Autophagy is the process whereby cytoplasmic cargo (e.
g., protein and organelles) are sequestered within a double membrane-enclosed transport vesicle and degraded after vesicle fusion with the vacuole/lysosome. Current evidence suggests that the
Vps34 phosphatidylinositol 3-kinase is essential for macroautophagy, a
starvation-induced autophagy pathway (Kihara et al., 2001). Here, we characterize a requirement for Vps34 in constitutive autophagy by the cytoplasm-to-vacuole targeting (Cvt) pathway. First, we show that transient disruption of
phosphatidylinositol (
PtdIns) 3-phosphate (
PtdIns[3]P) synthesis through inactivation of temperature-sensitive Vps34 or its upstream activator, Vps15, blocks the Cvt and macroautophagy pathways. Yet, PtdIns(3)P-binding FYVE domain-containing
proteins, which mediate
carboxypeptidase Y (CPY) transport to the vacuole by the CPY pathway, do not account for the requirement of Vps34 in autophagy. Using a genetic selection designed to isolate PtdIns(3)P-binding effectors of Vps34, we identify Etf1, an uncharacterized type II transmembrane
protein. Although Etf1 does not contain a known 3-phosphoinositide-binding domain (i.e., FYVE or
Phox), we find that Etf1 interacts with
PtdIns(3)P and that this interaction requires a
basic amino acid motif (KKPAKK) within the cytosolic region of the
protein. Moreover, deletion of ETF1 or mutation of the KKPAKK motif results in strong sorting defects in the Cvt pathway but not in macroautophagy or in CPY sorting. We propose that Vps34 regulates the CPY, Cvt, and macroautophagy pathways through distinct sets of PtdIns(3)P-binding effectors and that Vps34 promotes protein trafficking in the Cvt pathway through activation/localization of the effector
protein Etf1.