Analysis of the molecular bases that mediate the interplay between Multisubunit Tethering Complexes and P4-ATPases

Abstract

Vesicle trafficking is fundamental to distribute correctly lipids and proteins among cellular compartments. A complex network of interconnected pathways transport vesicles between organelles to satisfy the cellular demand. Multisubunit Tethering Complexes (MTCs) form a group of 9 protein assemblies that are conserved from yeast to human and that are essential for intracellular trafficking: Exocyst, Dsl1, COG, GARP, HOPS, Corvet, TRAPPI, TRAPPII and TRAPPIII. Each MTC recognizes a specific type of vesicle and tether it to the appropriate acceptor membrane, providing specificity and directionality to the trafficking. My research aimed to do a comparative analysis among all MTCs to shed light on the mechanism of action of these complexes. In my group we have developed PICT (Protein interaction from Imaging of Complexes after Translocation), a high sensitivity technique that allows us to study interactions between two proteins using live-cell imaging. We first conducted an in silico genome-wide screen to identify those proteins that could be functionally related to MTCs. We then used PICT for the protein interactions screen with the proteins selected in the in silico analysis. Given the high sensitivity of the assay and that the experiments were done directly in living cells, this method was prompt to detect interactions that have been overlooked by previous analysis, such as indirect, transient or low abundant interactions. I found that MTCs establish a network of interactions with P4-ATPases, lipid flippases that translocate phospholipids to the cytosolic leaflet of the membranes. P4-ATPases maintain the asymmetric distribution of phospholipids and they also play essential roles in vesicle trafficking. In yeast, there are 5 different P4-ATPases: Dnf1, Dnf2, Dnf2, Drs2 and NeoI. Dnf1 and Drs2 are the ones that concentrate most of the interactions with MTCs, mainly GARP and the TRAPPs. Up to date, no study had observed that MTCs and P4-ATPases might function together. In this Thesis I investigated the molecular mechanism that mediates the interplay in this network of essential components for the trafficking machinery. I found that GARP is responsible to transport Dnf1 from late endosomes to trans-Golgi network during the recycling of Dnf1 to the plasma membrane, a transport step that is essential to maintain the correct distribution of PE in the plasma membrane. Furthermore, I have found that Drs2 is required for the proper functioning of the Cytoplasm-to-Vacuole Targeting (Cvt) pathway, a model historically used to study selective autophagy. When the Cvt pathway is triggered, both TRAPIII and a transmembrane protein named Atg9 reach the phagophore assembly site (PAS) to start the process. The results of my thesis show that, in response to a temperature decrease, the cell induces the binding between TRAPPIII and Drs2 and that this interaction is required to sustain the adequate transport of Atg9 to the PAS. In line with this, I have seen Drs2 is fundamental for the biogenesis of vesicles loaded with Atg9 from endocytic compartments. Altogether, this data evidences a new

function of this flippase in the Cvt pathway that might have important implications in other types of autophagy. Additionally, I identified a short motif in the N-terminal tail of Drs2 that is required for the interaction TRAPPIII-Drs2. This is a motif highly conserved among P4-ATPases and other proteins that bind MTCs, what suggests that it might have a more general role in regulating the trafficking of vesicles that remains to be elucidated.