Tic component of the interenvelope space

Tic22 is apperently the only soluble Tic subunit which is located in the interenvelope space between the outer- and innermost envelope membrane of higher plant

chloro-24http://pfam.sanger.ac.uk/

25http://www.ebi.ac.uk/Tools/InterProScan/

plasts. So far, there is very little experimental evidence for soluble components of the translocation machinery within the intermembrane space. Tic22 seems to be responsi-ble for the coordination of Toc and Tic translocons and/or the guidance of imported proteins to the Tic pore [84, 140]. In this approach we have investigated the cellular localisation of Tic22 protein of P. tricornutum and studied its interaction. First we expressed the presequence and full length proteins fused to GFP (PtTic22pre:GFP and PtTic22full:GFP). We observed a GFP fluorescence within the plastid and in the case of the full length protein we additionally 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]. It has been suggested that the BLS phenotype results from targeting of GFP to the periplastidic space [51, 53]. The same phenotype as received when we co-transformed PtTic22full:GFP11 with PtPlSP1full:GFP1-10 (Figure 2.4B), show-ing that a BLS in general can also be observed in the interenvelope space and not only in the periplastidic space. To check whether PtTic22 is soluble, a peripheral membrane or an integral membrane protein we made a carbonate extraction. We assumed a signal in the soluble or the peripheral protein fraction because no trans-membrane domains are predicted, but we observed one in the soluble fraction with the wrong size suggesting this could be GFP (Figure 2.8B). One problem is that the Tic22full:GFP construct still possesses the GFP start codon which might therefore be separately or additionally expressed. Compared to the control experiment the signal we get in the integral membrane fraction might be also an unspecific signal because PtTic22 should not contain any transmembrane helices (Figure 2.8Band Figure 2.9A, B).

In order to investigate protein-protein interactions in vivo we used again the split-GFP system. In this approach we fused PtTic22 to the C-terminal part of the split-GFP and co-transformed it with the PtTic21:NGFP fusion protein, resulting in GFP fluores-cence in the stroma but also in accumulation of GFP in dots (Figure 2.11B), similar to the localisation experiment of PtTic21full:GFP (Figure 2.16C). In higher plants Tic22 was isolated by label transfer experiments that identified proteins in direct contact to presequences during import [84]. We could not observe such an interaction between PtTic22 and a presequence:GFP fusion protein (PtOEE1pre) using split-GFP. But this could be also due to the fact that the P. tricornutum transformants do not ex-press the GFP fragments. Another explanation might be that the interaction between the presequence:GFP fusion protein and PtTic22 is too short and therefore we could

not observe any GFP fluorescence. For this reason it is still thinkable that PtTic22 might act as a guidance protein for preproteins.

A maximum-likelyhood tree drawn from 17 sequences with bootstrap values for 100 replicate trees shows that the interenvelope space located Tic22 form two distinct clades: a red and a green clade. In particular, Tic22 from the diatoms P. tricornu-tum and T. pseudonana branch together with secondary algae (from the red lineage;

containing secondary plastids) like the red alga C. merolae.

Conclusion

The focus of chloroplast protein import research has developed from the identification of components and the aim to study where these subunits are localised within the chloroplast, how these subunits interact and how they operate in regulating protein translocation and biogenesis. In this work we took our focus on identification and first characterisation of translocons at the inner envelope membrane of chloroplasts (Tic) in the diatom P. tricornutum and partly T. pseudonana (Figure 2.14). There is evidence that these proteins are involved in protein targeting across the innermost plastid envelope membrane.

-100 1 100 200 300 400 500 600 800 1000 1200

PtTic20

Figure 2.14.: Schematic representation of all identified Tics inPhaeodactylum tricornutum.The amino acid numbers can be seen on the x-axis at the bottom of the figure. The first residue of the mature protein is labeled as 1, and the bipartite presequence is in negative numbers. Transit peptides are shown as solid lines. Domains in mature proteins are colored. The legend to this is listed on the right side of the figure. Putative domains are marked with a question mark.

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.

PtTic20pre:GFP

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site enhanced green fluorescent protein

Figure 2.15.: Localisation of PtTic20. (A) PtTic20 GFP fusion constructs. (B) expression of Pt-Tic20pre:GFP in Phaeodactylum tricornutum, GFP is imported into the plastid; (C) expression of PtTic20full:GFP inP. tricornutum, GFP labels the silhouette of the plastid and accumulates in dots close to the chloroplast; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluores-cence, GFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.

01 05 10 15 20 25 30 35 40 45 50

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site enhanced green fluorescent protein

Figure 2.16.: Localisation of PtTic21. (A) PtTic21 GFP fusion constructs. (B) expression of Pt-Tic21pre:GFP in Phaeodactylum tricornutum, GFP is imported into the plastid; (C) expression of PtTic21full:GFP in P. tricornutum, GFP labels the silhouette of the plastid and accumulates in dots close to the chloroplast; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluores-cence, GFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.

(A)

01 05 10 15 20 25 30 35 40 45 50

51 55 60 65 70 75 100

01 05 10 15 20 25 30 35 40 45 50

51 55 60 65 70 75 409

PtTic22pre:GFP

PtTic22full:GFP

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site enhanced green fluorescent protein

MVRVSMLVFLSLVGQGTA

MVRVSMLVFLSLVGQGTA

SVEVARPATPTRAAAVSRSLPSKVWRQSRKVL NLSSTFGSATKASPYDRIAGHPVFS... 21aa...VQE

SVEVARPATPTRAAAVSRSLPSKVWRQSRKVL NLSSTFGSATKASPYDRIAGHPVFS... 331aa...RAW

p:

p:

BOLD

UNDERLINED ITALIC

eGFP

eGFP

BLACK:

DIC Chlorophyll GFP Chl

GFP

(B)

PtTic22pre:GFP

DIC Chlorophyll GFP Chl

GFP

(C)

PtTic22full:GFP

Figure 2.17.: Localisation of PtTic22. (A) PtTic22 GFP fusion constructs. (B) expression of Pt-Tic22pre:GFP inPhaeodactylum tricornutum, GFP is imported into the plastid; GFP labels the silhou-ette of the plastid and accumulates in dots close to the chloroplast Nomarski’s differential interference contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence and merged image showing the respec-tive channels in the indicated colours are shown from left to right; scale bars represent10µm.

(A)

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site enhanced green fluorescent protein

Figure 2.18.: Localisation of PtTic32. (A) PtTic32 GFP fusion constructs. (B) expression of Pt-Tic32pre:GFP in Phaeodactylum tricornutum, GFP is imported into the plastid; (C) expression of PtTic32full:GFP in P. tricornutum, GFP is imported into the plastid; 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.

(A)

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site enhanced green fluorescent protein

Figure 2.19.: Localisation of PtTic55. (A) PtTic55 GFP fusion constructs. (B) expression of Pt-Tic55pre:GFP in Phaeodactylum tricornutum, GFP is present in the cytosol; (C) expression of Pt-Tic55full:GFP inP. tricornutum, GFP is imported into the plastid; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence and merged image showing the respec-tive channels in the indicated colours are shown from left to right; scale bars represent10µm.

(A)

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site enhanced green fluorescent protein

DIC Chlorophyll GFP GFPChl

GFP

(B)

PtTic62pre:GFP

DIC Chlorophyll GFP Chl

GFP

(C)

PtTic62full:GFP

Figure 2.20.: Localisation of PtTic62. (A) PtTic62 GFP fusion constructs. (B) expression of Pt-Tic62pre:GFP in Phaeodactylum tricornutum, GFP is imported into the plastid; (C) expression of PtTic62full:GFP in P. tricornutum, GFP is imported into the plastid; 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.

(A)

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

Figure 2.21.: Localisation of PtTic110. (A) PtTic110 GFP fusion constructs. (B) expression of PtTic110pre:GFP in Phaeodactylum tricornutum, GFP is present in the cytosol; (C) expression of PtTic110full:GFP inP. tricornutum, GFP labels the silhouette of the plastid and accumulates in dots close to the chloroplast; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluores-cence, GFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.

Im Dokument Protein Targeting into Diatom Plastids (Seite 53-65)