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Title

Regulation of VPS4 Activity during Endosomal Maturation by CHMPs and LIP5

Date of Award

Spring 5-15-2011

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Transmembrane protein degradation in the lysosome requires an endosomal intermediate known as the multivesicular body, where transmembrane proteins are concentrated to areas of the limiting membrane that bud into the lumen of the endosome to form small intra-lumenal vesicles. This molecular budding machinery requires a set of related CHMPs (charged multi-vesicular body proteins) and the VPS4 ATPase. How the two human VPS4 isoforms disassemble the membrane-associated lattice formed by at least 11 different CHMPs is unknown, but is believed to involve conformational remodeling of CHMP proteins. The VPS4 cofactor, LIP5 is thought to contribute to this remodeling.

Here I show that LIP5 binds with high affinity to VPS4 and to a subset of the CHMPs. CHMPs use a common interface to bind both LIP5 and VPS4. All tested CHMP proteins stimulate VPS4 ATPase activity, suggesting that VPS4 engages each of the CHMPs when it disassembles the membrane-associated CHMP lattice in vivo. The ability of CHMPs to stimulate VPS4 activity requires both a specific binding motif and additional acidic residues found in the CHMP C-termini. Consistent with this, engineered acidic substrates stimulate VPS4 ATPase activity. Further experimentation shows that novel VPS4 N-terminal regions regulate its activity through autoinhibition and enable its response to acidic substrates.

Autoinhibition also regulates engagement between LIP5 and VPS4. LIP5 is capable of binding CHMP1B and CHMP5 simultaneously. Binding of CHMPs 1B, 2A, and 3 release LIP5 autoinhibition and enable LIP5 to function as an adaptor protein that also interacts with VPS4, but CHMP5 binding does not. To confirm the role of LIP5 as a scaffold between VPS4 and particular CHMPs, an engineered construct shows simultaneous engagement of VPS4 via MIT and β-domains greatly enhances enzymatic activity.

The work in this thesis shows that VPS4 engages all CHMP proteins once CHMP acidic residues relieve its intrinsic autoinhibition. LIP5 contributes to this process by simultaneously engaging VPS4 and two different CHMPs. This may effectively tether VPS4 to the CHMP lattice while the lattice is being dismantled by VPS4. Control of lattice disassembly is provided by the many layers of autoinhibition present in CHMPs, LIP5, and VPS4.

Language

English (en)

Chair and Committee

Phyllis Hanson

Committee Members

Ron Bose, Kendall Blumer, Stuart Kornfeld, Petra Levin, Maurine Linder, Philip Stahl

Comments

Permanent URL: https://doi.org/10.7936/K7ZK5DMQ

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