Nitrogen starvation-mediated reduction of Ypk1 is suggested to suppress translational initiation, possibly in parallel with the target of rapamycin complex 1 (TORC1) signaling. However, the molecular mechanism that regulates Ypk1 in nitrogen-starved cells is poorly understood. Here we report that Ypk1 is a novel selective substrate for nitrogen starvation-triggered proteolysis requiring autophagy system. Among various nutrient starvation methods used to elicit autophagy, rapid Ypk1 degradation was specific to nitrogen starvation. In screening genes required for such nitrogen starvation-specific vacuolar proteolysis, we found that autophagy-related degradation of Ypk1 depended on the endosomal sorting complex required for transport (ESCRT) machinery, which is conventionally thought to function in endosomal trafficking. In microscopic analyses, the disruption of ESCRT subunits resulted in the accumulation of both Ypk1 and autophagosomal Atg8 at a perivacuolar site that was distinct from conventional endosomes. ESCRT machinery was not involved in autophagic flux induced by the TORC1 inhibitor rapamycin, thus suggesting that ESCRT represents an exclusive mechanism of nitrogen starvation-specific proteolysis of Ypk1. Overall, we propose a novel regulation of Ypk1 that is specific to nitrogen limitation.Cells control their proliferation and growth by modulating anabolic and catabolic processes in response to extracellular nutrient availability. Nutrient starvation stops cellular anabolic processes by rapidly inhibiting ribosome biogenesis and protein translation to conserve limited nutrients. Concomitantly, starvation induces a cellular catabolic process, autophagy, to supply limited nutrients. The cellular response to starvation is regulated, at least in part, by the evolutionally and structurally well conserved target of rapamycin complex 1 (TORC1), 3 which plays pivotal roles in the regulation of cellular proliferation and growth in nutrient-responsive signaling (1-4). Both nutrient limitation and blocking of TORC1 by rapamycin induce autophagy (5, 6); therefore, rapamycin-mediated autophagy is considered to mimic that of nutrient starvation, and a clear distinction between the two is not well documented.YPK1 encodes a serine/threonine protein kinase belonging to the cyclic AMP-dependent protein kinase/protein kinase G/protein kinase C (AGC) kinase family. Although a direct substrate of this yeast protein kinase has not been identified, loss of Ypk1 causes several cellular deficiencies in actin cytoskeletal organization (7) and endocytosis (8) as well as resistance to ISP-1/myriocin, a potent inhibitor of sphingolipid biosynthesis (9). As a common feature of AGC kinases, Ypk1 kinase activity is regulated by three phosphorylations at the T-loop, turn motif, and hydrophobic motif. Phosphorylation of Ypk1 at the T-loop is regulated by Pkh1/2 kinases, yeast homologs of 3-phosphoinositide-dependent kinase (PDK1) (10), and those at the turn and hydrophobic motifs are mediated by rapamycininsensitive TORC2 (11).Although Y...

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