Engineered AAA+ proteases reveal principles of proteolysis at the mitochondrial inner membrane

Hui Shi,Anthony J. Rampello,Steven E. Glynn

doi:10.1038/ncomms13301

Hui Shi, Anthony J. Rampello + Show 1 more

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https://doi.org/10.1038/ncomms13301

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Journal: Nature Communications	Publication Date: Oct 27, 2016
Citations: 44	License type: CC BY 4.0

Affiliation: Stony Brook University

Abstract

The human YME1L protease is a membrane-anchored AAA+ enzyme that controls proteostasis at the inner membrane and intermembrane space of mitochondria. Understanding how YME1L recognizes substrates and catalyses ATP-dependent degradation has been hampered by the presence of an insoluble transmembrane anchor that drives hexamerization of the catalytic domains to form the ATPase active sites. Here, we overcome this limitation by replacing the transmembrane domain with a soluble hexameric coiled coil to produce active YME1L hexamers that can be studied in vitro. We use these engineered proteases to reveal principles of substrate processing by YME1L. Degradation by YME1L requires substrates to present an accessible signal sequence and is not initiated simply by substrate unfolding. The protease is also capable of processively unfolding substrate proteins with substantial thermodynamic stabilities. Lastly, we show that YME1L discriminates between degradation signals by amino acid composition, implying the use of sequence-specific signals in mitochondrial proteostasis.

Highlights

The human YME1L protease is a membrane-anchored AAA þ enzyme that controls proteostasis at the inner membrane and intermembrane space of mitochondria
We identified a stable human YME1L construct containing the AAA þ ATPase and protease domains but lacking the N-terminal domain and transmembrane span (Fig. 1b; Supplementary Fig. 1). This catalytic core protein (YME1L-AP; residues 317 to 773) was expressed in Escherichia coli, purified to near homogeneity, and migrated by size exclusion chromatography (SEC) at a volume approximately corresponding to a monomer (Fig. 2b)
The mitochondrial AAA þ proteases are anchored into the inner membrane by insoluble transmembrane spans

Summary

Introduction

The human YME1L protease is a membrane-anchored AAA þ enzyme that controls proteostasis at the inner membrane and intermembrane space of mitochondria. Understanding how YME1L recognizes substrates and catalyses ATP-dependent degradation has been hampered by the presence of an insoluble transmembrane anchor that drives hexamerization of the catalytic domains to form the ATPase active sites. We overcome this limitation by replacing the transmembrane domain with a soluble hexameric coiled coil to produce active YME1L hexamers that can be studied in vitro. Two homologous AAA þ proteases are anchored to the mitochondrial inner membrane where they regulate the composition and quality of the mitochondrial proteome in the intermembrane space (IMS), inner membrane and matrix (Fig. 1a). How this diverse group of substrates are recognized and engaged by YME1L is unclear

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