Molecular Dissection of Novel Trafficking and Processing of the Toxoplasma Gondii Rhoptry Metalloprotease toxolysin-1

Traffic. 2012 Feb;13(2):292-304. doi: 10.1111/j.1600-0854.2011.01308.x. Epub 2011 Nov 29.

Abstract

Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted rhoptry metalloprotease with homology to insulysin that we named toxolysin-1 (TLN1). Through genetic ablation and molecular dissection of TLN1, we have identified the smallest rhoptry targeting domain yet reported and expanded the consensus sequence of the rhoptry pro-domain cleavage site. In addition to removal of its pro-domain, TLN1 undergoes a C-terminal cleavage event that occurs at a processing site not previously seen in Toxoplasma rhoptry proteins. While pro-domain cleavage occurs in the nascent rhoptries, processing of the C-terminal region precedes commitment to rhoptry targeting, suggesting that it is mediated by a different maturase, and we have identified residues critical for proteolysis. We have additionally shown that both pieces of TLN1 associate in a detergent-resistant complex, formation of which is necessary for trafficking of the C-terminal portion to the rhoptries. Together, these studies reveal novel processing and trafficking events that are present in the protein constituents of this unusual secretory organelle.

Publication types

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

MeSH terms

  • Amino Acid Substitution / physiology
  • Catalytic Domain / genetics
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Enzyme Precursors / metabolism
  • Gene Knockout Techniques
  • Insulysin
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Metalloendopeptidases / chemistry
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / metabolism*
  • Molecular Sequence Annotation
  • Peptide Fragments / metabolism
  • Protein Multimerization / physiology
  • Protein Processing, Post-Translational / physiology*
  • Protein Sorting Signals / physiology
  • Protein Structure, Tertiary / physiology
  • Protein Transport / physiology*
  • Proteolysis
  • Proteomics
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Toxoplasma / genetics
  • Toxoplasma / growth & development
  • Toxoplasma / metabolism*
  • Vacuoles / metabolism
  • Virulence / physiology

Substances

  • DNA, Complementary
  • Enzyme Precursors
  • Membrane Proteins
  • Peptide Fragments
  • Protein Sorting Signals
  • Protozoan Proteins
  • ROP 1 protein, Toxoplasma gondii
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Metalloendopeptidases
  • Insulysin