SNAREs pp 289-301 | Cite as

A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion

Part of the Methods in Molecular Biology book series (MIMB, volume 1860)


Endocytosis is a fundamental process underlying diverse eukaryotic physiology. The terminal stage of this process is membrane fusion between the perimeter membrane of a late endosome filled with intraluminal vesicles, or multivesicular body (MVB), and the lysosome membrane to facilitate catabolism of internalized biomaterials or surface polytopic proteins. To comprehensively understand the mechanisms underlying MVB-lysosome membrane fusion, we developed a quantitative, cell-free assay to study this SNARE-mediated event in molecular detail using Saccharomyces cerevisiae and its vacuolar lysosome, or vacuole, as models. This involves separately isolating organelles from two yeast strains each expressing a different complementary fusion probe targeted to the lumen of either MVBs or vacuoles. Isolated organelles are mixed in vitro under fusogenic conditions. Upon MVB-vacuole membrane fusion, luminal contents mix to facilitate probe interaction, reconstituting β-lactamase activity recorded by a colorimetric enzyme activity assay. This method accommodates a multitude of approaches (e.g., genetics, addition of purified protein reagents) to study this process in isolation, and in theory could be repurposed to study other SNARE-mediated fusion events within cells.

Key words

β-Lactamase Content mixing assay Lysosome Membrane fusion Multivesicular body (MVB) Soluble NSF-associated protein receptor (SNARE) Vacuole 



We thank W.T. Wickner for plasmids. E.K. McNally and T. Kazmirchuk provided invaluable discussions and useful feedback. D.R.S. is a postdoctoral scholar funded by the Stiftelson Olle Engkvist Byggmästare. This work was supported by Natural Sciences and Engineering Research Council of Canada grants RGPIN/403537-2011 and RGPIN/2017-06652 to C.L.B.


  1. 1.
    Babst M (2011) MVB vesicle formation: ESCRT-dependent, ESCRT-independent and everything in between. Curr Opin Cell Biol 23:452–457CrossRefGoogle Scholar
  2. 2.
    Henne WM, Buchkovich NJ, Emr SD (2011) The ESCRT pathway. Dev Cell 21:77–91CrossRefGoogle Scholar
  3. 3.
    Huotari J, Helenius A (2011) Endosome maturation. EMBO J 30:3481–3500CrossRefGoogle Scholar
  4. 4.
    Schmidt O, Teis D (2012) The ESCRT machinery. Curr Biol 22:R116–R120CrossRefGoogle Scholar
  5. 5.
    Piper RC, Katzmann DJ (2007) Biogenesis and function of multivesicular bodies. Annu Rev Cell Dev Biol 23:519–547CrossRefGoogle Scholar
  6. 6.
    Luzio JP, Gray SR, Bright NA (2010) Endosome-lysosome fusion. Biochem Soc Trans 38:1413–1416CrossRefGoogle Scholar
  7. 7.
    Kümmel D, Ungermann C (2014) Principles of membrane tethering and fusion in endosome and lysosome biogenesis. Curr Opin Cell Biol 29:61–66CrossRefGoogle Scholar
  8. 8.
    Wickner W (2010) Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. Annu Rev Cell Dev Biol 26:115–136CrossRefGoogle Scholar
  9. 9.
    Wickner W, Rizo J (2017) A cascade of multiple proteins and lipids catalyzes membrane fusion. Mol Biol Cell 28:707–711CrossRefGoogle Scholar
  10. 10.
    Karim MA, Mattie S, Brett CL (2018) Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay. Traffic 19:138–149CrossRefGoogle Scholar
  11. 11.
    Jun Y, Wickner W (2007) Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing. Proc Natl Acad Sci U S A 104:13010–13015CrossRefGoogle Scholar
  12. 12.
    Haas A (1995) A quantitative assay to measure homotypic vacuole fusion in vitro. Methods Cell Sci 17:283–294CrossRefGoogle Scholar
  13. 13.
    Cao Q, Zhong XZ, Zou Y, Murrell-Lagnado R, Zhu MX, Dong XP (2015) Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion. J Cell Biol 209:879–894CrossRefGoogle Scholar
  14. 14.
    Pryor PR, Mullock BM, Bright NA, Lindsay MR, Gray SR, Richardson SC, Stewart A, James DE, Piper RC, Luzio JP (2004) Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events. EMBO Rep 5:590–595CrossRefGoogle Scholar
  15. 15.
    Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH (1992) Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 3:1389–1402CrossRefGoogle Scholar
  16. 16.
    Robinson JS, Klionsky DJ, Banta LM, Emr SD (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol 8:4936–4948CrossRefGoogle Scholar
  17. 17.
    Morvan J, Köchl R, Watson R, Collinson LM, Jefferies HB, Tooze SA (2009) In vitro reconstitution of fusion betweem immature autophagosomes and endosomes. Autophagy 5:676–689CrossRefGoogle Scholar
  18. 18.
    Vida T, Gerhardt B (1999) A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome. J Cell Biol 146:85–98CrossRefGoogle Scholar
  19. 19.
    Karim MA, Brett CL (2018) The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion. Mol Biol Cell 29:317–325CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiologyConcordia UniversityMontréalCanada
  2. 2.Department of Cell BiologyUniversity of AlbertaEdmontonCanada

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