Disección genética de transporte de proteínas a vacuolas

Resumen

Vacuoles have essential and specific roles in plants. However, our mechanistic knowledge of how trafficking to, and biogenesis of these organelles occurs in plants is very limited. To gain insight into these matters, we have undertaken a genetic approach to identify the cellular machinery involved in transporting proteins to the vacuole. Using a genetic screen for modified transport to the vacuole (mtv) mutants that secrete the chimeric vacuolar cargo VAC2, we have isolated and characterized three novel genes involved in vacuolar trafficking, MTV1, MTV3 and MTV4. MTV1 encodes for a protein with an Epsin N‐terminal homology (ENTH) domain for binding to phosphoinositides (PIs) and several clathrin‐binding motifs in the C‐terminal part of the protein. MTV4 encodes for the AGD5 protein that contains an Arf GTPase activating domain. MTV1 and MTV4 colocalize in a specific subdomain of the trans Golgi Network (TGN), together with clathrin. Moreover, both MTV1 and MTV4 interact directly with clathrin and are found in clathrin coated vesicles (CCVs). Combined mutations in MTV1 and MTV4 severely disrupt vacuolar trafficking and development. These trafficking defects are coupled to altered TGN morphology and defective distribution of vacuolar sorting receptors (VSRs) that transport vacuolar cargo from the TGN to the prevacuolar compartment (PVC) in CCVs. Altogether, our results support that MTV4 and MTV1 mediate formation of CCVs at the TGN for transport of VSRs and associated vacuolar cargo to the PVC. MTV3 encodes the PTEN2a phosphatase that dephosphorylates the 3' position in phosphatidylinositol 3‐phosphate (PI3P), phosphatidylinositol 3,4 biphosphate and phosphatidylinositol 3,5 biphosphate, which are the PIs that specifically label organelles in the route to the vacuole. MTV3 distributes between the cytosol and the TGN, and the mtv3‐ 1 mutation, which co‐dominantly disrupts vacuolar trafficking, provokes a decrease in TGN association of the protein. We show that concurrent loss of function of MTV3/PTEN2a and its paralogue PTEN2b also results in abnormal secretion of VAC2 and of endogenous vacuolar proteins, indicating that these two phosphatases have a redundant function in transport of these cargoes. Moreover, overexpression of MTV3 fused to GFP or RFP also causes alterations in trafficking and secretion of vacuolar cargo. The vacuolar trafficking defects in the over expressing plants can be ascribed to the missorting of proteins that are essential for transport in this route, such as the VSRs and the TGN localized SNARE VTI12. Moreover, we show that over expression of MTV3 and of a PI3P binding protein (2X FYVE) results in PVC enlargement and altered distribution of PI3P, supporting that MTV3 regulates the allocation of PI species in membranes in vivo, and that this activity is essential for a correct functioning of the vacuolar trafficking pathway.