Vacuolin-1 Potently and Reversibly Inhibits Autophagosome-Lysosome Fusion by Activating RAB5A

Autophagy. 2014;10(11):1895-905. doi: 10.4161/auto.32200. Epub 2014 Oct 30.


Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca(2+) content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.

Keywords: ATG, autophagy-related; BAF, bafilomycin A1; CQ, chloroquine; CTSB, cathepsin B; CTSL, cathepsin L; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; GPN, glycyl-l-phenylalanine 2-naphthylamide; LAMP1, lysosomal-associated membrane protein 1; Leup, leupeptin; MAP1LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin; RAB5A; RFP, red fluorescent protein; autophagosomes; endosomes; lysosomes; pH; tfLC3, tandem fluorescence-tagged LC3; vacuolin-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Autophagy*
  • Calcium / metabolism
  • Cell Proliferation
  • Cell Survival
  • Chloroquine / chemistry
  • Endosomes / metabolism
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Hep G2 Cells
  • Heterocyclic Compounds, 4 or More Rings / chemistry*
  • Homeostasis
  • Humans
  • Hydrogen-Ion Concentration
  • Inhibitory Concentration 50
  • Lentivirus / genetics
  • Lysosomes / metabolism*
  • Metabolism
  • Microscopy, Electron, Transmission
  • Mutation
  • Phagosomes / metabolism*
  • rab5 GTP-Binding Proteins / metabolism*


  • Heterocyclic Compounds, 4 or More Rings
  • vacuolin-1
  • Green Fluorescent Proteins
  • Chloroquine
  • Adenosine Triphosphatases
  • RAB5C protein, human
  • rab5 GTP-Binding Proteins
  • Calcium