A lysosomal biogenesis map reveals the cargo spectrum of yeast vacuolar protein targeting pathways

Abstract

The lysosome is the major catabolic organelle in the cell that has been established as a key metabolic signaling center. Mutations in many lysosomal proteins have catastrophic effects and cause neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many evolutionary conserved proteins. Proteins reach vacuoles via the Vps10-dependent endosomal vacuolar protein sorting pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. A systematic understanding of the cargo spectrum of each pathway is completely lacking. Here, we use quantitative proteomics of purified vacuoles to generate the yeast lysosomal biogenesis map. This dataset harbors information on the cargo–receptor relationship of almost all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data show how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis.

An ESCRT-dependent pathway coordinates Nuclear and Cytoplasmic Spatial Protein Quality Control at Nuclear Vacuolar Junctions

“…7c, e). Interestingly, we also observed a range of GFP-positive degradation products in the WT and +Bz cells that disappear when the vacuolar proteases were inhibited (Figure 7g), which is a hallmark of vacuolar targeting of GFP-tagged proteins 64,65,83,[86][87][88][89] . Quantification of the intensity of degradation fragments relative to initial full-length protein confirmed the reduction in clearance when vacuolar proteases are inhibited (Fig.…”

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro

“…A possible reason was that the overall deletion of the VPS10 genes caused the inability of some vacuolar proteins to be transported into the vacuole, and the macromolecular circulation, protein homeostasis and detoxification in cells were disrupted. 29 Effect of N-glycosylation modification on the expression of LIPR in P. pastoris GS115 It is well known that N-glycosylation modification is involved in protein synthesis, folding and transport as well as contributes to the efficient expression and secretion of proteins in P. pastoris. 11,30 As shown in Fig.…”

“…However, knocking out VPS10‐01 and VPS10‐02 genes (strain P17, GS115/Mss‐PDI‐LIPR‐ VPS10‐01 / 02 Δ) simultaneously would reduce the expression level of LIPR. A possible reason was that the overall deletion of the VPS10 genes caused the inability of some vacuolar proteins to be transported into the vacuole, and the macromolecular circulation, protein homeostasis and detoxification in cells were disrupted 29 …”

When less is more – A fast TurboID KI approach for high sensitivity endogenous interactome mapping

“…Aside from a few cargo proteins that are often used as model cargos, little is known about which proteins are sorted by which adaptor protein in mammalian cells 39 . Recent proteomic studies have shed light on cargo sorting pathways in yeast cells 40 , however such a comparative and comprehensive study is still lacking for mammalian cells. We also do not fully understand the mechanisms of recruitment of AP complexes to different membranes.…”