Molecular determinants of ER–Golgi contacts identified through a new FRET–FLIM system

Abstract

ER–TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.

Structure, functions and regulation of CERT, a lipid‐transfer protein for the delivery of ceramide at the ER–Golgi membrane contact sites

“…). De Matteis and colleagues recently demonstrated that the knockdown of CERT in HeLa cells unexpectedly promotes ER‐Golgi MCS formation . However, CERT might be involved in the formation and maintenance of MCS under the specific conditions described below.…”

Illuminating the membrane contact sites between the endoplasmic reticulum and the trans‐Golgi network

“…Although this was instrumental in identifying one of the functions of ER–TGN contact sites, namely the counter‐exchange of PI4P for cholesterol (see below), this tool artificially induces stable ER–TGN contact sites and is not suitable for screening possible physiological components that establish and maintain the contacts. There was a major breakthrough in ER–Golgi contact site biology very recently as a result of work conducted by Venditti et al ,. which combined the classical electron microscopy approach with innovative fluorescence‐based techniques, thus providing a powerful tool for ER–TGN contact site visualization and study.…”

“…At the level of ER–TGN contact sites, VAPs represent the ER‐anchor for proteins required for ER–TGN contact site formation and maintenance, not only for phosphatidylserine (PS) transfer protein ORP10 and the tethering factors oxysterol‐binding protein 1 (OSBP1) and ORP9 , but also for all the lipid transfer proteins (LTPs) that take advantage of ER–TGN contact sites to mediate non‐vesicular trafficking of lipids between the two membranes, such as ceramide transfer protein (CERT), Nir2 and OSPB1 . As a consequence, the role of VAPs at ER–TGN contact sites is crucial and cells lacking VAPs show a strong impairment in ER–TGN contact site establishment ().…”

“…A few proteins have been described as acting as molecular tethers at ER–TGN contact sites: the ER‐resident VAP proteins (both VAPA and VAPB) and OSBP1 and ORP9 LTPs . VAP (VAMP‐associated proteins, as well as their yeast homologs Scs2 and Scs22) proteins represent a hallmark of other ER‐mediated contact sites in cells .…”

“…(A) CERT acts at the ER–TGN interface driving non‐vesicular trafficking of ceramide from the ER to the TGN . (B) OSBP1 and ORP9 act as redundant tethering factors at ER–TGN contact sites . Additionally, OSBP1 operates a counter‐transport of cholesterol from ER to the TGN with PI4P in the opposite direction to drive in cis dephosphorylation of the latter by its phosphatase Sac1 .…”