IEM stroma
2.3. Materials and Methods
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. tricornutum transfor-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
: 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
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
: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain
: mature protein
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)
: 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
spacer domain and amino acids introduced by cloning fragments of green fluorescent protein
GFP1-10
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
: 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
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 autofluorescinterfer-ence, GFP fluorescinterfer-ence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.
Localisation experiments using carbonate extraction
The full length:GFP fusion proteins from PtTic20, PtTic21, PtTic55 and PtTic110 show that these Tics are located in the plastid (Figure 2.15C, 2.16C and 2.21C), but it is predicted that they are membrane proteins with one to four transmembrane spanning helices (Table A.2). In order to demonstrate that these Tic proteins are indeed membrane integrated as it is known for Tic20 [84], Tic21 [149], Tic55 [22] and Tic110 [77] in Pisum sativum and to see where the remaining Tics (Tic22, Tic32 and Tic62) are located in detail, we employed carbonate extraction. Carbonate extraction is a approach for fractionation of integral from membrane associated and soluble proteins. The method is based on the high alkalinity of sodium hydrogen carbonate solution (pH 11,5) to solubilise and strip loosely peripheral associated proteins from insoluble (in the carbonate solution) integral membrane proteins.
First, we checked the fractionation efficiency by western blot analyses with antibod-ies against the D1 and RuBisCo protein. For this, we used protein fractions (including a fraction with soluble proteins, a fraction with peripheral associated membrane pro-teins and a fraction with integral membrane propro-teins) which we get from carbonate extraction of transformed P. tricornutum cells (PtTic20full:GFP, PtTic22full:GFP, PtTic32full:GFP, PtTic55full:GFP, PtTic62full:GFP) and wildtype cell (Figure 2.7).
Because D1 is an integral membrane protein we assumed the strongest signal in the fraction with the integral membrane proteins. In the case of PtTic55full-, PtTic62full-and the wildtype extract we get only one bPtTic62full-and in the integral membrane fraction (Figure 2.7 middle and bottom), which indicates an effective purification. For the samples PtTic20full, PtTic22ful and PtTic32full (Figure 2.7top andmiddle) bands were also observed in the soluble protein and peripheral membrane fraction but with a lower intensity compared to the fraction with the integral membrane protein. Western blot assays using a α-RuBisCo antibody showed the strongest signal in the soluble protein fraction compared to the remaining fractions, which was expected (Figure 2.7).
The intensity of the bands diminishes with the purification steps, which means that we get the weakest signal in the integral membrane fraction.
To analyse whether Tics are soluble, peripheral membrane or integral membrane proteins, we performed western blot analyses using a polyclonal antibody against GFP.
Obtained signals with the expected size are marked with an arrow in Figure 2.8A-D. P. tricornutum transformants expressing the PtTic110full:GFP (∼149 kDa), Pt-Tic55full:GFP (∼ 78 kDa) fusion construct show the strongest signal in the integral membrane fraction with the size we assumed (Figure 2.8C and D). Fusion proteins
PtTic20full PtTic22full sol. per. int. sol. per. int.
10070
sol. per. int. sol. per. int.
10070 sol. per. int. sol. per. int.
10070
sol. per. int. sol. per. int.
10070 sol. per. int. sol. per. int.
10070
sol. per. int. sol. per. int.
10070
Figure 2.7.: Carbonate extraction efficiency test (A)Western blot analyses from carbonate extracted fractions using a D1 antibody. Strongest signal can be observed in the integral membrane fraction (∼
32 kDa). (B)Western blot analyses from carbonate extracted fractions using a RuBisCo antibody.
Strongest signal can be observed in the soluble protein fraction (∼53kDa); soluble protein fraction (sol.), peripheral membrane protein fraction (per.), integral membrane protein fraction (int.)
PtTic21full PtTic20full
sol. per. int. sol. per. int.
100 sol. per. int. sol. per. int.
100
sol. per. int. sol. per. int.
( )A ( )B
Figure 2.8.: Western blot analyses from Tic protein fractions using carbonate extraction (A) PtTic20full:GFP∼48kDaand PtTic21full:GFP∼54kDa;(B)PtTic22full:GFP and PtTic32full:GFP
∼61kDa(C)PtTic55full:GFP∼78kDa, PtTic62full:GFP∼54kDa(D)PtTic110full:GFP∼149 kDa; unspecific signals are indicated with a star; bands with right size are indicated with an arrow;
soluble protein fraction (sol.), peripheral membrane protein fraction (per.), integral membrane protein fraction (int.)
from transformed P. tricornutum cells expressing PtTic20full:GFP (∼ 48 kDa) and PtTic21full:GFP (∼ 54 kDa), which should possess three to four transmembrane he-lices show bands in the integral membrane fraction. In case of PtTic20full:GFP we obtained one band with the expected size and for PtTic21full:GFP a smaller band as expected (Figure 2.8A). As indicated in Figure 2.8C a signal from cell lysates of PtTic62full:GFP (∼ 54kDa) expressing P. tricornutum cells was detected in the soluble protein fraction. Another fusion protein we suggest either in the soluble or peripheral protein fraction, is PtTic22full:GFP (∼ 61 kDa). However, we only ob-served a single signal in the integral fraction, which might be too small (Figure 2.8B).
Furthermore we could detect a band with the expected size of∼61kDain the periph-eral membrane fraction obtained from PtTic32full:GFP expressing cells. Compared to the approach with the control proteins (Figure 2.9AandB) received from wildtype cells, PtTic110pre:GFP and GFP (transformants which express GFP in the cytosol of the cell) expressing P. tricornutum cells, we can exclude several unspecific signals in the western blots, labeled with a star in Figure 2.9A-D. In the protein fractions, we obtained from wildtype cells we observed a band in the soluble and the integral membrane fraction (Figure 2.9A). These bands should be unspecific signals because these cells do not express GFP. In the approach with PtTic110pre:GFP and cell lysate fromP. tricornutum cells, which express GFP in the cytosol, we detected besides the
GFP (27 kDa), which was the only expected signal, further unspecific bands in the
GFP (27 kDa), which was the only expected signal, further unspecific bands in the