Structural and functional regulation of endoplasmic reticulum-Golgi contact sites
Despite their early identification, many unknowns regarding the functions and composition of ER-Trans Golgi Network (TGN) membrane contact sites (MCSs) lay unanswered for a long time, due to the lack of visualization methods with optical microscopy. To overcome those limitations, we have developed a FRET-based strategy to follow the ER-TGN MCSs (ERTGoCS) that involves a donor fluorophore (GFP) conjugated to a TGN membrane protein (TGN46) that, when excited, will transfer energy to an acceptor fluorophore (mCherry) conjugated to an ER membrane protein (the C-tail of cytochrome b5, Cb5) provided that the two fluorophores are within a distance of 1-10 nm. We adopted this strategy to run a siRNA-based High Content Screening looking for components and regulators of these structures. We identified, among other components, the VAPA and VAPB proteins as structural determinants for this class of contact sites. Intriguingly, the VAP proteins are not only scaffolding components of ERTGoCS but also play an active role in the regulation of Golgi PI4P levels by interacting directly with the ER-located Sac1 4-phosphatase at ERTGoCS. By contrast, we found that proteins possessing a dual targeting motif to the Golgi and to the ER, such as the oxysterol binding proteins OSBP1 and ORP9, have a redundant role for the maintenance of the ERTGoCS, as only their combined depletion results in the destabilization of ERTGoCS. In addition, depleting ORP10 results in massive ERTGoCS disassembly, due to its ability to transfer phosphatidylserine. Intriguingly, we also observed that ERTGoCS destabilization affects PI4P levels at the TGN. Altering PI4P levels through ERTGoCS selectively regulates the export of some class of cargoes (i.e. ApoB100) compared to others (i.e. albumin or alpha 1 anti-trypsin), providing evidences that ERTGoCS act as “gate-keepers” for selective exiting from the TGN, as well as the clear demonstration that certain PI4P amounts are a crucial requirement for the TGN export of specific cargoes. Next, we also identified regulators (in terms of kinases and phosphatases) of ERTGoCS formation, showing the high complexity of the system. All together, these findings define a novel scenario where ERTGoCS couple lipid homeostasis with trafficking events, as TGN sorting and export.