Transport processes across multiple membranes
Zusammenfassung der Projektergebnisse
To study mechanisms of protein import in the diatom Phaeodactylum tricornutum, molecular tools, biochemical approaches, sequence information, bioinformatic analyses as well as electron microscopy were combined. Via immunoelectron microscopy we were able to display the structure of the membranes surrounding the plastids especially in the areas where the periplastidic space is enlarged. However we were not able to localize GFP intracellularly on the ultrastructural level via photooxidation of diaminobenzidine (DAB). Although we tried to optimize the procedure background staining also in wild type cells was too strong to allow a clear identification of GFP within the periplastidic space. In order to characterize the Tic complex in diatoms, we have demonstrated the presence of several components within the inner membrane of the P. tricornutum plastids. We showed that PtTic20, PtTic55 and PtTic110 are integral membrane proteins, while PtTic32 is a peripheral membrane protein and PtTic62 a soluble protein. Results obtained in orientation studies (using a self-assembling GFP system), 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 inter-envelope space) as an integral membrane protein localized in the innermost membrane. By utilizing different native and artificial presequence constructs we were able to show that within the periplastidic space a cleavage enzyme might be located which does not distinguish between native and modified bipartite topogenic signals without “ASAFAP”-motif. GFP targeting experiments with heterologous presequences indicate that bipartite presequences from other diatoms, from brown algae, dinoflagellates, cryptophytes and haptophytes are functional as plastid import signals indicative for a common import system across the phyla borders. Furthermore we used transit peptides from a glaucophyte, a chlorophyte and a red alga, after addition of an N-terminal signal peptide domain, the resulting presequences from all these phyla were found to be functional for protein transport into P. tricornutum plastids. In order to investigate whether protein targeting across the periplastidic space might occur via pores, we used a self-assembling GFP system. We fused the GFP1-10 and GFP11 fragments to a CER-lumenal marker protein and an inter-envelope 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, indicating that the GFP1-10 and GFP11 fusion proteins are located in two separate compartments that are not connected to each other. Finally we have studied plastidic nucleotide translocators in P. tricornutum. We identified a high number of putative nucleotide transporters (NTTs) in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum and characterized the first 2 isoforms (NTT1 and NTT2). We found that heterologously expressed NTT1 acts as a proton-dependent adenine nucleotide importer, whereas NTT2 facilitates the counter exchange of (deoxy-)nucleoside triphosphates. Therefore, these transporters functionally resemble NTTs from obligate intracellular bacteria with an impaired nucleotide metabolism rather than ATP/ADP exchanging NTTs from primary plastids.
Projektbezogene Publikationen (Auswahl)
- (2008) A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis. PLoS ONE, 3, e1426
Kroth PG, Chiovitti A, Gruber A, Martin-Jezequel V, Mock T, Parker MS, Stanley MS, Kaplan A, et al.
- (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature, 456, 239-244
Bowler C et al.
- (2009) Diatom plastids depend on nucleotide import from the cytosol. Proc. Natl. Acad. Sci. USA 106: 3621-3626
Ast M, Gruber A, Schmitz-Esser S, Neuhaus H-E, Kroth PG, Horn M, Haferkamp I
- (2009) Distribution and localization of thioredoxins in the diatom Phaeodactylum tricornutum. Molecular Plant 2: 468-477
Weber, T, Gruber, A. and Kroth, PG
- (2009) Intracellular distribution of the reductive and oxidative pentose phosphate pathways in two diatoms. J. Basic Microbiol. 49: 58-72
Gruber A, Weber T, Río Bártulos C, Vugrinec S, Kroth PG
