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Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay.

Author(s): Karim MA, Samyn DR, Mattie S, Brett CL

Traffic. 2018 02;19(2):138-149 Authors: Karim MA, Samyn DR, Mattie S, Brett CL

Article GUID: 29135058
Rab-Effector-Kinase Interplay Modulates Intralumenal Fragment Formation during Vacuole Fusion.

Author(s): Karim MA, McNally EK, Samyn DR, Mattie S, Brett CL

Dev Cell. 2018 10 08;47(1):80-97.e6 Authors: Karim MA, McNally EK, Samyn DR, Mattie S, Brett CL

Article GUID: 30269949
A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion.

Author(s): Karim MA, Samyn DR, Brett CL

Methods Mol Biol. 2019;1860:289-301 Authors: Karim MA, Samyn DR, Brett CL

Article GUID: 30317513
Title:A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion.
Authors:Karim MA,  Samyn DR,  Brett CL
Link:https://www.ncbi.nlm.nih.gov/pubmed/30317513?dopt=Abstract
DOI:10.1007/978-1-4939-8760-3_19
Category:Methods Mol Biol
PMID:30317513
Dept Affiliation: BIOLOGY
1 Department of Biology, Concordia University, Montréal, QC, Canada.
2 Department of Cell Biology, University of Alberta, Edmonton, AB, Canada.
3 Department of Biology, Concordia University, Montréal, QC, Canada. christopher.brett@concordia.ca.

Description:

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

Methods Mol Biol. 2019;1860:289-301

Authors: Karim MA, Samyn DR, Brett CL

Abstract

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 ß-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.

PMID: 30317513 [PubMed - indexed for MEDLINE]