Functional Analysis of ENTH Proteins in Vacuolar Transport

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Vacuoles of plant cells are essential for a multitude of physiological processes, but the molecular mechanisms of vacuolar protein transport are complex and not fully understood. I showed for the first time that vacuolar cargo can be transported via clathrin coated vesicles (CCVs) from the trans-Golgi network (TGN), and that this route requires the novel protein MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) (Sauer et al., 2013). MTV1 contains an ENTH domain and is structurally similar to EPSIN proteins, which interact with subunits of the adaptor complexes (APs) and form accessory proteins required in early stages of CCV generation. However, MTV1 is phylogenetically very distant from the EPSINs, forming a unique outgroup that is conserved throughout the plant kingdom (Zouhar and Sauer, 2014). Preliminary functional and biochemical data suggest that MTV1 defines a special CCV generating mechanism at the TGN with an important role in stress tolerance, growth and senescence (Sauer, unpublished). Moreover, interaction data indicate an unexpected direct participation also in late events of CCV genesis and vesicle scission (Sauer, unpublished). Because of the apparently unique properties of the MTV1 pathway and its strict conservation within the plant kingdom, an in depth functional and molecular characterization will yield important insights into plant vacuolar transport and, moreover, give novel molecular details about the general process of CCV generation. The proposed project aims to elucidate the unique properties of the MTV1 pathway and define it molecularly. It consists of largely independent modules, so it can be adjusted flexibly depending on the experimental course to ensure a publishable outcome. In a functional part, we will address the requirement for MTV1 for specific physiological functions applying genetic and physiological approaches, with a particular emphasis on stress responses and the functional relation of MTV1 with the other three EPSINs in Arabidopsis. Further, the functional differences and potential redundancy among MTV1 and the EPSINs will be addressed. So far, only limited physiological studies have been performed on EPSIN1, while EPSIN2 and 3 have not been functionally analyzed at all. On the molecular side, the hypothetical interaction of MTV1 with the AP-4 complex will be tested and, if positive, characterized in greater detail, as the AP-4 complex is still little understood in plants. Furthermore, the putative direct interactions of MTV1 with components of the vesicle scission machinery will be verified and analyzed in detail, using molecular and genetic approaches, also involving high and super-resolution microscopy. A direct interaction between EPSIN-like proteins and vesicle scission components has not been reported so far, therefore such a finding would be of broad general interest also beyond the plant field.