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The Molecular Mechanisms Underlying Fusion Events at the Lysosome Membrane

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The Molecular Mechanisms Underlying Fusion Events at the Lysosome Membrane

Abdul Karim, Mahmoud (2017) The Molecular Mechanisms Underlying Fusion Events at the Lysosome Membrane. PhD thesis, Concordia University.

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Abstract

Lysosome membrane fusion represents the last step of the endocytic pathway. During endocytosis, internalized surface proteins are delivered to endosomes where they are packaged into intraluminal vesicles (ILVs). Multiple rounds of ILV formation produce mature multivesicular body (MVB) that fuses with lysosomes to expose protein-laden ILVs to lumenal acid hydrolases for catabolism. Lysosomes also undergo homotypic membrane fusion for remodeling in response to cellular stress, or aging, or for organelle inheritance. A unique feature of homotypic lysosome fusion is the formation of IntraLumenal Fragments (ILF), whereby portions of the membranes are internalized and degraded upon merging. Although critical for homeostasis and remodeling, it is not clear how ILFs are formed during the homotypic lipid bilayer fusion reaction, nor is it clear whether similar molecular mechanisms orchestrate MVB-lysosome fusion.
Using Saccharomyces cerevisiae and its vacuolar lysosome as a model, I show that coordinated interactions between the fusion protein machinery (Rab-GTPase Ypt7 and its effector Vps41), and the protein kinase Yck3, target hemifusion intermediates to control ILF formation upon vacuolar lysosome fusion. To do so, I introduced a point mutation in Ypt7 (D44N) that permits activation but impairs binding to Vps41 causing it to be phosphorylated by Yck3. Phosphorylated Vps41 causes the multisubunit tethering complex HOPS to dissociate from vacuolar lysosome membranes preventing efficient trans-SNARE pairing required for lipid bilayer pore formation. This stall in pore formation allows hemifusion diaphragms to expand across docked organelle interfaces causing fewer ILFs to form. Knocking out YCK3 stabilizes Vps41 and HOPS on membranes restoring fusion defects and ILF formation.
With a better understanding of homotypic vacuolar lysosome fusion and ILF formation, I next determined if MVB-vacuolar lysosome fusion relies on a similar mechanism. By developing a new luminal β-lactamase complementation assay to measure MVB-vacuolar lysosome fusion in vitro, I show that both fusion events require Ypt7 and HOPS, but heterotypic fusion is distinct in that it uses a unique non-canonical Q-SNARE bundle composed of the endosomal Qa-SNARE Pep12, Qb-SNARE Vti1, and soluble Qc-SNARE Vam7, that complexes with the lysosomal R-SNARE Nyv1 to drive lipid bilayer merger. Loss-of-function mutations that impair MVB maturation-deleting the endosomal Na+(K+)/H+ exchanger NHX1 or components of the ESCRT machinery that drive ILV formation blocks heterotypic membrane fusion. Correcting luminal pH rescues the fusion impairment caused by deleting NHX1, whereas activating the Rab-GTPase Ypt7 rescues fusion defects caused by ESCRT dysfunction.
Using new insights from these results, I present refined working models describing the molecular underpinnings of homotypic vacuolar lysosome and MVB-vacuolar lysosome fusion. Because all of the underlying machinery is conserved in all eukaryotic phyla, I anticipate that they will be used to help design strategies to treat human disorders linked to mutations in genes implicated in these pathways.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Thesis (PhD)
Authors:Abdul Karim, Mahmoud
Institution:Concordia University
Degree Name:Ph. D.
Program:Biology
Date:16 May 2017
Thesis Supervisor(s):Brett, Christopher
ID Code:982557
Deposited By: MAHMOUD ABDULKARIM
Deposited On:08 Nov 2017 20:27
Last Modified:15 May 2019 00:00
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