Tricalbins Are Required for Non-vesicular Ceramide Transport at ER-Golgi Contacts and Modulate Lipid Droplet Biogenesis

Abstract

Lipid composition varies among organelles, and the distinct lipid composition is important for specific functions of each membrane. Lipid transport between organelles, which is critical for the maintenance of membrane lipid composition, occurs by either vesicular or non-vesicular mechanisms. In yeast, ceramide synthesized in the endoplasmic reticulum (ER) is transported to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed. Here we show that a fraction of Tcb3p, a yeast tricalbin protein, localizes to ER-Golgi contact sites. Tcb3p and their homologs Tcb1p and Tcb2p are required for formation of ER-Golgi contacts and non-vesicular ceramide transport. Absence of Tcb1p, Tcb2p, and Tcb3p increases acylceramide synthesis and subsequent lipid droplet (LD) formation. As LD can sequester excess lipids, we propose that tricalbins act as regulators of ceramide transport at ER-Golgi contact sites to help reduce a potentially toxic accumulation of ceramides.

Protocol for measuring sphingolipid metabolism in budding yeast

“…This protocol was used in a recent publication ( Ikeda et al., 2020 ) to study the roles of tricalbins in the transport of ceramide in living Saccharomyces cerevisiae cells. Ceramide is delivered to the luminal side of the Golgi apparatus and converted into IPC by IPC synthase catalyzing the transfer of phosphoinositol from phosphatidylinositol to ceramide.…”

“…After extraction from the silica, the lipids were analyzed by TLC using solvent system III (right). (A–C) The figures are reprinted with permission from Ikeda et al., 2020 . …”

“… (A–C) The graphs show the quantified results as described in the Quantification and Statistical Analysis section. The figures are reprinted with permission from Ikeda et al., 2020 . …”

“…This protocol for metabolic labeling can be used to investigate ceramide transport in an in vivo environment. For complete details on the use and execution of this protocol, please refer to Ikeda et al. (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to Ikeda et al. (2020) .…”