The Vacuole Lipid Droplet Contact Site vCLIP

Abstract

Lipid droplets frequently form contact sites with the membrane of the vacuole, the lysosome-like organelle in yeast. These vacuole lipid droplet (vCLIP) contact sites respond strongly to metabolic cues: while only a subset of lipid droplets is bound to the vacuole when nutrients are abundant, other metabolic states induce stronger contact site formation. Physical lipid droplet-vacuole binding is related to the process of lipophagy, a lipid droplet-specific form of microautophagy. The molecular basis for the formation and function of vCLIP contact sites remained enigmatic for a long time. This knowledge gap was filled when it was found that vCLIP is formed by the structurally related lipid droplet tether proteins Ldo16 and Ldo45, and the vacuolar surface protein Vac8. Ldo45 additionally recruits the phosphatidylinositol transfer protein Pdr16 to vCLIP. Here, we review the literature on the lipid droplet-vacuole contact site in light of the progress in our understanding of its molecular basis and discuss future directions for the field.

Keywords: LDAF1; Ldo16; Ldo45; Pdr16; Vac8; contact site; lipid droplet; lipophagy; lysosome; seipin; vCLIP; vacuole.

Figure 1.

The vacuole lipid droplet contact site machinery vCLIP. (Left) Schematic representation of lipid droplet (LD) contacts with endoplasmic reticulum (ER) and vacuole membranes depending on metabolic states. At nutrient repletion, most lipid droplets are not bound to the vacuole. Early in glucose exhaustion, lipid droplets accumulate at the nucleus-vacuole junction NVJ by forming vacuole lipid droplet (vCLIP) contact sites. vCLIPs expand when starvation conditions persist, eventually leading to lipid droplet internalization into the vacuole by lipophagy. (Right) Schematic representation of the proteins involved in lipid droplet accumulation at the NVJ. Nvj1- Vac8 and Ldo16/45-Vac8 form the physical basis of the NVJ and the vCLIP, respectively. The two Ldo proteins are structurally related and share the same Vac8 binding domain. Ldo45 additionally comprises an N-terminal extension which recruits the phosphatidylinositol transfer protein Pdr16. The NVJ tether protein Mdm1 resides in the periphery of the NVJ, where it demarcates sites of lipid droplet formation.

Figure 2.

The Lipid Droplet Organization proteins localize to sites of tight vacuole-lipid droplet contact. (A) Cells expressing Ldo-GFP and the lipid droplet (LD) marker Erg6-mCherry were cultured overnight in synthetic medium with 2% glucose, and then transferred to glucose-free medium containing 0.2% oleic acid for 24 hours. Cells were stained with the vacuole lumen dye CMAC (7-amino-4-chloromethylcoumarin) and imaged on a ZEISS LSM980 Airyscan microscope. The Ldo proteins act as lipid droplet-vacuole tethers and are visible as defined foci at vacuole lipid droplet (vCLIP) contact sites. Scale bar, 5 µm. (B) Cells expressing Ldo-GFP and the vacuole membrane marker Vph1-mCherry were treated and analyzed as described in (A). Lipid droplets were visualized using the neutral lipid dye MDH (monodansylpentane). An untethered lipid droplet (red arrowhead), a vCLIP-engaged lipid droplet (yellow arrowhead), and a lipid droplet internalized into the vacuole (white arrowhead) are visible. Scale bar, 5 µm.