ESCRTing during barley endosperm development
Abstract
The endosomal sorting complex required for transport (ESCRT)-0, I, II and III promotes ubiquitin-regulated endocytosis where cargo proteins are sorted into multi vesicular bodies (MVB), finally fusing with the vacuole and releasing their content into its lumen. ESCRT proteins are highly conserved and have been shown to carry out a wide range of functions in plant cells: they are required for cytokinesis, development, vacuolar organization and participate in the ER-Golgi mediated protein sorting machinery.In particular ESCRT-III mediates thebiogenesis of multivesicular bodies (MVBs) by exerting membrane bending, scissionand fusion,and functions in controllingautophagosome fusions, vacuolar integrity and peroxisomal invaginations. The endomembrane system of endosperm tissue is characterized by a high structural plasticity and endosomal activity. Protein trafficking inthese cells is complicated by the presence of several different storage organelles including dynamic protein storage vacuoles (PSVs) and protein bodies (PBs) derived from the endoplasmic reticulum (ER). In addition, trafficking may follow a number of different routes, depending on cell type, developmental stage and environment, showing that the endomembrane system is capable of massive reorganization. In view of the highly dynamic endomembrane system in cereal endosperm cells it is reasonable to expect that ESCRT-III participates in cellular processes in this tissue. First bioinformatics results underline this assumption as there are orthologs of the ESCRT-III proteins present in cereals. In addition, it has been shown that SAL1 (ESCRT-III associated complex protein) is indispensable for cellular processes in maizealeurone, indicating a fuctional significance of ESCRT-III in endosperm tissue. This project therefore aims to further our knowledge about the functional organization of ESCRT-III in barley endosperm tissue andits protein sortingrole in seed storage protein trafficking in barley endospermby cell biological, molecular biological, bioinformatic and biochemical studies. As trafficking pathways of proteins and their final storage in vacuoles are still a matter of debate in cereal endosperm, the results of this project will shed light onhow ESCRT-III will sort seed storage proteins in barley endosperm. Therefore, the focus of this work is to characterize the spatial distribution of ESCRT-IIIwithin the tissue and at the subcellular level and to understand how ESCRT-III will contribute to the sorting of SSPs in barley endosperm. To this end, I will study the co-localization of ESCRT-III with endomembrane markers, identifying possible interactions with parts of the cereal endomembrane system. In addition, I will analyze the barley SSP hordein in ESCRT-III knock down barley seeds. IPs of HosESCRT-III proteins will identify new interaction proteins.