Novel targeting assay uncovers targeting information within peroxisomal ABC transporter Pxa1

Renate L.M. Jansen, Marco van den Noort, Arjen M. Krikken, Chen Bibi, Astrid Böhm, Maya Schuldiner, Einat Zalckvar, Ida J. van der Klei*

*Corresponding author for this work

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Abstract

The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist. Here, we studied sorting of yeast peroxisomal ABC transporter Pxa1. Co-localisation analysis of Pxa1-GFP in a collection of 86 peroxisome-related deletion strains revealed that Pxa1 sorting requires Pex3 and Pex19, while none of the other 84 proteins tested were essential. To identify regions with peroxisomal targeting information in Pxa1, we developed a novel in vivo re-targeting assay, using a reporter consisting of the mitochondrial ABC transporter Mdl1 lacking its N-terminal mitochondrial targeting signal. Using this assay, we showed that the N-terminal 95 residues of Pxa1 are sufficient for retargeting this reporter to peroxisomes. Interestingly, truncated Pxa1 lacking residues 1–95 still localised to peroxisomes. This was confirmed via localisation of various Pxa1 truncation and deletion constructs. However, localisation of Pxa1 lacking residues 1–95 depended on the presence of its interaction partner Pxa2, indicating that this truncated protein does not contain a true targeting signal.

Original languageEnglish
Article number119471
Number of pages11
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1870
Issue number5
DOIs
Publication statusPublished - Jun-2023

Keywords

  • Fluorescence microscopy
  • Membrane proteins
  • Peroxisomes
  • Protein targeting
  • Pxa1
  • Yeast

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