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<p>Peroxisomal matrix proteins are nuclear encoded and synthesized in the cytosol. It is well established that folded, cofactor-containing, and even oligomeric matrix proteins can pass the peroxisomal membrane. Here, we studied whether relatively long unfolded proteins can be imported into peroxisomes using the yeast <i>Hansenula polymorpha</i> as a model organism. First, we designed fusion proteins containing parts of the intrinsically disordered N-terminal region of 630 residues of <i>Saccharomyces cerevisiae</i> nucleoporin 1 (Nsp1). These included fusion proteins containing the first N-terminal 301 or 601 residues or two times the 601 residues region of Nsp1. Green fluorescent protein was added at the N-terminus, and a peroxisomal targeting signal 1 at the C-terminus. Fluorescence microscopy revealed that all three fusion proteins colocalized with a peroxisomal marker protein, indicating that large unfolded protein domains can be imported into peroxisomes. To obtain a fully unfolded protein, we replaced the folded GFP tag by a double human influenza hemagglutinin tag, which unlike GFP is unable to fold. An in vivo protease protection assay showed that a portion of the produced proteins localized to peroxisomes, which was confirmed by quantitative immuno-electron microscopy analysis. On the basis of our observations, we conclude that a fully unfolded protein of over 1200 residues (almost 500&#xa0;nm in length) can pass the peroxisomal membrane.</p>

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The peroxisomal importomer can accommodate an intrinsically disordered protein of 1247 residues

  • 期刊论文

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  • 发表日期：

    2026年5月12日

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  • Marc P. Pedersen,
  • Arjen M. Krikken,
  • Rinse de Boer,
  • Ida J. van der Klei

摘要

Peroxisomal matrix proteins are nuclear encoded and synthesized in the cytosol. It is well established that folded, cofactor-containing, and even oligomeric matrix proteins can pass the peroxisomal membrane. Here, we studied whether relatively long unfolded proteins can be imported into peroxisomes using the yeast Hansenula polymorpha as a model organism. First, we designed fusion proteins containing parts of the intrinsically disordered N-terminal region of 630 residues of Saccharomyces cerevisiae nucleoporin 1 (Nsp1). These included fusion proteins containing the first N-terminal 301 or 601 residues or two times the 601 residues region of Nsp1. Green fluorescent protein was added at the N-terminus, and a peroxisomal targeting signal 1 at the C-terminus. Fluorescence microscopy revealed that all three fusion proteins colocalized with a peroxisomal marker protein, indicating that large unfolded protein domains can be imported into peroxisomes. To obtain a fully unfolded protein, we replaced the folded GFP tag by a double human influenza hemagglutinin tag, which unlike GFP is unable to fold. An in vivo protease protection assay showed that a portion of the produced proteins localized to peroxisomes, which was confirmed by quantitative immuno-electron microscopy analysis. On the basis of our observations, we conclude that a fully unfolded protein of over 1200 residues (almost 500 nm in length) can pass the peroxisomal membrane.