Jcb_201404115 1..10

Lipid droplets (LDs) are ubiquitous organelles that store triacylglycerol and sterol esters for use by cells to produce energy and membranes (Martin and Parton, 2006; Walther and Farese, 2012). LDs emerge from the ER (Kassan et al., 2013; Pol et al., 2014), and their number, size, and distribution vary under different growth conditions (Yang et al., 2012). LDs are capable of interacting with many organelles (Liu et al., 2008; Pu et al., 2011), which is thought to facilitate lipid transfer. However, the exact mechanisms through which LDs form, grow, and mobilize their contents remain largely unknown. The breakdown of storage lipids within LDs is attributed to various lipid hydrolase activities (Ducharme and Bickel, 2008). Recent evidence suggests that autophagy provides an alternative route for LD breakdown in the hepatocyte (Singh et al., 2009; Singh and Cuervo, 2012). This lipophagy process uses macroautophagy machinery that is mediated by core autophagy-related (Atg) proteins (Xie and Klionsky, 2007; Mizushima et al., 2011) to sequester LDs into forming autophagosomes that subsequently fuse with lysosomes. Lipophagy was also found in yeast. When triacylglycerol is overloaded into LDs by oleate induction, autophagy machinery targets LDs to vacuoles for lipid mobilization (van Zutphen et al., 2014). The yeast lipophagy morphologically resembles microautophagy, which involves a direct modification on the vacuolar membrane that engulfs LDs. Although it seems likely that lipophagy is a selective process, the exact LD targets for lipophagy remain elusive. Most cells, including yeast, spend most of life dealing with many faces of stresses and nutrient deficiency in quiescence. Mimicking the conditions by culturing cells in nutrientrich medium for extended periods has been a valuable model to study cell physiology during stationary phase (stat-phase; Werner-Washburne et al., 1993). Herein, we analyze LD distribution and find that LDs enter the vacuole lumen along with a unique, hydrolase-resistant, membrane during stat-phase. The membrane is the liquid-ordered (Lo) vacuolar microdomain that forms only when cells enter stat-phase (Toulmay and Prinz, 2013). Our data reveal that LD entry into the vacuole requires vacuolar microdomain formation and that the pathway contributes to the vacuolar microdomain maintenance during stat-phase. Stationary phase (stat-phase) is a poorly understood physiological state under which cells arrest proliferation and acquire resistance to multiple stresses. Lipid droplets (LDs), organelles specialized for cellular lipid homeostasis, increase in size and number at the onset of stat-phase. However, little is known about the dynamics of LDs under this condition. In this paper, we reveal the passage of LDs from perinuclear endoplasmic reticulum association to entry into vacuoles during the transition to stat-phase. We show that the process requires the core autophagy machinery and a subset of autophagy-related (Atg) proteins involved in selective autophagy. Notably, the process that we term stat-phase lipophagy is mediated through a sterol-enriched vacuolar microdomain whose formation and integrity directly affect LD translocation. Intriguingly, cells defective in stat-phase lipophagy showed disrupted vacuolar microdomains, implying that LD contents, likely sterol esters, contribute to the maintenance of vacuolar microdomains. Together, we propose a feedforward loop in which lipophagy stimulates vacuolar microdomain formation, which in turn promotes lipophagy during stat-phase. A sterol-enriched vacuolar microdomain mediates stationary phase lipophagy in budding yeast