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Peroxisomes in human fibroblasts have a basic pH

Nature Cell Biology volume 2, pages 51–53 (2000)Cite this article

Peroxisomes are single-membrane-bound organelles found in nearly all eukaryotic cells. These organelles have a central function in lipid metabolism, including the beta-oxidation of very-long-chain and branched-chain fatty acids and the biosynthesis of ether phospholipids and cholesterol. Another characteristic of peroxisomes is their ability to degrade hydrogen peroxide by catalase1,2. A deficiency in one or more peroxisomal enzymes has been linked to at least 20 (often lethal) disorders3, showing the key role of this organelle in normal functioning of the human body. Peroxisomes are fragile structures that easily lose their integrity upon isolation4. This poses a serious problem for studying these organelles in vitro and explains why our knowledge about the properties of the peroxisomal membrane, including the change in pH across it, is limited. In vivo, peroxisomes have been shown to be closed structures that are impermeable to NADH and NADPH, implying the existence of NADP redox shuttles5,6. Here we study the pH in peroxisomes by targeting a pH-sensitive fluorescent reporter group to these organelles in living fibroblasts. We attained specific targeting by conjugating the fluorophore to a membrane-permeable peptide that contains a type-I peroxisome-targeting sequence (PTS1; amino-acid sequence AKL)7. Using this peptide probe, we establish that peroxisomes of human fibroblasts have a pH of 8.2 ± 0.3. Fibroblasts from RCDP (rhizomelic form of chondrodysplasia punctata) type 1 patients with severe mutations in the PEX7 protein, which result in an isolated defect in peroxisomal import of proteins with a PTS2 sequence8, are still capable of importing the probe into peroxisomes, but have a pH of 6.5 ± 0.3.

The dissipation of the pH gradient in the RCDP fibroblasts must be a consequence of their mutations in PEX7 and the resulting defect in PTS2-protein import. RCDP cells are deficient in the known PTS2-containing proteins alkyl-DHAP (dihydroxyacetone-phosphate) synthase and phytanoyl-CoA alpha hydroxylase, and also in the PTS1 protein DHAP acyltransferase3, the stability of which requires an interaction with alkyl-DHAP synthase12. To determine whether the basic peroxisomal pH in wild-type cells is related to one of the enzymes deficient in the RCDP type 1 patients, we measured the pH in fibroblasts from patients with an isolated defect in DHAP acyltransferase (RCDP type 2; ref. 13), alkyl-DHAP synthase (RCDP type 3; ref. 14) or phytanoyl-CoA alpha hydroxylase (Refsum disease15). All of these cells had a normal basic pH (data not shown). This indicates that the aberrant peroxisomal pH in RCDP type 1 cells must be due to a deficiency in some other enzyme that is not imported, destabilized, or deprived of its substrate. The other possibility is that the mutations in PEX7, the PTS2 receptor, give rise to the formation of a transient pore in the peroxisome membrane, allowing protons to leak back into the peroxisome.

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Figure 1: Peroxisomal localization of BODIPY–PTS1.
Figure 2: The peroxisomal pH in control and RCDP human fibroblasts.

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Acknowledgements

We thank P.A.W. Mooijer for help with tissue cultures.

Correspondence and requests for materials should be addressed to K.W.A.W.

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Authors and Affiliations

  1. Department of Biochemistry of Lipids, Institute of Biomembranes, Centre for Biomembranes and Lipid Enzymology, Utrecht University, Padualaan 8

    Tobias B. Dansen, Karel W. A. Wirtz & Eward H. W. Pap

  2. PO Box 80054, Utrecht, 3508 TB, The Netherlands

    Tobias B. Dansen, Karel W. A. Wirtz & Eward H. W. Pap

  3. Departments of Clinical Chemistry and Paediatrics, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, Amsterdam, NL-1105 AZ, The Netherlands

    Ronald J. A. Wanders

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  1. Tobias B. Dansen
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Correspondence to Karel W. A. Wirtz.

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Dansen, T., Wirtz, K., Wanders, R. et al. Peroxisomes in human fibroblasts have a basic pH. Nat Cell Biol 2, 51–53 (2000). https://doi.org/10.1038/71375

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