An ER‐Golgi tether facilitates ceramide transfer out of the ER and alleviates ceramide toxicity

Ceramide is key intermediate for synthesis of sphingolipids, It is also an important second messenger in signal transduction. However, overproduction of ceramide triggers apoptosis and causes cell death. Our goal is to understand how cell adapt itself from ceramide toxicity stress. Ceramide is synthesized in ER, but is transferred to Golgi to form complex sphingolipids. Such lipid trafficking events can be divided into vesicular and non‐vesicular transport. Non‐vesicular transport usually occurs at Membrane contact sites (MCSs), which are regions where two organelles come into close proximity and are thought to bridge the gap between compartments during lipid transport. We aimed to investigate the role of MCSs in non‐vesicular ceramide transfer between the ER and Golgi. We screened for proteins could serve as potential tethers or facilitate non‐vesicular lipid transport. One promising candidate we identified was Nvj2. In yeast, ER‐to‐Golgi ceramide transfer is partially blocked in mutant with defect in vesicular transfer, such as sec18‐1 ts . Ceramide transfer in this strain occurs only via non‐vesicular transport. Expression of Nvj2 resulted in increased non‐vesicular ceramide transfer in sec18‐1 ts cells. Conversely, knockout of Nvj2 in sec18‐1 ts cells exacerbated the block in ceramide transport. Importantly, Nvj2 is specifically localized at ER/medial‐Golgi junctions, closely juxtaposed to Golgi‐localized Aur1 (the enzyme required for the next step of ceramide conversion to sphingolipid), indicating that Nvj2 might be ER/medial‐Golgi tether that facilitate ceramide transfer by non‐vesicular manner. We further show that Nvj2 alleviates ceramide toxicity through non‐vesicular transfer of ceramide out of the ER. orm1Δ orm2Δ cells are hypersensitive to ER stress due to its unusual ceramide accumulation. Vesicular transport pathway is also slowed down in the strain. We would like to investigate if Nvj2 can alleviate ceramide accumulation by exporting excess ceramide from ER to Golgi. Indeed, overexpression of Nvj2 rescues hypersensitivity of ER stress and also reduces the abnormally high level of unfolded protein response in orm1Δ orm2Δ. Accordingly, we found that Nvj2 alleviates ceramide accumulation in orm1Δ orm2Δ by facilitating ceramide transfer out from ER. Interestingly, we found that Nvj2‐GFP accumulates at ER‐medial‐Golgi contact sites during ER stress, suggesting that Nvj2 is actively recruited to these sites to expedite ceramide export, Taken together, We prefer a model that cells accelerate non‐vesicular transport of ceramide out from ER to adapt from ceramide accumulation stress. Importantly, Nvj2 is conserved in higher eukaryotes and may share a similar function in ER‐to‐Golgi transport of ceramide, an intriguing possibility that warrants further investigation.