Abstract
Lipid droplets (LDs) are dynamic organelles for lipid storage and homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs and enzymes required for LD growth and turnover. The small size of LDs precludes the observation of their associated enzyme densities and dynamics with conventional fluorescence microscopy. Here we employ quantitative photo-activated localization microscopy to study the density of the fatty acid (FA) activating enzyme Faa4 on LDs in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs colocalize with the endoplasmic reticulum (ER) where their emergence and expansion are mediated by the highest observed Faa4 densities. During transition to the stationary phase, LDs with a ∼2-fold increased surface area translocate to the vacuolar surface and lumen and exhibit a ∼2.5-fold increase in Faa4 density. The increased Faa4 density on LDs further suggests its role in LD expansion, is caused by its ∼5-fold increased expression level, and is specific to exogenous FA chain-lengths. When lipolysis is induced by refreshed medium, Faa4 shuttles through ER- and lipophagy to the vacuole, where it may activate FAs for membrane expansion and degrade Faa4 to reset its cellular abundance to levels in the log phase.