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

Nat Commun. 2016 Oct 27:7:13301. doi: 10.1038/ncomms13301.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • ATPases Associated with Diverse Cellular Activities / chemistry
  • ATPases Associated with Diverse Cellular Activities / genetics
  • ATPases Associated with Diverse Cellular Activities / metabolism*
  • Amino Acid Sequence
  • Biocatalysis
  • Humans
  • Metalloendopeptidases / chemistry
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / metabolism*
  • Mitochondrial Membranes / metabolism*
  • Mitochondrial Proteins / chemistry
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Protein Engineering / methods
  • Protein Multimerization
  • Protein Unfolding
  • Proteolysis
  • Proteostasis
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Mitochondrial Proteins
  • Metalloendopeptidases
  • YME1L1 protein, human
  • ATPases Associated with Diverse Cellular Activities