Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins

Jennifer E. Kalish, Gilbert Andre Keller, James C. Morrell, Stephanie J. Mihalik, Barbara Smith, James M. Cregg, Stephen J. Gould

Research output: Contribution to journalArticlepeer-review

Abstract

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, > 98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix: membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the pexoisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.

Original languageEnglish (US)
Pages (from-to)3275-3285
Number of pages11
JournalEMBO Journal
Volume15
Issue number13
DOIs
StatePublished - Jul 1 1996

Keywords

  • Green fluorescent protein
  • Integral peroxisomal membrane protein
  • Peroxisome assembly
  • Protein translocation
  • RING finger

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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