PhD Oral Exam - Mahmoud Karim, Biology

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

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 merger. 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 models, 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 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 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 lysosome fusion and ILF formation, I next determined if MVB-lysosome fusion relies on similar mechanism. By developing a new luminal β-lactamase complementation assay to measure MVB-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 lysosome and MVB-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.