Abstract
Ykt6 is a conserved SNARE protein involved in multiple membrane trafficking pathways, including intra-Golgi transport and autophagic membrane fusion. We previously demonstrated that mammalian Ykt6 is uniquely modified with farnesyl and geranylgeranyl groups at two C-terminal cysteines through the sequential action of farnesyltransferase (FT) and geranylgeranyltransferase type 3 (GGT3). Although these two cysteines are strictly conserved in all eukaryotes, the evolutionary conservation of Ykt6 double prenylation remains unclear, as budding yeast appears to lack the α subunit of GGT3. In this study, we used structural predictions to identify the uncharacterized protein Ecm9 as the functional α subunit of yeast GGT3. Ecm9 forms a complex with Bet2 and transfers a geranylgeranyl group to mono-farnesylated Ykt6. MALDI-TOF/TOF mass spectrometry confirmed that budding yeast Ykt6 is doubly prenylated with farnesyl and geranylgeranyl groups in wild-type cells but not in ecm9Δ cells. Loss of Ecm9 resulted in fragile cell walls, likely due to mislocalization of Golgi mannosyltransferases. Furthermore, ecm9Δ cells exhibited impaired Ykt6 localization to organelle membranes including autophagosomes, leading to reduced autophagic activity. These findings establish that double prenylation is an evolutionarily conserved structural feature of Ykt6 and is essential for its membrane localization and function.