Unfortunately, the expectations that such effectors would be evolutionary conserved and share a common PtdIns(3,5)P2-binding motif of high affinity remain unfulfilled. For example, deletion of Atg18p, a protein involved also in autophagy in S. cerevisiae, causes enlarged vacuole and 10-fold elevation in PtdIns(3,5)P2. Atg18p binds PtdIns(3,5)P2 with high affinity and specificity. However, except for autophagy, the mammalian orthologs of Atg18p do not share similar functions. Two other yeast proteins (Ent3p and Ent5p) found in prevacuolar and endosomal structures are potential PtdIns(3,5)P2 effectors in MVB sorting. They contain a phosphoinositide-binding ENTH domain and their deletion causes MVB sorting defects resembling those reported for Fab1p deletion. However, neither Ent3p nor Ent5p possess preferential and high affinity binding specificity towards PtdIns(3,5)P2 in vitro. Mammalian VPS24 (a member of the charged multivesicular body proteins (CHMPs) family) is another putative PtdIns(3,5)P2 effector. Alas, surface plasmon resonance measurements do not support specific or high-affinity recognition of PtdIns(3,5)P2 for both mammalian and yeast VPS24. The human transmembrane cationic channel TRPML1 (whose genetic inactivation causes lysosomal storage disease) has been recently put forward as PtdIns(3,5)P2 effector, based on in vitro binding assays and its ability to rescue the vacuolation phenotype in fibroblasts from ArPIKfyve/Vac14 knockout mice. But the deletion of the orthologous protein in yeast does not cause vacuole enlargement, thus casting doubts about the evolutionary conservation of this effector mechanism. Further studies are needed to validate these or uncover yet unknown PtdIns(3,5)P2 effectors. References External links Phospholipids