Selektiver Transport zur lytischen Vakuole und Degradation von Membranproteinen

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Plant endosomal and prevacuolar compartments (EPVCs) are considered to be an intermediate compartment en route to the lytic vacuole. In analogy to what is known from animal cells, it is considered that EPVCs receive proteins from the Golgi- Apparatus in a receptor-mediated transport mode and subsequently deliver them to the vacuole. Whilst VSRs are shuttled back to the Golgi-Apparatus, membrane proteins destined for degradation are thought to be separated into the internal vesicles of these multivesicular structures, which are then discharged into the lumen of the vacuole (MVB pathway). In contrast to animals, plant cells possess different types of vacuolar compartments, suggesting complex sorting mechanisms for plant EPVCs. We propose a combined biochemical, genetical and ultrastructural approach to:1) analyse the role of retromer for transport of soluble vacuolar proteins as well as for the steady-state distribution of VSRs, using quantitative protein transport assays in combination with RNA interference (RNAi) to induce post-transcriptional silencing of genes encoding for retromer subunits Vps35, Vps29 and Vps26 in vivo.2) characterise plant ESCRT homologues as a contribution towards understanding the molecular machinery responsible for sorting of membrane proteins into luminal vesicles of the MVBs. Here, we will clone Arabidopsis ESCRT genes to prepare plant specific antibodies as a prerequisite to conduct ultrastructural analysis and immunolocalization studies. To establish the function of plant ESCRT genes, we will characterize available Arabidopsis T-DNA insertion mutants using quantitative protein transport assays in combination with functional in vivo complementation experiments.3) determine, whether VSRs are turned-over via the MVB pathway. Therefore, we will analyse the half-live time of VSRs using pulse-chase experiments in combination with transiently expressed dominant negative mutants of the ESCRT machinery.