title: Vps15p regulates the distribution of cup-shaped organelles containing the major eisosome protein Pil1p to the extracellular fraction required for endocytosis of extracellular vesicles carrying metabolic enzymes.
abstract: BACKGROUND
  Exosomes are small vesicles secreted from virtually every cell from bacteria to humans. Saccharomyces cerevisiae is a model system to study trafficking of small vesicles in response to changes in the environment. When yeast cells are grown in low glucose, vesicles carrying gluconeogenic enzymes are present as free vesicles and aggregated clusters in the cytoplasm. These vesicles are also secreted into the periplasm and account for more than 90% of total extracellular organelles, while less than 10% are larger 100-300 nm structures with unknown functions. When glucose is added to glucose-starved cells, secreted vesicles are endocytosed and then targeted to the vacuole. Recent secretomic studies indicated that more than 300 proteins involved in diverse biological functions are secreted during glucose starvation and endocytosed during glucose re-feeding. We hypothesised that extracellular vesicles are internalised using novel mechanisms independent of clathrin-mediated endocytosis.
  
  
  RESULTS
  Our results showed that vesicles carrying metabolic enzymes were endocytosed at a fast rate, whereas vesicles carrying the heat shock protein Ssa1p were endocytosed at a slow rate. The PI3K regulator Vps15p is critical for the fast internalisation of extracellular vesicles. VPS15 regulates the distribution of the 100-300 nm organelles that contain the major eisosome protein Pil1p to the extracellular fraction. These Pil1p-containing structures were purified and showed unique cup-shape with their centres deeper than the peripheries. In the absence of VPS15, PIL1 or when PIL1 was mutated, the 100-300 nm structures were not observed in the extracellular fraction and the rapid internalisation of vesicles was impaired.
  
  
  CONCLUSIONS
  We conclude that VPS15 regulates the distribution of the 100-300 nm Pil1p-containing organelles to the extracellular fraction required for fast endocytosis of vesicles carrying metabolic enzymes. This work provides the first evidence showing that Pil1p displayed unique distribution patterns in the intracellular and extracellular fractions. This work also demonstrates that endocytosis of vesicles is divided into a fast and a slow pathway. The fast pathway is the predominant pathway and is used by vesicles carrying metabolic enzymes. Cup-shaped Pil1p-containing structures are critical for the rapid endocytosis of vesicles into the cytoplasm.
  
  
  CONCLUSIONS
  This work provides the first evidence showing that Pil1p displayed unique distribution patterns in the intracellular and extracellular fractions. This work also demonstrates that endocytosis of vesicles is divided into a fast and a slow pathway. The fast pathway is the predominant pathway and is used by vesicles carrying metabolic enzymes. Cup-shaped Pil1p-containing structures are critical for the rapid endocytosis of vesicles into the cytoplasm.
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA, 17033, USA.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Stein
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA, 17033, USA.
authors.forename: Chelsea
authors.initials: C
authors.lastname: Winters
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA, 17033, USA.
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Chiang
mesh_terms: D000199:Actins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D000067128:Extracellular Vesicles / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D005947:Glucose / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D015388:Organelles / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D058571:Vacuolar Sorting Protein VPS15 / Q000378:metabolism*
chemical_list: D000199:Actins; C487327:PIL1 protein, S cerevisiae; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; C546854:VPS15 protein, S cerevisiae; D058571:Vacuolar Sorting Protein VPS15; D005947:Glucose
keywords: Eisosomes; Extracellular vesicles; Gluconeogenic/glycolytic enzymes; Vacuole import and degradation; Vps15p