Sascha Vugrinec
∗, Ansgar Gruber, Peter G. Kroth
Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany
∗Author for correspondence. E-mail: sascha.vugrinec@uni-konstanz.de
5.1. Abstract
Plastids of diatoms are surrounded by four membranes, with the outermost being continuous with the endoplasmic reticulum. This complex ultrastructure complicates the transport of nucleus-encoded proteins to the plastid compared to higher plants (where only two membranes have to be crossed by proteins that enter the plastid stroma from the cytosol).
At the moment, several models for the import of proteins into the complex plastids of diatoms are discussed. All models postulate co-translational transport into the chloroplast endoplasmic reticulum (CER) via the Sec Translocon as the first step of protein transport to the plastid, and all models postulate transport via a translocator similar to the translocon of the inner chloroplast membrane (Tic) of higher plant plas-tids as the final import step across the innermost membrane. The models differ in the way they explain transport out of the CER lumen and into the interenvelope space:
The translocator model proposes that proteins are transferred from the CER lumen to the interenvelope space by additional translocators in the second and third mem-branes counted from outside. Alternatively, the vesicular shuttle model proposes that proteins are transported from the CER lumen across the periplastidic space not by translocators, but via vesicles, from which they are released directly into the interen-velope space. Alternatively, it might be possible, that proteins cross the periplastidic space through a pore, formed by a connection between the CER lumen and the in-terenvelope space. To find out whether such a connection between the CER-lumen and the interenvelope space exists, we used the self-assembling GFP system. We fused the GFP1-10 and GFP11 fragments to a CER-lumenal marker protein and a interen-velope space marker protein, respectively. The two GFP fragments self-assemble to a fluorescing unit when present in the same compartment. However, we could not observe fluorescence, in our transformed cell lines expressing the two GFP fragments.
This means that the GFP1-10 and GFP11 fusion proteins are localised in two sepa-rate compartments that are not connected to each other in a way that the two GFP fragments can complement each other for fluorescence.
Keywords
chloroplast· protein import· diatom
5.2. Introduction
Diatoms likePhaedactylum tricornutum or Thalassiosira pseudonana possess a more complex ultrastructure than higher plants. This can be explained with the endosym-biotic origin of plastids. Plastids in general most likely evolved by a process called endocytobiosis, the uptake of a free living cyanobacterium into a heterotrophic eukary-otic cell, followed by the reduction of the endosymbiont to an organelle (see chapter 1, Figure 1.1). The resulting primary plastids are monophyletic and found in the recent glaucophytes, rhodophytes, chlorophytes and land vascular plants [100, 111].
Diatoms and other groups of algae possess secondary plastids which originate from a secondary endocytobiosis, which means the uptake of a eukaryotic alga possessing primary plastids into another eukaryotic host cell. This endosymbiotic alga again was subsequently reduced, now functioning as a photosynthetic organelle within the host cell. Diatom plastids are surrounded by four membranes, with the outermost being continuous with the endoplasmic reticulum [24, 105, 25]. Compared to higher plants, nucleus-encoded plastid proteins from diatoms need to cross four envelope membranes, instead of two, which is more complicated.
While the general transport machinery of primary plastids has mostly been solved, the process of protein targeting into complex plastids of diatoms and related algae is still unclear. A “translocator model” [27, 49, 87] and a “vesicular shuttle model” [47]
are discussed (Figure 5.1). Both models have in common that nucleus encoded plas-tid proteins are proposed to cross the lumen of the chloroplast endoplasmic reticulum (CER) membrane co-translationally and to cross the innermost envelope membrane by a translocon similar to the Tic apparatus (translocon at the inner envelope mem-brane of chloroplasts) found in higher plants. The two models differ in the way how preproteins are proposed to be transported across the second and the third outermost membrane and through the periplastidic space. The “translocator model” postulates import into the periplastidic space by an additional translocator in the second mem-brane (counted from outside), followed by transport into the plastid stroma via two additional translocators similar to the Toc and Tic translocators in land plant chloro-plasts (Figure 5.1). The “vesicular shuttle model” postulates, that proteins cross the periplastidic space via vesicles and are released into the inter-envelope space (Fig-ure 5.1), where they might cross the innermost plastid membrane by a Tic-related translocon. It was speculated by Gruber [52] that co-translational protein transport across the CER membrane and Tic mediated transport across the innermost plastid
en-CER lumen
pps
ies
stroma cytosol
Tic
Tic Tic
Omp85?
SELMA?
Sec61 Sec61
SP
SP FTP mature protein
FTP mature protein
mature protein FTP
(A) (B) (C)
Sec61
Figure 5.1.: Hypothetic import pathways into complex plastids. (A)In the “vesicular shuttle model”, proteins are transported co-translationally into the chloroplast endoplasmic reticulum (CER) lumen, then cross the periplastidic space (pps) via vesicles and are released into the interenvelope space (ies).
Final import occurs via Tic translocator. (B)In the “translocator model”, proteins are transferred into the pps by an additional translocator in the second outermost membrane followed by transport into the plastid stroma via two additional translocators similar to the Toc and Tic in higher plants;(C)In the “pore model”, proteins cross the pps via a pore.
velope alone would be sufficient for the import of nucleus encoded proteins into diatom plastids, if a direct connection between the CER and the inter-envelope space should exist (Figure 5.1). This assumption contradicts the current view of the evolutionary origin of the plastid membranes (compare to [25]). However, it should be noted that to our knowledge for secondary plastids, no complete high resolution three dimensional model of the surrounding membranes is available and that membrane topologies result-ing in a compartment that appears to be surrounded by four membranes in ultrathin sections are also possible with less than four membranes [83]. Despite the advances in characterisation of the targeting signals that mediate transport of pre proteins into the plastids [82, 53, 43, 124], not all mechanisms involved in protein targeting into diatom plastids are clarified experimentally. There is experimental evidence for the
co-translational ER import step [92], while the mechanisms of the remaining steps remain hypothetical in all cases. On the one hand, the observation of vesicular struc-tures between the second and the third plastid membrane of various algae led to the hypothesis that these vesicles might be involved in the transport of proteins from the CER to the interenvelope space [47]. This hypothesis is additionally supported by the finding that the amount of vesicular structures in the periplastidic space de-creases when biosynthesis of nucleus encoded proteins is inhibited, possibly due to a lower abundance of transport substrates [47]. Furthermore, it has been shown that application of Brefeldin A blocks transport of preproteins to the stroma [82]. On the other hand, it has been shown that the transit peptide-like domains of presequences from diatoms and cryptophytes are recognised by the Toc system of plant plastids in heterologous in vitro import experiments [92, 156], which indicates that the complete transport of proteins to the stroma involves additional translocators. Furthermore a duplicated plastid associated ERAD system (also referred to as symbiont specific ERAD-like machinery, SELMA) has been identified in algae with secondary plastids, and it has been proposed that this system might be involved in transporting proteins from the CER-lumen to the periplastidic space [142, 61, 62]. Recently, a Toc75 re-lated protein (Omp85) has been identified in the diatom P. tricornutum [160]. This protein is possibly located in the third plastid membrane (counted from outside) and was proposed to be involved in protein translocation across this membrane [20].
To study whether there is a direct connection between the CER-lumen and the pps we used a self-assembling GFP system. A specific feature of this system is that the C-terminal β-strand of GFP (GFP11) is separated from the rest of the GFP protein (GFP1-10) and neither fragment alone is fluorescening. If, however, both fragments are localised in the same compartment they can spontaneously reassemble, resulting in GFP fluorescence. For that we fused the presequence from a CER-lumenal marker protein (PtBIP) to a small GFP fragment (GFP11) and a protein which is located in the interenvelope space (PtPlSP1) to the complementary GFP part (GFP1-10).
5.3. Materials and Methods
5.3.1. 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 (16.6g·L−1), 50 % concentration compared to natural seawater. Cells were grown in liquid culture in flasks under rigorous shaking (120 rpm) at 20◦C with continuous illumination at 35µmol·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 (BD, Sparks, MD, USA).
5.3.2. PCR and construction of the plasmids
For the construction of the self-assembling GFP fusion proteins, the GFP1-10 (GFP1-10 representing the ß-strands 1-(GFP1-10 of GFP) and GFP11 (GFP11 representing ß-strand 11 of GFP) were amplified from the plasmids delivered with the Mammalian Opti-mized Split GFP Detection System (Sandia BioTech, Inc. Albuquerque, NM, USA) [21], introducing recognition sites for the restriction enzyme EcoRI. A modified version of the shuttle vector pPha-T1 (GenBank: AF219942.1, [162]), in which the fcpA pro-moter was exchanged against the propro-moter of thePhaeodactylum tricornutum nitrate reductase ([62], kindly provided by Stefan Zauner/Philipps-Universitat Marburg), was linearised using EcoRI. The modified GFP1-10/GFP11 fragments were subsequently ligated into this plasmid and screened for correct orientation in relation to the nitrate reductase promoter. The resulting plasmids pPha-T1-GFP1-10 and pPha-T1-GFP11 were supplemented with spacer domains (derived amino acid sequence: GGSGGGS) via round circle PCRs. In the case of pPha-T1-GFP1-10 the sequence encoding the spacer domain contains one recognition site for AfeI. In the case of pPha-T1-GFP11, the spacer domain encoding sequence includes a frame shift stuffer with three transla-tional stops (one in each frame) and recognition sites for AflII and AfeI. Sequences of interest were PCR amplified fromP. tricornutumcDNA. The PtPlSP1full (Protein ID 17972 in theP. tricornutumgenome database JGI1, [16]) and PtOEE1pre (Protein ID 20331) inserts were cloned into the pPha-T1-GFP1-10 vector via the AfeI restriction
1http://www.jgi.doe.gov/
site, the PtBIPpre (Protein ID 54246) and PtATPCpre (Protein ID 20657) inserts were cloned into the pPha-T1-GFP11 vector via the AfeI and AflII restriction site.
All constructs were sequenced (GATC Biotech AG, Konstanz, Germany) from their 5’ end, to ensure correct cloning.
5.3.3. Nuclear transformation
Nuclear transformation of P. tricornutum has been performed using a Bio Rad Bi-olistic PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA, USA) fitted with 1350 psi rupture discs as described previously [2, 88]. For the selection and cul-tivation of P. tricornutum transformants 75 µg·mL−1 Zeocin (Invitrogen, Carlsbad, CA, USA) was added to the solid medium. The plates were incubated at 20◦C under continuous illumination (35µmol·photons·m−2 ·s−1) for three weeks.
5.3.4. 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.
5.4. Results and Discussion
The genomes of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudo-nana encode putative components of the Tic apparatus (see chapter 2), but inter-estingly no Toc subunits were identified so far [150, 107]. Although an ERAD-like machinery and an Omp85 protein have been identified and were proposed to be lo-cated in the second and third outermost membrane, respectively, their actual function has not been proven yet [20, 61, 142]. Furthermore no gene encoding proteins that might be involved in vesicular transport through the pps, could be detected up to now. It can therefore also be proposed that new or modified systems account for the protein transport across the second and the third outermost membrane and neither vesicles nor translocons are involved. At the moment, there is little doubt about the co-translational import via the Sec61 complex across the outermost plastid membrane, and since we characterised Tic components in diatoms (see chapter 2), it seems that they route proteins across the innermost membrane. Based on the assumption that these two steps would be enough for protein import and a direct connection between the chloroplast endoplasmic reticulum and the ies exists, Gruber [52] suggested a third import way: the “pore model”. The special feature of this model is that nucleus-encoded plastid proteins cross the pps through a pore, which developed from the fusion of the periplastid membrane [25] with the second innermost membrane which is the former red algal cytosol.
To investigate whether there is such a connection we used the self-assembling GFP system. To check the functionality of this system we made control constructs. As a positive control for this system 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 5.2C). PtOEE1 is normally targeted into the thylakoids; for a better visualisation of GFP fluorescence, the third targeting domain responsible for thylakoid targeting has been deleted [82]. As a negative 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 5.2D).
Here we could not observe any GFP fluorescence, indicating that both GFP fragments are localised in different compartments. P. tricornutum transformants expressing Pt-BIPpre:GFP11 (PtBIP: CER-lumenal marker protein) and the interenvelope space marker protein PtPlSP1full:GFP1-10 lead to no detectable GFP fluorescence. If there is a pore within the periplastidic space, we should observe GFP fluorescence. But
P. tricornutum transformants expressing PtBIPpre:GFP1-10 fusion construct and Pt-PlSP1full:GFP11 lead to no GFP fluorescence within the plastid (Figure 5.2B). Based on the fact that we could only observe a fluorescence when both GFP fragments are expressed in the same compartment, it is possible that no connection exists between the CER and the ies. However, it is also thinkable that only one of the GFP fragments
(A)
signal peptide predicted by SignalP´s hidden Markov models estimated transit peptide domain
mature protein
spacer domain and amino acids introduced by cloning conserved motif at signal peptide cleavage site fragments of green fluorescent protein
MPTISDHTHACRAANLASSPQRRMVLLLALCLSLVAPSVTA
Figure 5.2.: Pore-test using self-assembling GFP system. (A) PtPlSP1full:GFP1-10 and PtBIP-pre:GFP11 fusion constructs; (B)no GFP fluorescence can be observe, GFP1-10 and GFP11 fusion proteins might be lokalised in two separate compartments;(C) GFP fluorescence, including GFP self-assembly when both fragments are localised in the same compartment, was observed when combining PtOEE1pre:GFP1-10 with PtATPCfull:GFP11; (D)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 dif-ferential interference contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence, CFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right;
scale bars represent 10µm.
is expressed. To check this we made a control experiment where we tried to detect both GFP pieces with a GFP antibody, but without success (data not shown). The
GFP antibody could only bind to one GFP piece. It might as well be possible, that only a small amount of PtBIPpre:GFP1-10 and PtSP1:GFP11 was expressed inP. tri-cornutum which might be not enough for a detectable GFP fluorescence. Although we repeated the experiment three times (at an average of twenty transformants, where we should expect at least one positive clone) it is not sure whether a pore exists within the pps or not.
Another argument against the presence of a pore could be, that if a connection between the CER-lumen and the ies would exist, the pps would not enclose the whole plastid, which means that the cellular integrity of the secondary endosymbiont would not be given. However, there is no reason to suppose that the endosymbiont was retained as a whole during secondary endocytobiosis. It might be also thinkable that organelles of the endosymbiont were acquired subsequently or individually similar to kleptoplasts [52]. Kleptoplasty (or “stealing” of chloroplasts) is an event which describes the behavior of a group of organisms which are able to engulf algal cells and degrade the cells, but not the chloroplasts contained within the cells [131]. The chloroplasts remain functional for some period of time during which the photosyn-thetic products generated by the sequestered chloroplasts are utilised by the new
“host” [131].
A model of secondary plastids which is surrounded by four envelope membranes was derived mostly from electron microscopy producing two dimensional images of ultrathin sections. Continuity of the periplastid membrane with the outer envelope membrane or a connection between the pps and ies can miss our attention, if they might mistakenly be interpreted as preparation artifacts or the resulting clear struc-tures are too small to be included within ultrathin sections frequently. In theory it is possible to imagine an “almost completely” four membrane bound plastid, that is surrounded by four, three or two membranes, as outlined by Köhler [83]. The prob-lems in determining membrane structure of secondary plastids by electron microscopy are described by the discussion about the exact number of membranes surrounding the non-photosynthetic apicoplast inToxoplasma gondii [155]. Three or four envelope membranes were reported to surround the apicoplast in ultrathin sections, but this does not necessarily mean that this is the actual number of membranes that have to be overbear from the cytosol into the apicoplast. Electron microscopical investigation of serial ultrathin sections of the apicomplex Toxoplasma gondii tachyzoites, made by Köhler [83], discovered thatT. gondii apicoplasts are not entirely surrounded by four membranes. Extensive sectors of the apicoplast were also separated from the
cytosol by two membranes [83]. Up to now, no complete three dimensional model of secondary plastids with a high resolution of the surrounding membranes is available, and therefore, local membrane fusions of the four surrounding membranes cannot be completely ruled out.
Conclusion
Based on the fact that neither the “vesicular shuttle model” nor the “translocator model” are finally experimentally proven, leaves space for a further model the “pore model”, which Gruber proposed [52]. Our results show, that it is possible that no connection between the chloroplast endoplasmic reticulum lumen and the interenve-lope space exists. This still leaves space for further speculations whether vesicles, translocons or pores are involved in the import of nucleus-encoded plastid targeted proteins.
Acknowledgements
This work was supported by the Universität Konstanz and the DFG. We thank D.
Ballert for technical assistance as well as Uwe G. Maier group (Philipps-Universität Marburg, Marburg, Germany) for kindly providing templates for NR-Vector.