this sinking organic matter accumulated at the sea bottom as sediments during pre-historic periods and contributed to recent petroleum reserves. Because of the important role of diatoms in global CO2-fixation, it has been tried to change atmospheric carbon dioxide concentrations by “Iron Fertilization” of the ocean [34]. The principle of this approach is to enhance biological productivity, which can benefit the marine food chain and remove the greenhouse gas carbon dioxide from the atmosphere. Diatoms also have a potential as factories for the production of a wide range of metabolites (including oils, fatty acids, and pigments) [94] that may be of great importance for biotechnology.
Diatoms are unicellular or colonial members of the taxonomy phylum Bacillario- phyta [1, 106]. Their name is derived from the Greek word diatomos, meaning “cut in half”, a indirectness to the design of their silicified cell wall (frustules), which sur- round the cells in two parts (hypotheca and epitheca), comparable to a petri dish.
Characteristic for this algal group is for example a girdle lamella (peripheral stacks of three thylakoids, which lie beneath the plastid membrane), chlorophylls a/c and fucoxanthin as the major light-harvesting pigments for photosynthesis and chloroplast DNA, which is usually concentrated within a ring-shaped nucleoid at the periphery of the plastid [151]. Besides, plastids of diatoms are surrounded by four (higher plant plastids possess “only” two envelope membranes) membranes, the outermost of which is studded with ribosomes and continuous with the endoplasmic reticulum (ER), therefore called chloroplast ER (CER) [48].
This specific cell design can be explained with the endosymbiotic theory. There
heterotrophic eukaryotic cells, followed by the reduction of the endosymbiont to an organelle [135, 98, 108]. This process, called primary endocytobiosis (Figure 1.1), re- sulted in monophyletic plastids surrounded by two membranes that are characteristic for glaucophytes, rhodophytes, and chlorophytes including their higher plant progeny [111, 129]. Fossils of green algal origin have been found in Central Australia that are about 900 million years old, while fossils from a red alga occurred about 590 million years ago [106]. Compared to algae with primary plastids, diatoms are comparatively young in an evolutionary context. The earliest fossil records of diatoms date back 190 million years before present [137].
Diatoms and other groups originated from a process called secondary endocytobiosis, where a eukaryotic alga with primary plastid was taken up by another eukaryotic heterotrophic cell (Figure 1.1). This engulfed alga again was subsequently reduced to a “green organelle” within the eukaryotic cell. Secondary endocytobiosis occurred at least twice in evolution, involving a green or a red alga as endosymbiont, respectively, according to the photosynthetic pigmentation of algae that evolved that way [87, 25]. It is still under debate whether the secondary plastids of the red algal lineage originate from a single [24] or multiple endosymbiotic events [10] and whether the resulting chromalveolates (including the chromists: stramenopiles, cryptophytes and haptophytes; as well as the alveolates: ciliates, apicomplexa and dinoflagellates) are finally monophyletic or polyphyletic.
Research on diatoms extremely profited from the advances in molecular techniques and the increasing amount of sequence data. Especially for two diatom model organ- isms, the pennate Phaeodactylum tricornutum Bohlin and the centric Thalassiosira pseudonana Hasle & Heimdal (Figure 1.2), a variety of molecular tools and extensive sequence information are available. Both diatom genomes have been sequenced as part of a program at the Joint Genome Institute in California. The sequence informa- tion from the centric diatom T. pseudonana was the first to be reported [6], and it was the first of any eukaryotic marine phytoplankton species to be sequenced with a database of 13,000 expressed sequence tags (ESTs). The P. tricornutum genome was subsequently completed [17]. Both species contain around 11,000 predicted genes in approximately 30 million base-pair (Mbp; 32 Mbp forT. pseudonana and 27 Mbp for P. tricornutum) genomes [132]. The availability of a variety of molecular tools and the continuously increasing amounts of sequence data provide new opportunities to study diatoms not only with biochemical and physiological approaches, but also using
Primary endocytobiosis
Secondary endocytobiosis
Cyanobacterium Mitochondrium Nucleus
Endoplasmic reticulum
Gene transfer
Plastid
Protein targeting
N
N
N
N
N
N
N
Figure 1.1.: Plastid evolution by endocytobiosis. Primary endocytobiosis: a photoautotrophic prokary- ote was engulfed by a heterotrophic eukaryotic cell and subsequently reduced to a primary plastid surrounded by two envelope membranes; secondary endocytobiosis: a eukaryotic primary alga was taken up by another eukaryotic cell and subsequently evolved into a secondary plastid surrounded by four (or three) membranes; protein targeting (black arrow) and gene transfer (green arrow) co-evolved.
Modified after McFadden [106].
molecular techniques. The information from genome data of the two diatom species (P. tricornutum and T. pseudonana [6, 16]), together with molecular tools such as genetic transformation [2, 88, 126, 127, 162], can be used to investigate molecular as- pects of protein import in diatoms. Protein targeting into organelles often depends on N-terminal presequences that allow prediction of the intracellular localisation [87, 53].
Because of the more complex structure of diatom plastids (four envelope membranes
Figure 1.2.: Phaeodactylum tricornutumandThalassiosira pseudonana, model organisms for pen- nate and centric diatoms. (A)Nomarski’s differential interference contrast (DIC) illumination show- ing the three morphotypes ofPhaeodactylum tricornutum: left, fusiform; top right, triradiate; bottom right, oval. (B)Thalassiosira pseudonana, valve (top) and girdle (bottom) view, DIC; all scale bars represent 10µm
surround the plastid, instead of at least two envelope membranes in higher plants) nucleus-encoded plastid targeted proteins possess a bipartite presequence consisting of a signal and a transit peptide, and a special motif (“ASAFAP”) at the signal peptide cleavage site.
In this work, molecular tools, biochemical approaches, sequence informations and bioinformatic analyses were combined to study mechanisms of protein import in the diatom P. tricornutum. We characterised the Tic (translocon at the inner envelope membrane of chloroplasts) complex in the innermost plastid envelope membrane (chap- ter 2) and looked for further possible import pathways for nucleus-encoded plastid proteins (chapter 5). Furthermore, we tested several presequences from different or- ganisms with primary as well as secondary plastids for their ability to direct GFP into P. tricornutum plastids, to draw conclusions from possible conserved targeting routes and signals (chapter 3). Finally, we observed the behaviour from native and artificial proteins which are possibly targeted into the periplastidic space (chapter 4). Taken together, these characterisations of import mechanisms help to better understand protein targeting into diatom plastids.
diatom P. tricornutum
Sascha Vugrinec
∗, Ansgar Gruber, Peter G. Kroth
Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany
∗Author for correspondence. E-mail: sascha.vugrinec@uni-konstanz.de
2.1. Abstract
The chloroplast is the site of photosynthesis, and harbours a large array of biosynthetic pathways needed for normal growth and development. These biosynthetic pathways rely on the import of numerous nucleus-encoded proteins. Plastids of diatoms have a more complex ultrastructure and a partially different physiology compared to higher plants and green or red algae: As a consequence of their evolution by secondary endo- cytobiosis diatom plastids are surrounded by four membranes, representing additional barriers for protein import.
We identified seven genes encoding putative proteins belonging to the inner enve- lope membrane translocons of chloroplasts (Tic) in the genomes of the diatomsPhaeo- dactylum tricornutumandThalassiosira pseudonana. We were able to identify several conserved protein domains within these Tic proteins. To test the localisation of the putative P. tricornutum Tics, we constructed presequence:GFP and full length:GFP fusion proteins and expressed them in the diatom P. tricornutum. We found GFP fluorescence within the plastid indicating a localisation of these Tic proteins in the plastid membrane. Further investigations on the subcellular localisation showed that PtTic20, PtTic55 and PtTic110 are integral membrane proteins, with PtTic32 being a peripheral membrane protein and PtTic62 a soluble protein. Results we observed in orientation studies, support the role of PtTic20 (N- and C-terminus faces into the stroma) and PtTic21 (N-terminus faces into stroma and C-terminus probably into the interenvelope space) as an integral membrane protein localised in the innermost membrane. Interestingly, in interaction studies we observed an interaction between PtTic21 and PtTic22 using a split-GFP system. Here we show first experimental evi- dence for the existence of Tic components inP. tricornutum, which might be involved in protein import of nucleus-encoded plastid proteins through the innermost envelope membrane of the plastid.
Keywords
chloroplast· protein import· translocon ·Tic complex · diatom
2.2. Introduction
Chloroplasts evolved by a process termed primary endocytobiosis: a free living pho- toautotrophic cyanobacterium has been engulfed by a eukaryotic heterotrophic cell and was subsequently transformed into a plastid. The primary endosymbiotic event in plastid evolution gave rise to glaucophytes, rhodophytes, chlorophytes and land vascular plants lineages [128]. Plastids of these three algal groups have two envelope membranes (the glaucophytes still possess a residual murein sacculus between the en- velope membranes [145]), but a rather different type of photosynthetic pigmentation and thylakoid structure. During the reduction of the primary and secondary endosym- biotic cells, most of the genes of the endosymbiont were either lost, replaced by genes of the host or transferred to the nucleus of the host cell [33, 101]. Therefore an efficient plastid protein import system had to be established in order to provide the organelles with plastid proteins now encoded in the nucleus [66, 87]. Protein targeting across the two envelope membranes of the primary plastids of land plants such as Arabidopsis thaliana is well characterized. Most chloroplast proteins from rhodophytes, glauco- phytes and the viridiplantae are encoded by the nuclear genome and possess cleavable transit peptides (TP), which direct them to the “green organelle” [69, 139]. Translo- cation into chloroplasts appears posttranslationally and involves binding of proteins to the Toc/Tic (translocon at the inner/outer envelope membrane of chloroplasts) apparatus in the chloroplast envelope membrane [140].
Diatoms and other groups of algae possess secondary plastids which originated from a secondary endocytobiosis, which means the uptake of a eukaryotic alga possessing primary plastids into another eukaryotic host cell followed by the reduction of the endosymbiotic alga to a plastid. This event occurred at least twice in evolution, involving a green or a red alga as endosymbiont, respectively [25, 26]. Plastids, derived from the secondary endocytobiosis, possess up to four envelope membranes and are therefore called “complex plastids” [24, 25, 107]. In diatoms, the outermost membrane is continuous with the host´s ER membrane and studded with 80S ribosomes. Nucleus- encoded plastid proteins are imported co-translationally into the lumen of the ER, which was proven by in vitro and in vivo experiments [93, 13, 92]. This shows that the signal peptide (SP) is a necessary component for the import into the ER-lumen via the Sec61-translocon, which was further confirmed by in vivo experiments, in which signal peptides were fused to GFP and shown to be targeted to the ER-lumen or to the secretory system [3, 82, 50]. In the ER-lumen, the signal peptide is cleaved off, thus
exposing the transit peptide which mediates protein transport over the remaining three envelope membranes. It is supposed that from the ER-lumen, proteins may cross the second membrane by a machinery which evolved from the endosymbionts ER-associated degradation (ERAD) machinery, called symbiont-specific ERAD-like machinery (SELMA) [62, 49], and are released into the periplastidic space (pps), which is the former cytoplasm of the secondary endosymbiont. Further transport across the last two envelope membranes depends on the first amino acid of the transit peptide.
An aromatic amino acid at the first position directs the protein into the stroma, without such an amino acid the destination might be the pps [53]. The two innermost envelope membranes of diatom plastids are thought to represent the plastid envelope of the secondary symbiont. This, and the necessity of a transit peptide as an import signal may imply that nucleus-encoded plastid proteins cross this barrier through a translocon similar to the Tic/Toc complex from higher plants. However, analyses of the genomes of the diatomsT. pseudonana andP. tricornutum did not yield putative components of the Toc apparatus. It was proposed that a component derived from the cyanobacterial outer membrane protein Omp85, which is related to the Toc75 protein in higher plants, could act as a phenylalanine specific receptor and membrane channel across the third outermost membrane [160, 20]. In higher plants seven or eight proteins have been proposed to form the Tic complex: Tic110 and Tic20 as putative constituents of a translocon channel; the co-chaperone Tic40; and the translocon- associated Tic55, Tic32 and Tic62 subunits [147]. Tic21 is the most recently added putative component of the Tic translocon [149] (Figure 2.1). Whether or not it belongs to the Tic complex is not clear yet, as it might also represent a metal permease [38].
Here we report experimental hints for the localization of Tic components in the innermost membrane of the diatom P. tricornutum. P. tricornutum homologs of different Tic subunits can be found in vascular plants, moss, green-, red algae, stra- menopiles and cyanobacteria. For the Tic components in the diatomP. tricornutum we could show their localisation, orientation of the intermembrane parts or interac- tions with other Tics byin vivo, in vitro and in silico studies.
Toc complex
Tic22
Tic110 Tic40 Tic62 TPR
Tic55 Tic32
Tic20 Tic21
FNR Hip/Hop
IMS OEM
IEM stroma
CaM
Figure 2.1.: Schematic illustration of the Tic components in higher plants. Tic110, Tic20 and Tic21 were proposed to form a channel in the inner envelope membrane (IEM) of chloroplasts. Tic110 is in contact with the Toc complex and also associated with Tic40 via its TPR domain. The only component located in the intermembrane space (IMS) is Tic22. Tic62, Tic55 and Tic32 form the redox regulon. The calmodulin (CaM) binding site of Tic32 and the FNR-interacting domain of Tic62 are indicated. OEM: outer envelope membrane
2.3. Materials and Methods
2.3.1. Culture conditions
Phaeodactylum tricornutum Bohlin (University of Texas Culture Collection, strain 646) was cultivated in Provasoli’s enriched seawater [144] using ”Tropic Marin” (Dr.
Biener GmbH, Wartenberg, Germany) salt (16.6 g·L−1), at a final concentration of 50 % compared with natural seawater. Cells were grown in liquid culture in flasks under rigorous shaking (120 rpm) at 20◦C with continuous illumination at 35 µmol· photons·m−2·s−1. Solid media contained 1.2 % (w/v) Bacto Agar (BD, Sparks, MD, USA).Escherichia coli strain XL-1 Blue (Stratagene, La Jolla, CA, USA) was grown over night at 37◦C in Luria Broth medium, using a shaker for liquid cultures [133].
Solid media contained 1.5 % Bacto Agar.
2.3.2. Sequence analysis and prediction programs
To identify putative Tics we searched the current US Department of Energy Joint Genome Institute (JGI1) diatom genome sequencing project for the diatoms P. tri- cornutum [16] and T. pseudonana [6] as well as the publicly available database the National Center for Biotechnology Information (NCBI2) for sequences of interest. Re- sulting hits were screened for the presence of signal peptides by help of the program SignalP3 [11]. For cleavage site predictions the results of SignalP’s Neuronal net- works (NN) [118] or Hidden Markov Models (HMM) [119] were used, for prediction of chloroplast transit peptide-like domains, the programs ChloroP4 [41] and TargetP5 [40] were used. Transmembrane helices were predicted using TMHMM6 Server v.2.0 [86, 143], TMMOD7 [72], MINNOU8 [23] and SPLIT9 [71]. Transmembrane predic- tion programs sometimes interpret hydrophobic regions of signal and transit peptides as transmembrane helices [39, 109]. In order to avoid missprediction of signal and transit peptide-like domains as transmembrane helices in the N-terminal region of the Tics, sequences were sent to these prediction sites without presequences. The transit
1http://www.jgi.doe.gov/
2http://www.ncbi.nlm.nih.gov/
3http://www.cbs.dtu.dk/services/SignalP/
4http://www.cbs.dtu.dk/services/ChloroP/
5http://www.cbs.dtu.dk/services/TargetP/
6http://www.cbs.dtu.dk/services/TMHMM/
7http://liao.cis.udel.edu/website/servers/TMMOD/scripts/frame.php?p=submit
8http://minnou.cchmc.org/
9http://split.pmfst.hr/split/4/
peptide-like domains of bipartite plastid targeting sequences often attain poor predic- tion scores and their length is difficult to predict, so we removed the signal peptide as predicted by SignalP [11], plus 15 additional amino acids to account for the estimated transit peptide-like domain. Protein motifs and domains were identified using the protein family database PFAM10 [45], InterProScan11 [4] and from supplementation of alignments from Kalanon and McFadden (2008) [73]. Protein IDs from diatom sequences and the corresponding databases are included in Table 2.1 and Table A.1 and A.2, .
2.3.3. PCR and construction of the plasmids
Generation of eGFP constructs
Standard cloning procedures were used [133]. Polymerase chain reaction (PCR) was performed with a Master Cycler Gradient (Eppendorf, Hamburg, Germany) using recombinant Pfu polymerase (Fermentas GmbH, St. Leon-Rot, Germany) or Ka- paHifi Polymerase (Peqlab Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s instructions. Presequences and full-length sequences used in this work were cloned from cDNA derived from Phaeodactylum tricornutum and cloned into the pPha-T1-StuI-GFP vector as described in [53] via a StuI restriction site or into the pPha-T1-HpaI-GFP vector via a HpaI restriction site, upstream of and in frame with the enhanced Green Fluorescent Protein (eGFP) (BD Bioscience, Palo Alto, CA, USA) coding sequence. This leads to the derived artificial amino acid pro- line and asparagine, respectively between the presequences and GFP (indicated in Figure 2.15A, 2.16A, 2.17A, 2.18A, 2.19A, 2.20A, 2.21A).
Generation of self-assembling GFP constructs
The self-assembling GFP (sa-GFP) system was established earlier [21, 62] and used previously to specify protein localization [62]. For self-assembling GFP assays a mod- ified pPha-T1 vector was used which is under the control of the endogenous nitrate reductase promoter of P. tricornutum. For the construction of the self-assembling- GFP fusion proteins, the GFP1-10 (GFP1-10 representing theβ-strands 1-10 of GFP) and GFP11 (GFP11 representing β-strand 11 of GFP), respectively, were amplified from an exceptionally well folded variant of GFP termed “superfolder GFP” [125],
10http://pfam.sanger.ac.uk/
11http://www.ebi.ac.uk/Tools/InterProScan/
introducing the recognition site for EcoRI. The shuttle vector pPha-T1-NR was lin- earised using EcoRI and the modified GFP1-10/GFP11 fragment was ligated stick- yend into the plasmid in the orientation of the NR promoter, resulting in the plasmid pPha-T1-GFP1-10 and pPha-T1-GFP11, respectively. In a last step the spacer (pro- tein sequence: GGSGGGS), which contains the recognition site for AfeI was inserted via a round circle PCR into the pPha-T1-GFP1-10-vector. In the case of pPha-T1- GFP11 the spacer, which was also inserted via a round circle PCR (protein sequence:
GGSGGGS) possess a frameshift stuffer with three translational stops (one in each frame), for preventing false positives from self-religated plasmid. Furthermore there is a AflII restriction site at the N-terminus and a AfeI restriction site at the C-terminus of the spacer. Genes of interest derived from P. tricornutum cDNA were cloned into the pPha-T1-GFP1-10 vector via a AfeI restriction site and into the pPha-T1-GFP11 vector, via an additional AflII restriction site.
Generation of split-GFP constructs
The principle of split-GFP was described previously [158, 76, 62]. For split-GFP assays a modified pPha-T1 vector was used which is under the control of the en- dogenous nitrate reductase promoter of P. tricornutum. For the construction of the split-GFP fusion proteins, the NGFP (NGFP representing aminoacid 156-245 of GFP) and CGFP (CGFP representing aminoacid 1-155 of GFP), respectively, has been am- plified in a first step from the eGFP (BD Bioscience, Palo Alto, CA, USA), with primers introducing the recognition site for EcoRI. The shuttle vector pPha-T1-NR was linearised using EcoRI and the modified NGFP/CGFP fragment was ligated stick- yend into the plasmid in the orientation of the NR promoter, resulting in the plasmid pPha-T1-NGFP and pPha-T1-CGFP, respectively. In a last step the spacer (protein sequence: GGSGGGS), which contains the recognition site for AfeI was inserted via a round circle PCR into the pPha-T1-NGFP- and pPha-T1-CGFP-vector, respectively.
Phosphoribulokinase (PRK) (Protein ID: 50773), Tic20 (Protein ID: 56631), Tic21 (Protein ID: XM_002183811), Tic22 (Protein ID: EEC47673) full-length sequences and OEE1 (oxygen evolving enhancer protein 1; Protein ID: XP_002180309) prese- quence derived from P. tricornutum cDNA were cloned into the pPha-T1-CGFP and pPha-T1-NGFP, respectively, via a AfeI restriction site upstream of and in frame with the CGFP and NGFP, respectively, coding sequence.
2.3.4. Phylogenetic analyses
Tic sequences were extracted from the NCBI12database, or the corresponding genome projects. The data set includes available sequences from the JGI13 of green algae (Chlamydomonas reinhardtii,Ostreococcus tauri [121], Ostreococcus lucimarinus), the moss Physcomitrella patens and the stramenopile Thalassiosira pseudonana. The sequences from the red algae Cyanidioschyzon merolae [103] (http://merolae.biol.s.u- tokyo.ac.jp/) was obtained from the respective genome project. Tic components from the vascular plantArabidopsis thaliana were identified in theArabidopsis Information Resource (TAIR14) protein database. For amino acid sequence alignments we used the web-based ClustalW15 program at GenomeNet16, the given settings for slow/accurate alignment were chosen, with the output format Phylip. The alignment was manually refined. Maximum likelihood analyses were done using PhyML17 online [55], the substitution model LG was selected. Bootstrap analyses with 100 replicates were performed.
2.3.5. SDS-PAGE and western blots
Carbonate extracted proteins were separated by SDS-PAGE in 12 % acrylamide gel [90]. Proteins were transferred electrophoretically onto a PVDF membrane (HybondT M- P, Amersham Bioscience UK Limited, Buckinghamshire, UK) and incubated with an antiserum against GFP (Anti-GFP serum, Invitrogen, Eugene,Oregon, USA). For the control experiments antibodies against D1 (Anti-PsbA global antibody, AS05084, Agrisera, Sweden) and RuBisCo (RuBisCo large subunit, AS03037, Agrisera, Swe- den), respectively, were used. Detection was performed using the chemiluminescence detection system from Roche Diagnostics (BM Chemiluminescence Blotting Substrate POD; Roche Diagnostics GmbH, Mannheim, Germany).
2.3.6. Carbonate extraction
Phaeodactylum tricornutum cells were harvested by centrifugation (10 min, 2,500 g), resuspended in solubilization buffer (6-aminohexanoic acid, 50 mM NaCl, 1 mM
12http://www.ncbi.nlm.nih.gov/
13http://www.jgi.doe.gov/
14http://www.arabidopsis.org/
15http://www.ebi.ac.uk/clustalw/
16http://align.genome.jp/
17http://atgc.lirmm.fr/phyml/
EDTA, 50 mM imidazole/HCl ph 7.2, 8.5 % sucrose, Proteinase Inhibitor Cocktail (PIC)), and disrupted with a French press (800 psi or 107.5 M P a, four repeats). In- tact cells were removed by centrifugation (10 min, 8,000 g), and membranes within the supernatant were collected by centrifugation for 1h at 100,000 g. The membrane pellet was solubilized in carbonate buffer (100 mM NaHCO3 pH 11.5, 1 mM, PIC) and incubated on ice for 0.5 h. Membranes were again collected by centrifugation (1 h, 100,000 g) and proteins of membrane and supernatant fractions were treated with 15 % trichloroacetic acid (TCA) to precipitate proteins. Precipitated proteins were solubilised in equal volumes of urea-Buffer (8 mM urea, 200 mM Tris/HCl pH 7.5, 0.1 mM EDTA, 5 % (w/v) SDS, 0.03 % (w/v) bromephenol blue, 1 % (v/v) β-mercaptoethanol). All fractions were loaded on SDS-PAGE and analyzed by West- ern blot using specific antibody against green fluorescent protein (GFP) or an α-D1 and α-Ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo), respectively, for a fractionation efficiency test.
2.3.7. Nuclear transformation
Nuclear transformation of P. tricornutum was performed using the Particle Delivery System PDS-1000/He (Bio-Rad, Hercules, CA, USA; [79]), fitted with 1350 psi rupture discs as described previously [2] and recently in more detail [88]. Tungsten particles (M10, 0.7 µm median diameter, Bio-Rad) were coated with 5 µg of plasmid DNA in the presence of CaCl2 and spermidine (Sigma-Aldrich Corporation, St. Louis, MO, USA), according to Bio-Rad’s recommendations. One day prior to bombardment approximately 108 cells were spread in the centre of a plate containing 20mLof solid medium and allowed to recover. After bombardment, cells were allowed to recover again for 24h, before being suspended in 600 µLof sterile 50 % Provasoli’s enriched seawater medium. 150µLof this suspension were plated on solid medium containing 75µg·mL−1 Zeocin (Invitrogen, Carlsbad, CA, USA). The plates were incubated at 20◦C under continuous illumination (35µmol·photons·m−2·s−1) for three weeks.
2.3.8. Microscopy
Transformants were screened for the expression of GFP using an Olympus BX51 epi- fluorescence microscope equipped with a Zeiss AxioCam MRm digital camera system (Olympus Europe, Hamburg, Germany). Nomarski’s differential interference contrast (DIC) illumination was used to view transmitted light images (100x UplanFL objec-
tive, Olympus). Chlorophyll autofluorescence and GFP fluorescence of the transfor- mants were dissected using the mirror unit U-MWSG2 (Olympus) and the filter set 41020 (Chroma Technology Corp, Rockingham, VT, USA) respectively.
2.4. Results
2.4.1. Localisation
Sequence analysis
A genome wide search for genes encoding proteins homologous to translocons at the inner envelope membrane of chloroplasts (Tic) genes resulted in the identification of seven putative Tic genes in the genome of the pennate diatom P. tricornutum (Pt) and the centric diatomT. pseudonana(Tp). The presequences from these diatom Tics mostly possess a full bipartite presequence including an “ASAFAP”-motif (PtTic20, PtTic21, PtTic55, TpTic55, TpTic62 and TpTic110) (Table A.1). PtTic32 and Tp- Tic21 also contain bipartite presequences but instead of a phenylalanine (F) there is a tryptophane (W) or a tyrosine (Y), respectively, within the “ASAFAP”-motif, which is necessary for successful protein import. In the case of PtTic22 only a signal and a transit peptide without an “ASAFAP”-motif are predicted. Signatures that can be in- terpreted as instances of the “ASAFAP”-motif are also present in PtTic110, TpTic22 and TpTic32, but they do not coincide with the predicted signal peptide cleavage site.
For Tic20 ofT. pseudonana we get two possibilities for the signal peptide cleavage site depending on the prediction model (SignalP´s NN or HMM) used. Prediction with SignalP´s NN model predicted a bipartite presequence without an “ASAFAP”-motif, whereas the prediction with HMM resulted in a presequence consisting “ASAFAP”.
The previously detected Tic20, Tic21, Tic55 and Tic110 from P. tricornutum and T. pseudonanapossess 1-4 predicted transmembrane domains (Table A.2), depending on, the prediction program. In order to avoid missprediction of signal and transit peptide-like domains as transmembrane helices in the N-terminal region of the Tics, sequences were sent to TMHMM18Server v.2.0, TMMOD19, MINNOU20and SPLIT21 without presequences.
To identify several conserved protein domains and motifs we used the programs PFAM22 and InterProScan23or searched manually with the aid of alignments for char- acteristically regions (Figure A.1, A.2, A.3 and A.4). In silico studies on Tic proteins with predicted roles in redox regulation like Tic32, Tic55 or Tic62, showed that Pt-
18http://www.cbs.dtu.dk/services/TMHMM/
19http://liao.cis.udel.edu/website/servers/TMMOD/scripts/frame.php?p=submit
20http://minnou.cchmc.org/
21http://split.pmfst.hr/split/4/
22http://pfam.sanger.ac.uk/
23http://www.ebi.ac.uk/Tools/InterProScan/
Tic32 and PtTic62 possess an NAD binding domain consisting of a G-x-x-G-x-x-G motif for PtTic62 (Figure A.4) and a G-x-x-x-G-x-G motif for PtTic32 (Figure A.2), respectively, which is characteristic for short-chain dehydrogenase (SDR) proteins [8, 120, 73]. Furthermore it is possible that PtTic32 possess a calmodulin-binding site (CaM) (FigureSuppl A.2). Sequence analyses in PtTic55 revealed several conserved regions (Figure A.3). At the N-terminus we could detect a Rieske-type iron sulfur (2Fe-2S) cluster, as well as a mononuclear iron (Fe2+) binding site. Beside these two binding sites we found two additional motifs at the C-terminus (Figure A.3). On the one hand a pheophorbide a oxygenase (PAO) motif, which belongs to the non-heme iron-binding proteins and on the other hand a conserved region with a characteristical C-x-x-C motif. As indicated in Figure A.1 we could identify two conserved regions in PtTic22, which might be a protein from the interenvelope space. In the case of the putative channel components PtTic20, PtTic21 and PtTic110 we found beside the previously mentioned transmembrane helices only a particularly leucine-zipper-like motif (L-x-x-L-x-x-x-L-G) in the PtTic110 protein (data not shown).
We also tried to identify Toc genes in the diatomsP. tricornutumandT. pseudonana but similarly to Armbrustet al. (2004) [6] and McFadden and van Dooren (2004) [107]
we were not able to detect any Toc subunits .
Localisation experiments using GFP fusion proteins
To study the intracellular localisation of the Tic genes, we constructed presequence:GFP and full length:GFP fusion proteins and expressed them in the diatom P. tricornu- tum. The PtTic20, PtTic21, PtTic32, PtTic62 presequences all containing classical
“ASAFAP”- and “ASAWAP”-motifs, respectively, enabling GFP import into the plas- tids, based on the co-localisation of GFP fluorescence with the chlorophyll autofluo- rescence of the plastid (Figure 2.15B, 2.16B, 2.18B, 2.20B).
Although the PtTic22 presequence:GFP and full-length:GFP constructs do not con- tain a classical “ASAFAP”-motif, GFP is found in the plastid of P. tricornutum transformants expressing PtTic22pre:GFP and PtTic22full:GFP (Figure 2.17B, C).
Additionally in PtTic22full:GFP transformants we obtained a phenotype similar to a
“blob”-like structure (BLS), the accumulation of GFP in a small reticular structure tightly associated to the plastid but outside the stroma, probably between the plastid membranes [82].
Although we expected a localisation within the plastid (because of the bipartite presequence and the localisation of the full length fusion proteins), P. tricornutum
transformants expressing the PtTic55pre:GFP and PtTic110pre:GFP fusion construct show a GFP fluorescence within the cytosol of the cell (Figure 2.19B, 2.21B). A reason could be the still present start codon from GFP. It is possible that mainly the mature GFP without the PtTic55 and PtTic110 presequence have been translated and therefore we get a cytosolic GFP localisation.
The presence of a functional stroma targeting sequence of PtTic20full:GFP and PtTic21full:GFP in P. tricornutum leads to accumulation of the GFP within and around the plastid silhouettes and additional accumulation of GFP in dots close to the plastid probably composed of membrane bound fusion protein aggregates (Fig- ure 2.15C, 2.16C). PtTic20 and PtTic21 contain three to four predicted transmem- brane domains (Table A.2). The presence of a functional stroma targeting sequence together with the finding that only plastid silhouettes and dots at the plastid pe- riphery are labeled in full length GFP fusions, indicates that PtTic20 and PtTic21 insert into the innermost plastid membrane via the plastid stroma, where they might operate in aggregates.
Although PtTic110full:GFP possess a predicted presequence without a classical
“ASAFAP”-motif it shows a similar phenotype as PtTic20full:GFP and PtTic21full:GFP (Figure 2.21C).
The expression analysis of PtTic32full:GFP, PtTic62full:GFP and PtTic55full:GFP show a GFP accumulation in the plastid (Figure 2.18C, 2.20C, 2.19C), which also fits with the prediction of a signal-, transit peptide and a classical “ASAFAP”-motif at the signal peptide cleavage site.
Localisation experiments using the self-assembling GFP system
To determine the exact localisation of PtTic22 within the plastid, we used a system involving engineered self-assembling GFP fragments. As a specific feature of this sys- tem the C-terminalβ-strand of GFP (GFP11) is separated from the rest of the GFP protein (GFP1-10). Neither fragment alone is fluorescent. Only when both of the fragments are localised in the same compartment GFP will spontaneously associate, resulting in GFP folding and formation of the fluorophore (Figure 2.2). To check the functionality of this system we made control constructs. As a positive control, both GFP fragments were directed to the stroma using the topogenic signals of PtOEE1 and PtATPC, respectively, resulting in GFP fluorescence within the plastid (Figure 2.3B).
OEE1 is normally targeted into the thylakoids (Ammon and Kroth, unpublished); for a better visualisation of GFP fluorescence, in all of the following constructs the third
Co-localisation:
Compartment1
Compartment2 GFP-Fluorescence
Membrane-orientation:
Compartment1
Compartment2 GFP1-10
GFP11
Protein b Protein a
GFP1-10
N
C GFP1-10
Compartment1
Compartment2
GFP11
GFP-Fluorescence Compartment2
Compartment1
reference protein tested protein
Figure 2.2.: Principle of the self-assembling GFP system. Proteins of interest are fused to a small GFP fragment (GFP11) and to the complementary GFP part (GFP1-10). Neither fragment alone is fluorescent. But if both fragments are localised in the same compartment they can spontaneously reassemble, resulting in GFP fluorescence. Co-localisation: Co-localisation from soluble proteins getting tested; Membrane-orientation: Orientation from integral membrane proteins getting tested
targeting domain responsible for thylakoid targeting has been deleted [82]. As a nega- tive control, the GFP1-10 fragment was fused to a stromal marker (PtOEE1pre) and co-transformed with the smaller GFP part fused to the presequence of ER targeted BIP (Figure 2.3C). Here we could not observe any GFP fluorescence, indicating that both GFP fragments are localised in different compartments. P. tricornutumtransfor- mants expressing PtTic22full:GFP1-10 fusion construct and the interenvelope space marker protein PtPlSP1full [20] lead to a “blob”-like structure (BLS) and GFP fluo- rescence within the plastid (Figure 2.4B), indicating that PtTic22 is localised in the interenvelope space.
DIC Chlorophyll GFP Chl GFP (C)PtOEE1pre:GFP1-10 + PtBIPpre:GFP11
(B)PtOEE1pre:GFP1-10 + PtATPCpre:GFP11
DIC Chlorophyll GFP Chl
GFP
01 05 10 15 20 25 30 35 40 45
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01 05 10 15 20 25 30 35 40 45 50
61 65 70 75 80
01 05 10 15 20 22
01 05 10 15 20 22
PtOEE1pre:GFP1-10
PtATPCpre:GFP11
PtBIPpre:GFP11
: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain
: mature protein
lower case: spacer domain and amino acids introduced by cloning GREY: conserved motif at signal peptide cleavage site
fragments of green fluorescent protein MKFTAACSLALVASASA
MRSFCIAALFAVASA MMFMRIAVAALALLAAPSIRA
FAPIPSVSRTTDLSMSLQKDLANVGKS
FTTQPTSFTVKTANVGERASGVFPEQSSAHRTRKA TIVMDGKANAIRDRITSVKNTRKITMAMKLVR
MLEaggsgggs: R
a :
a :
BOLD UNDERLINED ITALIC
GFP1-10
GFP11 GFP11
BLACK
ggsgggs ggsgggs
:
(A)
Figure 2.3.: Control constructs for self-assembling GFP system. (A)Control self-assembling-GFP fusion constructs. (B) GFP fluorescence, including GFP self-assembly when both fragments are lo- calised in the same compartment, was observed when combining the stromal markers PtOEE1pre:GFP1- 10 with PtATPCfull:GFP11;(C)no GFP fluorescence, including no spontaneously GFP self-assembly when GFP fragments are not localised in the same compartment, was observed when combining the stromal marker PtOEE1pre:GFP1-10 with the ER marker PtBIP-GFP11; Nomarski’s differential inter- ference contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.
DIC Chlorophyll GFP Chl GFP (B)PtTic22full:GFP1-10 + PtPlSP1full:GFP11
(A)
01 05 10 15 20 25 30 35 40 45 50
51 55 60 65 69 417
01 05 10 15 20 25 30 35 40 45 50
51 55 60 65 69 330
PtTic22full:GFP1-10
PtPlSP1full:GFP11
: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain
: mature protein
lower case:
MVRVSMLVFLSLVGQGTA
MPTISDHTHACRAANLASSPQRRMVLLLALCLSLVAPSVTA
SVEVARPATPTRAAAVSRSLPSKVWRQSRKVL NLSSTFGSATKASPYDRIA
FRPSTPSAV RSSVFVSSRTKISWADASS
... 337aa...RAW ... 250aa...RHV
a :
a :
BOLD UNDERLINED ITALIC
ggsgggs
ggsgggs
spacer domain and amino acids introduced by cloning fragments of green fluorescent protein
GFP1-10 GFP11
BLACK:
Figure 2.4.: Localisation of PtTic22 using the self-assembling GFP system. (A)PtTic22 and Pt- PlSP1 self-assembling GFP fusion constructs. (B) GFP fluorescence, including GFP self-assembly when both fragments are localised in the same compartment, was observed when combining the PtTic22:GFP1-10 with the interenvelope space marker PtPlSP1full:GFP11; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence and merged image show- ing the respective channels in the indicated colours are shown from left to right; scale bars represent 10µm.
Orientation experiments using the self-assembling GFP system
It is supposed that PtTic20 and PtTic21 are integrated in the innermost membrane of the complex plastid and possesses three to four transmembrane domains. To ver- ify this assumption and to exclude a possible localisation in another membrane, we used the self-assembling GFP system. We combined GFP11 C-terminally with the PtTic20 protein (PtTic20N:GFP11). When P. tricornutum cells are co-transformed with the stroma targeted PtOEE1pre:GFP1-10 fusion protein we detected GFP flu- orescence, which indicates that the C-terminus faces into the stroma (Figure 2.5B first row). The same result was observed when we made another fusion protein with the exception that the small GFP fragment (GFP11) was fused between the PtTic20 presequence and the PtTic20 mature protein (PtTic20C:GFP11), and co-transformed it with the stromal marker protein (PtOEE1pre:GFP1-10) (Figure 2.5C first row).
To verify these findings we expressed PtTic20C:GFP11 and PtTic20N:GFP11, respec- tively, with PtPlSP1:GFP1-10 in P. tricornutum. In this case we did not detect GFP fluorescence, which fits with the previous results reported (Figure 2.5B and C second row). Similar results were obtained for the orientation experiments with PtTic21. However, we observed GFP fluorescence only when PtTic21N:GFP11 was co- transformed together with PtOEE1pre:GFP1-10 and not with PtSP1:GFP1-10, which means that the N-terminus faces into the stroma (Figure 2.6B and Cfirst row). No GFP expressing cell lines could be obtained yet after co-transformation of P. tricor- nutum with the vectors containing the PtTic21C:GFP11 fusion gene, together with PtOEE1pre:GFP1-10 and PtPlSP1:GFP1-10, respectively.
(A)
01 05 10 15 20 25 30 35 40 45
46 50 56
01 05 10 15 20 25 30 35 40 45 50
51 55 60 65 69 330
01 05 10 15 20 25 30 35 40
50 100 105 110 115 294
01 05 10 15 20 25 30 35 40 45 50
51 55 60 65 70 231
PtOEE1pre:GFP1-10
PtPlSP1full:GFP1-10
PtTic20Cfull:GFP11
PtTic20Nfull:GFP11
: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain
: mature protein
lower case:
GREY: conserved motif at signal peptide cleavage site MKFTAACSLALVASASA
MPTISDHTHACRAANLASSPQRRMVLLLALCLSLVAPSVTA
MGTLPTMKRSLMSIAIAVSVFKGSYA :
MGTLPTMKRSLMSIAIAVSVFKGSYA
FAPIPSVSRTTDLSMSLQKDLANVGKS
FRPSTPSAV RSSVFVSSRTKISWADASS
FVRDLQQKTPTRSA RLR
FVRDLQQKTPTRSARLR MLE R
... 250aa...RHV
SSSAFASQIS...175aa...GPF
SSSAFAS QISRSTNLQKPVGTLYGMWS... 157aa...GPFa
a :
a :
:
: BOLD
UNDERLINED ITALIC
ggsgggs
ggsgggs
ggsgggs
ggsgggs
spacer domain and amino acids introduced by cloning fragments of green fluorescent protein
GFP1-10
GFP11 GFP11
GFP11
BLACK:
DIC Chlorophyll GFP Chl
GFP (B)PtOEE1pre:GFP1-10 + PtTic20Cfull:GFP11
(C)PtOEE1pre:GFP1-10 + PtTic20Nfull:GFP11
DIC Chlorophyll GFP Chl
GFP
DIC Chlorophyll GFP Chl
GFP
DIC Chlorophyll GFP Chl
GFP PtPlSP1full:GFP1-10 + PtTic20Cfull:GFP11
PtPlSP1full:GFP1-10 + PtTic20Nfull:GFP11
Figure 2.5.: Orientation of PtTic20 using the self-assembling GFP system. (A)PtOEE1pre, Pt- PlSP1 and PtTic20 self-assembling GFP fusion constructs. (B)While GFP11 was inserted between the presequence and mature protein of PtTic20, GFP1-10 was fused to the C-terminus of the interenvelope marker PtPlSP1 and to the stromal localised PtOEE1pre protein. GFP fluorescence, including GFP self-assembly when both fragments are localised in the same compartment, was observed when com- bining the PtTic20C:GFP1-10 with the stromal marker PtOEE1pre:GFP11; (C)Whereas GFP11 was fused C-terminally to PtTic20 GFP1-10 was fused to the C-terminus C-terminus of the interenvelope marker PtPlSP1 and to the stromal localised PtOEE1pre protein. GFP fluorescence was observed when combining the PtTic20C:GFP1-10 with the stromal marker PtOEE1pre:GFP11; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence and merged image show- ing the respective channels in the indicated colours are shown from left to right; scale bars represent 10µm.
(B)PtOEE1pre:GFP1-10 + PtTic21Nfull:GFP11
DIC Chlorophyll GFP Chl
GFP
DIC Chlorophyll GFP Chl
GFP PtPlSP1full:GFP1-10 + PtTic21Nfull:GFP11
(A)
01 05 10 15 20 25 30 35 40 45
46 50 56
01 05 10 15 20 25 30 35 40 45 50
51 55 60 65 69 330
01 05 10 15 20 25 30 35 40
50 100 105 110 115 294
01 05 10 15 20 25 30 35 40 45 50
51 55 60 65 70 231
PtOEE1pre:GFP1-10
PtPlSP1full:GFP1-10
PtTic21Cfull:GFP11
PtTic21Nfull:GFP11
: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain
: mature protein
lower case:
GREY: conserved motif at signal peptide cleavage site MKFTAACSLALVASASA
MPTISDHTHACRAANLASSPQRRMVLLLALCLSLVAPSVTA MVNFHSTRIPYGVAAFLCLHQITGA : MVNFHSTRIPYGVAAFLCLHQITGA
FAPIPSVSRTTDLSMSLQKDLANVGKS
FRPSTPSAV RSSVFVSSRTKISWADASS
FQVSYGDRRWTR
FQVSYGDRRWTR MLE R
... 250aa...RHV
QSTVVPNSPERRVTLRVPYSRSTVQRLTTPLLQT...175aa...GPF QSTVVPNSPERRV TLRVPYSRSTVQRLTTPLLQT... 157aa...GPFa
a :
a :
s :
: BOLD
UNDERLINED ITALIC
ggsgggs
ggsgggs
ggsggg
ggsgggs
spacer domain and amino acids introduced by cloning fragments of green fluorescent protein
GFP1-10
GFP11 GFP11
GFP11
BLACK:
Figure 2.6.: Orientation of PtTic21 using the self-assembling GFP system. (A)PtOEE1pre, PtSP1 and PtTic21 self-assembling GFP fusion constructs. (B)While GFP11 was inserted between the pre- sequence and mature protein of PtTic21, GFP1-10 was fused to the C-terminus of the interenvelope marker PtSP1 and to the stromal localised PtOEE1pre protein. GFP fluorescence, including GFP self- assembly when both fragments are localised in the same compartment, was observed when combining the PtTic21C:GFP1-10 with the stromal marker PtOEE1pre:GFP11; Nomarski’s differential interfer- ence contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.
