Die zentrale Vakuole von Phaeodactylum tricornutum:Charakterisierung und Untersuchung des Proteintransports

Protein transport to vacuoles or lysosomes takes place via the endomembrane system (EMS). The EMS is composed of the endoplasmic reticulum (ER), the Golgi apparatus and endosomes in its simplest constellation. The compartments of the EMS are connected by vesicles. These vesicles bud off the EMS and allow to transfer cargo proteins among the EMS. To find their final destination proteins need specific targeting signals located at the N-terminus, C-terminus or within the protein sequence. Those targeting signals can be recognized either by receptors or adaptor protein complexes, which mediates the transport of cargo proteins via clathrin coated vesicles in the late secretory pathway. However, Targeting of proteins to the central vacuole of the diatom Phaeodactylum tricornutum is poorly investigated. As part of this study the protein composition of the central vacuole was analyzed by using in silico and in vivo approaches. Putative vacuolar proteins were investigated by bioinformatic analyses regarding to N-terminal targeting sequences, trans-membrane domains, conserved protein domains and putative targeting signals. Subsequently, the candidates were expressed as eGFP fusion proteins for investigating their subcellular localization. Beside the already known vacuolar membrane proteins in P. tricornutum, this screen resulted in the identification of nine proteins in total new vacuolar membrane proteins, which might be involved in import or export of secondary metabolites, cations and carbohydrates. Apart from storing chrysolaminarin, the results of this work indicate that the central vacuole is a lyctic compartment in respect to protein degradation and detoxification of xenobiotics. A further part of this study dealt with the investigation of protein targeting to the central vacuole as well as the identification of targeting motifs. By generating mutated versions of the vacuolar membrane located proteins, the results indicate that tyrosine and dileucine motifs are relevant for vacuolar transport. Furthermore, the results suggest that the protein transport to the central vacuole of P. tricornutum are comparable to those in plants or mammals.