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J. Biol. Chem., Vol. 276, Issue 52, 48748-48753, December 28, 2001
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From the Peroxisomes are metabolically active
organelles that participate in the oxidation of long-chain fatty acids
and in the biosynthesis of bile acids, cholesterol, and ether
phospholipids. Even though maintenance of a stable acid-base milieu is
essential for proper peroxisomal function, the determination of the
peroxisomal pH (pHp) remains inconclusive, and little
is known about its regulation. To measure the pH of intact peroxisomes
in situ, we used the peroxisome-specific carboxyl-terminal
targeting sequence, SKL, to deliver a pH-sensitive mutant of the green
fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper
targeting was verified by colocalization with the peroxisomal marker
catalase. Peroxisomes were visualized by imaging fluorescence
microscopy, and ratiometric measurements were combined with calibration
using ionophores or a null-point method to estimate pHp.
The pHp was between 6.9 and 7.1, resembling the cytosolic
pH. Manipulation of the cytosolic pH in intact cells or after
permeabilization of the plasmalemma with streptolysin O revealed that
pHp changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H+ (equivalents). We
conclude that peroxisomes do not regulate their pH independently, but
instead their large H+ permeability effectively connects
them with the buffer reservoir of the cytoplasm and with the
homeostatic mechanisms that control cytosolic pH.
In Situ Measurements of the pH of Mammalian
Peroxisomes Using the Fluorescent Protein pHluorin*
§,,,,
Cell Biology Programme, Research Institute,
The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada and
¶ Department of Anatomy and Cell Biology, University of Western
Ontario, London, Ontario N5X 2Y8, Canada
*
This work was supported in part by the Canadian Cystic
Fibrosis Foundation and by the Canadian Institutes for Health Research (CIHR).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
International Scholar of the Howard Hughes Medical Institute,
recipient of a CIHR Distinguished Scientist Award, and holder of the
Pitblado Chair in Cell Biology. To whom correspondence should be
addressed: Cell Biology Program, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5727; Fax: 416-813-5028; E-mail: sga@sickkids.ca.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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