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First published online August 17, 2004
doi: 10.1242/10.1242/jcs.01305

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The peroxisomal lumen in Saccharomyces cerevisiae is alkaline

Carlo W. T. van Roermund^1,*, Mark de Jong¹, Lodewijk IJlst¹, Jan van Marle², Tobias B. Dansen^3,, Ronald J. A. Wanders¹ and Hans R. Waterham¹

¹ Department of Clinical Chemistry, University of Amsterdam, Academic Medical Centre, PO Box 22700, 1100 DE, Amsterdam, The Netherlands
² Department of Electronmicroscopy, University of Amsterdam, Academic Medical Centre, PO Box 22700, 1100 DE, Amsterdam, The Netherlands
³ Department of Biochemistry of Lipids, Institute of Biomembranes, Centre for Biomembranes and Lipid Enzymology, Utrecht University, PO Box 80054, 3508 TB Utrecht, The Netherlands

^* Author for correspondence (e-mail: c.vanroermund{at}amc.uva.nl)

Accepted 5 May 2004

Peroxisomes have a central function in lipid metabolism, including the ß-oxidation of various fatty acids. The products and substrates involved in the ß-oxidation have to cross the peroxisomal membrane, which previously has been demonstrated to constitute a closed barrier, implying the existence of specific transport mechanisms. Fatty acid transport across the yeast peroxisomal membrane may follow two routes: one for activated fatty acids, dependent on the peroxisomal ABC half transporter proteins Pxa1p and Pxa2p, and one for free fatty acids, which depends on the peroxisomal acyl-CoA synthetase Faa2p and the ATP transporter Ant1p. A proton gradient across the peroxisomal membrane as part of a proton motive force has been proposed to be required for proper peroxisomal function, but the nature of the peroxisomal pH has remained inconclusive and little is known about its generation. To determine the pH of Sacharomyces cerevisiae peroxisomes in vivo, we have used two different pH-sensitive yellow fluorescent proteins targeted to the peroxisome by virtue of a C-terminal SKL and found the peroxisomal matrix in wild-type cells to be alkaline (pH_per 8.2), while the cytosolic pH was neutral (pH_cyt 7.0). No pH was present in ant1 cells, indicating that the peroxisomal pH is regulated in an ATP-dependent way and suggesting that Ant1p activity is directly involved in maintenance of the peroxisomal pH. Moreover, we found a high peroxisomal pH of >8.6 in faa2 cells, while the peroxisomal pH remained 8.1±0.2 in pxa2 cells. Our combined results suggest that the proton gradient across the peroxisomal membrane is dependent on Ant1p activity and required for the ß-oxidation of medium chain fatty acids.

Key words: Peroxisomes, ß-oxidation, pH, Fatty acid transport, Carrier, Yeast

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