Lipid droplets (LDs) are central to cellular energy homeostasis, but the pathways governing their selective degradation are not fully defined. We identify a degradation route for a specific LD subpopulation marked by the phosphatidylinositol transfer protein Pdr16. Under nutrient limitation, the Mon1–Ccz1 GEF complex, through an amphipathic helix in Ccz1, directs its Rab GTPase substrate Ypt7 to Pdr16 positive LDs, initiating their vacuolar uptake. Ypt7 activation subsequently recruits the Retromer complex to vacuole LD (vCLIP) contact sites. Crucially, we find that Retromer, likely via its cargo receptor Vps10, promotes vacuolar membrane curvature to facilitate LD invagination. Vps10, a sorting receptor that cycles between the Golgi and endosomes, is recruited to these sites and appears to directly participate in shaping the vacuolar membrane during LD internalization. However, the identity of the Vps10 cargo involved in this process remains unknown.
Here, we hypothesize that Vps10 functions together with its cargo to generate the membrane curvature surrounding LDs, thereby enabling LD degradation through vacuolar invagination. To identify potential Vps10 cargoes, we employed label free vacuolar proteomics (Eising et al., 2018; Gao et al., 2022), which allows comprehensive identification of vacuolar proteins for comparative analyses. Our preliminary data show that impaired Vps1 function in a mutant leads to accumulation of Vps10 cargo on the vacuole. To ensure data reliability, we analyzed vacuolar proteomes from wild type and vps1Δ cells during stationary phase using both Orbitrap Eclipse and timsTOF Pro mass spectrometers. We then plotted the accumulated cargoes, identified by comparing vps1Δ to wild type ratios from the Orbitrap Eclipse (x axis) and timsTOF Pro (y axis). The candidate proteins identified in this manner will be validated through co localization and co immunoprecipitation with Vps10, and their roles in LD internalization will be tested.