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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

View larger version (18K): [in a new window]   Fig. 1. Subcellular localisation of EYFP, EYFP(H148G), EYFP-SKL and EYFP(H148G)-SKL expressed in S. cerevisiae and assessed by confocal laser scanning microscopy. Expression of EYFP-SKL (a) and EYFP (H148G)-SKL (b) in wild-type cells results in a peroxisomal localisation of EYFPs, whereas expression of EYFP (c) and EYFP(H148G) (d) results in a cytosolic localisation. Expression of EYFP-SKL (e) and EYFP (H148G)-SKL (f) in pex5 cells results in a cytosolic localisation, whereas in pex7 cells both EYFP-SKL (g) and EYFP(H148G)-SKL (h) are localised to peroxisomes. Bar: 5 µM.

View larger version (11K): [in a new window]   Fig. 2. Determination of cytosolic (pHcyt) and peroxisomal (pHper) pH. Wild-type cells were transformed with plasmids containing the coding sequence of EYFP (A), EYFP(H148G) (B), EYFP-SKL (C) and EYFP(H148G)-SKL (D), and were grown in oleate-containing medium. Calibration pH curves were generated by determining the fluorescence of digitonin-permeabilized transformants incubated in buffers with pH values ranging from 4.2 to 9.2 The emission was determined by confocal microscopy and the median of the fluorescence of at least 100 different cells was calculated (see Materials and Methods). To determine the pHcyt, the fluorescence was measured in at least 100 non-permeabilized cells expressing EYFP and EYFP(H148G) [green circles in (A) and (B)]. To determine the pHper, the fluorescence was measured in at least 100 non-permeabilized cells expressing EYFP-SKL and EYFP(H148G)-SKL [green circles in (C) and (D)]. Each experiment was performed at least three times and the standard deviations from the mean values are indicated by error bars.

View larger version (22K): [in a new window]   Fig. 3. Putative scheme of peroxisomal fatty acid ß-oxidation with the free fatty acid and the acyl-CoA uptake route plus the role of Ant1p in pH generation. FFA, free fatty acid; Fox1p, Fatty acyl-Coenzyme A oxidase; Fox2p, 3-hydroxyacyl-CoA dehydrogenase; Fox3p, 3-oxoacyl-CoA thiolase; Ant1p, Adenine nucleotide transporter; Pxa1p, peroxisomal ATP-binding cassette (ABC) transporter 1; Pxa2p, peroxisomal ATP-binding cassette (ABC) transporter 2, putative ATPase (postulated).

View larger version (19K): [in a new window]   Fig. 4. Octanoate ß-oxidation (A) and oleate ß-oxidation (B) in wild-type yeast cells. The medium-chain fatty acid ß-oxidation was completely deficient after 5 mM dinitrophenol had been added to dissipate the pH, whereas oleate ß-oxidation remained the same. Arrows indicate the time point were DNP was either added (+) or not (-).

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