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J. Biol. Chem., Vol. 276, Issue 52, 48748-48753, December 28, 2001

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In Situ Measurements of the pH of Mammalian Peroxisomes Using the Fluorescent Protein pHluorin^*

Andrzej Jankowski^§, Jae Hong Kim, Richard F. Collins, Richard Daneman, Paul Walton^¶, and Sergio Grinstein

From the  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

Received for publication, September 18, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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 (pH_p) 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 pH_p. The pH_p 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 pH_p 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Peroxisomes are small vesicular organelles found in almost all eukaryotic cells. They are biochemically diverse, with more than 50 different enzymes identified. The role of peroxisomes in cellular metabolism is dependent on cell type and varies with the environmental conditions. They are primarily responsible for the -oxidation of fatty acids, particularly those bearing long acyl chains, but also participate in the synthesis of bile acids, cholesterol, and ether lipids (1-3). Peroxisomes are also involved in reactions unrelated to lipid metabolism, such as catabolism of purines and polyamines, metabolism of amino acids and glyoxylate, and inactivation of reactive oxygen species (1). Over the past decade, much has been learned about the mechanisms of peroxisomal protein import and peroxisomal biogenesis. A set of peroxisomal genes was identified in yeast, and their mammalian homologues were recognized subsequently (4). Together with earlier subcellular fractionation and analytical studies (5, 6), the identification of these gene products greatly improved our knowledge of the biochemical constituents of peroxisomes.

Despite the importance of peroxisomes and the substantive knowledge of their biochemical properties, little is known about the ionic composition of the peroxisomal lumen, which is crucial for optimum enzymatic function. The initial insights into acid-base regulation were obtained by Douma et al. (7), who identified a putative proton-translocating ATPase in the yeast peroxisomal membrane. The functional role of this ATPase appeared to be validated by ³¹P NMR measurements (8), which suggested that the pH of the peroxisomal lumen is acidic.

Although internally consistent, the results obtained in yeast are not compatible with a recent publication where the pH of mammalian peroxisomes was found to be remarkably alkaline (9). Lastly, the existence of a proton (equivalent) gradient across the peroxisomal membrane (whether inward or outward) appears contrary to the notion that mammalian peroxisomes are highly permeable to small molecules (10, 11). The source of these apparent inconsistencies is not clear. They may be attributable to species or tissue differences, but the discrepancies are more likely to be of methodological origin (see "Discussion" for more details).

Regardless of the source of the reported differences, it is apparent that the pH of peroxisomes remains uncertain and is worthy of further analysis, preferably using alternative, improved techniques. In the present study we devised a strategy to measure the H⁺ activity of peroxisomes within living cells. To this end, we took advantage of endogenous cellular targeting processes to direct a pH-sensitive fluorescent protein specifically to the peroxisomal lumen. One such mechanism, which signals translocation of cytosolic proteins across the peroxisomal membrane, involves the carboxyl-terminal tripeptide sequence Ser-Lys-Leu or SKL. This peroxisomal targeting signal (PTS-1)¹ originally defined in firefly luciferase (12) suffices to target proteins to the peroxisomal interior in yeast, plant, insect, and mammalian cells (13). Importantly, even proteins like albumin or IgG, which are not normally targeted to peroxisomes, can be directed to accumulate within these organelles by attachment of PTS sequences (14-16). We therefore engineered a construct for expression in mammalian cells of pHluorin with a carboxyl-terminal SKL sequence. The fluorescence of pHluorin, a mutant form of the green fluorescent protein (GFP) of Aequora victoria (17), not only changes in intensity with changing pH but in addition undergoes a spectral shift. As a result, measurements of the ratio of the fluorescence intensity at two suitably chosen wavelengths provide accurate estimates of the pH, which are virtually insensitive to photobleaching or to alterations in the focal plane, an issue of great concern when imaging small, mobile organelles like peroxisomes. Using this approach, we measured the peroxisomal pH of Chinese hamster ovary (CHO) cells and studied its determinants.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Reagents and Antibodies-- Nigericin, 2',7'bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester, and rabbit anti-GFP antibody were from Molecular Probes Inc. (Eugene, OR). Streptolysin O was obtained from Dr. S. Bhakdi (Institute for Medical Microbiology, Johannes Gutenberg University, Mainz, Germany). Concanamycin A was obtained from Kamiya Biochemical Co. (Thousand Oaks, CA). CCCP (carbonyl cyanide m-chlorophenylhydrazone) and sodium butyrate were purchased from Sigma. FuGENE-6 transfection reagent was purchased from Roche Diagnostics GmbH. Polyclonal rabbit anti-catalase antibody was from Calbiochem (La Jolla, CA), and Cy3- and Cy5-labeled donkey anti-rabbit and anti-mouse antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). Monoclonal anti-giantin antibody was the kind gift of Dr. Hans-Peter Hauri of the Department of Physiology, University of Basel, Switzerland.

pHluorin-SKL Construct and Its Characterization-- pHluorin cDNA was the kind gift of Dr. J. Rothman and was modified using Stratagene's QuickChange site-directed mutagenesis kit to replace the two carboxyl-terminal amino acids (Tyr-Lys) with the peroxisomal targeting sequence Ser-Lys-Leu (SKL). The sense mutagenic oligomer was 5'-CAT GGC ATG GAT GAA CTA TCC AAA TTA TAA AGC GGA CGC GAC TCT-3', where SKL are underlined. The overlapping antisense oligomer was: 5'-AGA GTC GCG TCC GCT TTA TAA TTT GGA TAG TTC ATC CAT GCC ATG-3'. Mutants were screened for elimination of the EagI site.

Solutions-- The Na⁺-rich medium contained (in mM): NaCl 140, KCl 5, MgCl₂ 1, CaCl₂ 1, glucose 5, and Hepes 20, titrated to pH 7.3 at 37 °C. The pH 5.8 medium contained identical salt concentrations, but HEPES was substituted with MES, and pH was adjusted to 5.8 at 37 °C. The permeabilization medium contained (in mM) potassium glutamate 90, KCl 50, NaCl 10, MgCl₂ 1, CaCl₂ 2, EGTA 4, K₂HPO₄ 2, and HEPES 20, titrated to pH 7.0 at 37 °C. The K⁺-rich medium contained (in mM): KCl 140, glucose 5, Hepes/MES 15 with pH being adjusted to 7.8, 7.4, 6.91, or 6.41, as specified. RPMI 1640 medium (Mediatech Cellgro, Herndon, VA) was kept at 37 °C under 5% CO₂. PBS consisted of (in mM) NaCl 140, KCl 10, sodium phosphate 8, potassium phosphate 2, pH 7.4. Null-point calibration buffers contained (in mM): NaCl 50, Hepes 20, and a mixture of butyric acid and NH₄Cl (total concentration = 4 mM).

Cell Culture and Transient Transfections-- CHO cells obtained from the American Tissue Culture Collection were grown in minimum essential medium supplemented with 10% fetal bovine serum. Human foreskin fibroblasts were obtained from the Hospital for Sick Children tissue culture repository. Both cell lines were allowed to attain 50% confluency on 25-mm glass coverslips and then were transfected using FuGENE-6 transfection reagent with 1 µg/ml cDNA. Experiments were performed 48 h post-transfection.

Immunofluorescence and pHluorin-SKL Expression Characterization-- Cells were fixed for 45 min with 4% paraformaldehyde in PBS at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 60 min, and blocked with 5% milk in PBS for 1 h at room temperature. For localization of catalase coverslips were then incubated with a 1:100 dilution of the rabbit anti-catalase antibody followed by a 1:3000 dilution of Cy3-labeled anti-rabbit IgG. For localization of pHluorin and giantin, coverslips were fixed, permeabilized, and blocked as above and then incubated with a 1:200 dilution of anti-GFP antibody and a 1:500 dilution of anti-giantin antibodies, followed by incubation in a 1:3000 dilution of Cy3-labeled anti-mouse IgG and a 1:3000 dilution of Cy5-labeled anti-rabbit IgG. Alternatively, cells incubated for 5 min with 0.4 µg/ml streptolysin O at room temperature were incubated with a 1:200 dilution of rabbit anti-GFP antibody and a 1:500 dilution of anti-giantin antibody in cold permeabilization medium. Finally, samples were fixed and incubated with a 1:3000 dilution of Cy3-labeled anti-mouse IgG and Cy5-labeled anti-rabbit IgG.

Fluorescence Imaging-- Cells grown on coverslips were mounted in a thermostatted Leiden holder, bathed in a Na⁺-rich buffer, and placed on the stage of a Leica fluorescence microscope equipped with a PL Fluotar 100×/1.30 NA oil immersion objective. A Sutter filter wheel controller positioned excitation filters in front of a mercury lamp. A neutral density filter was used to reduce intensity of the excitation light reaching the cells, and each exposure was limited to 200-600 ms to minimize dye bleaching and photodynamic damage. Excitation was alternated at 480 nm and 400 nm using the Sutter filter wheel controller and directed to the cells through a 510 nm dichroic mirror. Emitted fluorescence was selected through a 535BP25 nm filter and captured with a cooled charge-coupled device camera (Princeton Instruments Inc., Princeton, NJ). Image acquisition was controlled by the Metafluor software version 3.5 (Universal Imaging Corp., West Chester, PA). The pH of individual peroxisomes was estimated by measuring the fluorescence of a peroxisomal region of interest. During initial experiments we found that the average pH of individual peroxisomes was not significantly different from that of the combined population of peroxisomes from each cell, estimated by defining a large region of interest encompassing all the peroxisomes within one cell and discarding non-peroxisomal background (cytosolic) fluorescence by thresholding. Therefore, all subsequent studies were performed with grouped peroxisomes from individual cells. To determine background, an area identical to the region of interest was selected outside the transfected cell, and fluorescence was acquired at both wavelengths. Background was subtracted prior to calculation of the ratio, using the Metafluor software.

The sample was continuously illuminated at >620 nm by placing a red filter in front of the transmitted incandescent source. By placing an additional 660 nm dichroic mirror in the light path, the red light was directed to a video camera, allowing continuous visualization of cell morphology by Nomarski microscopy.

Two independent methods of calibration were used. At the end of the experiment, a calibration curve of fluorescence ratio versus pH was obtained in situ by sequential perfusion with isotonic K⁺-rich medium buffered to predetermined pH values (between 7.8 and 6.41) containing 10 µg/ml nigericin. Calibration curves were constructed by plotting the extracellular pH, assumed to be identical to the peroxisomal/cytosolic pH under these conditions, against the corresponding fluorescence ratio. Alternatively, pH was validated by the null-point method using solutions containing varying ratios of weak acid (A)/base (B) (butyric acid and NH₄Cl). The null pH was calculated according to the following equation: pH = 1/2(pK_a+pK_b) + 1/2 log([B]/[A]). The ratio of acid/base was as follows: 1:2 for pH 6.76, 1:1 for pH 6.91, 2:1 for pH 7.06, and 3:1 for pH 7.15.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Characterization and Expression Profile of pHluorin-SKL-- We used pHluorin, a pH-sensitive mutant of GFP, to measure peroxisomal pH. This protein was shown by Miesenbrock et al. (17) to undergo reversible changes in fluorescence intensity when the pH is altered. Its pK_a is 6.5, making it suitable for the measurement of pH in the physiological range. To target this probe specifically to the peroxisomal lumen, we engineered a cDNA-encoding pHluorin modified to contain a carboxyl-terminal SKL tripeptide, which is an effective peroxisomal import sequence. The peroxisome-targeted pHluorin (called pHluorin-SKL hereafter) was transfected into CHO cells using FuGENE-6. The protein was found to be initially expressed in the cytosol but subsequently accumulated within punctate structures, likely peroxisomes. In cells with low or moderate expression levels most of the fluorescence detectable at 24-48 h was in the punctate vesicular compartment (Fig. 1A), whereas cytosolic fluorescence was more predominant in cells that overexpressed the protein. Only the former, which had a discrete punctate distribution of pHluorin-SKL, were used in the experiments described below.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Subcellular localization of pHluorin-SKL. A and B, CHO cells were transfected with pHluorin-SKL, fixed, and permeabilized. Peroxisomes were identified by immunostaining with antibodies to catalase. A, pHluorin-SKL fluorescence; B, catalase immunostaining. Dotted squares demarcate areas that are magnified in the inset at the bottom left corner of panels A and B. C and D, cells transfected with pHluorin, fixed, and permeabilized. Peroxisomes were identified by immunostaining with antibodies to catalase. C, pHluorin fluorescence. D, catalase immunostaining. Bars = 10 µm.

To confirm that under the conditions chosen for our experiments pHluorin-SKL is confined to the peroxisomes, cells were analyzed by immunofluorescence using organelle-specific markers. As shown in Fig. 1, A and B, the distribution of pHluorin-SKL is virtually identical to that of catalase, the hallmark of peroxisomes (see insets for detail). Targeting of pHluorin to peroxisomes was most likely mediated by the PTS-1 system because deletion of the SKL import peptide resulted in a diffuse cytosolic targeting of the fluorescent protein (Fig. 1C). The distribution of the untagged pHluorin differed drastically from that of catalase (cf. Fig. 1, C and D). Jointly, these results imply that pHluorin-SKL is targeted by the PTS-1 system to the lumen of peroxisomes where it can be used as a selective pH indicator.

Basal Peroxisomal pH-- Having identified the compartment labeled by pHluorin-SKL, we proceeded to measure its pH in situ by fluorescence ratio imaging. In otherwise untreated cells the ratio of fluorescence at 480/400 nm, indicative of the peroxisomal pH (pH_p), remained constant over extended periods of time (up to 20 min). To quantify pH_p, we calibrated the fluorescence ratio of pHluorin-SKL in situ using nigericin and K⁺-rich buffers, as described under "Experimental Procedures." A representative experiment is shown in Fig. 2A. Using this approach, pH_p in cells bathed in physiological (Na⁺-rich) medium averaged 7.12 ± 0.13 (n = 7). This pH differs markedly from that obtained in human fibroblasts, which were reported to be very alkaline (9). To find out whether this apparent discrepancy is attributable to differences in the biological system employed, we also measured the pH_p in human foreskin fibroblasts transfected with a pH-sensitive fluorescent protein bearing the carboxyl-terminal SKL sequence.² In these cells, pH_p was found to average 7.17 ± 0.22 (n = 7), which is not significantly different from that found in CHO cells (e.g. inset to Fig. 2B).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Estimation of peroxisomal pH. Cells were transfected with pHluorin-SKL, and the fluorescence emission was measured as described under "Experimental Procedures" with excitation at 480 and 400 nm. A, K+/nigericin calibration. After a base-line acquisition period, nigericin (10 µg/ml) was added, and the bathing medium was replaced by high K+ solutions of the pH indicated above the abscissa. B, null-point calibration. After a base-line acquisition period, the cells were sequentially perfused with solutions containing varying ratios of butyrate and ammonium chloride. The pH at which each combination is predicted to equilibrate is indicated above the abscissa. Inset, cytosolic pH and its calibration in CHO cells expressing cytosolic pHluorin. Ordinate, ratio of fluorescence emission at 480 nm/400 nm. These results are representative of seven experiments of each type.

The nigericin calibration method rests on the assumption that the luminal [K⁺] of peroxisomes is similar to that of the cytosol. Because the monovalent ion activity within peroxisomes has not been defined, we used an alternative pH calibration method that is independent of the prevailing ionic composition. The null-point procedure, proposed originally by Eisner et al. (18), employs a combination of two weak electrolytes of defined pK values and has been used successfully for other intracellular organelles (19). In this approach various ratios of weak acids and bases are used to search for a null point where the rates of protonation/deprotonation of the permeable species of the electrolytes are identical. For a given combination of acid and base, the null point is strictly a function of the luminal pH. A typical experiment is illustrated in Fig. 2B. Note that the fluorescence ratio increased when the cells were bathed in a solution predicted to equilibrate at 6.76 and decreased below the original baseline when using the 7.15 solution. Importantly, the original fluorescence ratio was virtually unaltered when using the solution predicted to equilibrate at 6.91, which must therefore approximate the resting pH_p. In eight similar experiments the resting pH_p averaged 6.92 ± 0.04 (mean ± S.E.). This value, which is similar yet not identical to that determined by the nigericin method, is more likely to be a reliable estimate of pH_p because it does not require any assumptions regarding the organellar alkali cation content. Jointly, these findings indicate that pH_p in CHO cells is near neutral.

Determinants of Peroxisomal pH-- Earlier biochemical and functional reports suggested the presence of proton pumps in peroxisomes (20, 21). Although no evidence of luminal acidification was obtained in our determinations, it is conceivable that proton extrusion processes exist in the peroxisomal membrane, which may offset the activity of the putative pumps. We therefore used concanamycin A to assess the contribution of V-ATPases to the flux of protons in peroxisomes. V-ATPases are the only known type of endomembrane proton pumps of mammalian cells, and concanamycin A is an effective and selective inhibitor of these pumps (22). Typical results are shown in Fig. 3A. Unexpectedly, we observed a transient acidification upon inhibition of the proton pump. This finding could mean that atypically oriented V-ATPases pump H⁺ out of the peroxisome and that their inhibition unmasks an underlying acidification process. However, this explanation appears unlikely, in that the observed acidification was transient. Alternatively, it is possible that inhibition of V-ATPases of other organelles (e.g. endosomes and lysosomes) results in a net release of acid equivalents into the cytosol. If V-ATPases are present in the plasma membrane of CHO cells, as suggested for other cell types, their inhibition in combination with ongoing metabolic production could contribute to cytosolic acidification. The resulting cytosolic acidification may in turn cause a secondary acidification of the peroxisomes.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of concanamycin and CCCP on peroxisomal pH. Cells were transfected with either pHluorin-SKL (A) or pHluorin (B) to measure peroxisomal or cytosolic pH, respectively, as described under "Experimental Procedures." After establishing the basal pH, the cells were exposed to concanamycin (100 nM) and CCCP (0.5 µM) where indicated. These results are representative of four experiments of each type.

The cytosolic pH was measured in parallel experiments to test the latter hypothesis. For this purpose, cells were transfected with soluble pHluorin, a cytosolic pH probe (see Fig. 1C). As reported using a variety of other methods, the cytosolic pH measured using pHluorin was close to neutrality (Fig. 3B and inset to Fig. 2B). Importantly, addition of concanamycin A induced a transient cytosolic acidification of magnitude and kinetics comparable with those recorded in the peroxisomes. These findings are most readily explained assuming that leakage of acid accumulated by V-ATPases in acidic organelles results in a transient cytosolic acidification, which in turn induces a secondary change in pH_p. This conclusion is supported by observations made using the protonophore CCCP. Increasing the passive permeability to protons, whether before (not shown) or after addition of concanamycin A (Fig. 3), accelerates the leakage of H⁺ from acidic organelles, as reflected by a transient cytosolic acidification. A mirroring acidification of pH_p was observed in parallel. Jointly, these observations suggest that the peroxisomal membrane is highly permeable to H⁺ equivalents, causing the pH_p to track closely the cytosolic pH.

Sidedness of pHluorin-SKL in Peroxisomes-- The similarity of pH_p to the cytosolic pH, both at rest and when concanamycin and CCCP are used, raises the concern that pHluorin-SKL may be mistargeted. Although the protein is clearly associated specifically with peroxisomes (Fig. 1), the possibility exists that translocation through the PTS-1 system may have been incomplete, leaving the pH-sensitive moiety of the protein exposed to the cytosolic face of the peroxisome. The precise location of pHluorin-SKL was probed using antibodies to GFP, which cross-react with the closely related pHluorin. As shown in Fig. 4, in cells permeabilized with Triton X-100, the GFP antibody reacts avidly with pHluorin (Fig. 4A), identifiable by its endogenous fluorescence (Fig. 4B). Under the conditions used, Triton permeabilizes both the plasma and peroxisomal membranes. In contrast, when the plasma membrane was selectively permeabilized using streptolysin O, the anti-GFP antibodies presumably entered the cytosol yet had no access to pHluorin-SKL (cf. Fig. 4, C and D), implying that the protein was sequestered within the lumen of peroxisomes. That streptolysin O effectively permeabilized the membrane allowing the antibodies access to the cytosolic aspect of the cells was shown directly, by adding immunoglobulins extracellularly and detecting their presence after fixation using a labeled secondary antibody (not shown). IgG was detected in the cytosol only in cells treated with streptolysin O but not in the untreated counterparts. That the amounts of IgG entering the cells were sufficient for immunostaining was shown using antibodies to giantin, a component of the Golgi complex that exposes epitopes to the cytosol. In the same cells where pHluorin-SKL was inaccessible to antibodies, giantin was readily immunostained (Fig. 4C). Unlike pHluorin-SKL, the staining pattern and intensity of giantin was comparable in streptolysin O- and Triton X-100-permeablized cells. The effective permeabilization of the plasmalemma by streptolysin O was confirmed by the observation that the cytosolically trapped marker BCECF was rapidly released from the cells upon addition of the pore-former antibiotic (Fig. 5C; see also Ref. 23).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Sidedness of pHluorin-SKL. Cells were transfected with pHluorin-SKL, and the topology of the protein was studied using antibodies to GFP (which cross-react with pHluorin) and to giantin. A and B, cells permeabilized with Triton X-100. A, endogenous pHluorin fluorescence. B, immunolabeling with anti-GFP antibodies. The inset shows anti-giantin immunofluorescence. C and D, selective permeabilization of the plasma membrane using streptolysin O (0.4 µg/ml). C, pHluorin fluorescence. D, anti-GFP immunofluorescence. Inset, corresponding anti-giantin immunofluorescence. Bar = 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Effect of changing extracellular pH on cytosolic and peroxisomal pH. Cells were transfected with either pHluorin (A) or pHluorin-SKL (B and D) to measure cytosolic or peroxisomal pH, respectively, as described under "Experimental Procedures." The cells were initially bathed in high Na+ buffer at pH 7.4. Where indicated, the solution was changed to Na+-free buffer, pH 5.8. In D, the plasma membrane was selectively perforated with streptolysin O in permeabilization buffer. Where indicated, the solution was changed to Na+-free buffer, pH 5.8. The effectiveness of the permeabilization was confirmed in C by monitoring the retention of BCECF. C, top panel, intact cells loaded with BCECF; lower panel, image of the same field 3 min after the addition of streptolysin O.

Relationship between pH_p and the Cytosolic pH-- Taken together, the results described above suggest that the peroxisomal membrane is highly permeant to acid equivalents. This concept was tested directly by measuring pH_p while manipulating the cytosolic pH in intact cells. The latter was accomplished by abruptly changing the extracellular pH while bathing the cells in Na⁺-free solutions. Under these conditions the Na⁺/H⁺ antiporter is unable to counteract the tendency of the cytosol to acidify and will in fact operate in reverse, contributing to the acidification. The effects of such a maneuver are illustrated in Fig. 5A, where cytosolic pH was measured using soluble pHluorin. The cytosol acidifies progressively and equilibrates near pH 5.9 over the course of 30 min. When pH_p was measured under comparable conditions, using pHluorin-SKL, a very similar change in pH was recorded (Fig. 5B), suggesting that the peroxisomal membrane is rather permeable to acid equivalents. This became even more patent when the plasmalemma was selectively permeabilized with streptolysin O (Fig. 5, C and D). A sudden drop of the extracellular (and hence, cytosolic) pH to 5.8 induced a nearly instantaneous acidification of the peroxisomal lumen.

Assessment of Peroxisomal Permeability to CO₂ and HCO-- The apparent discrepancy between our experiments and those of Dansen et al. (9), who recorded an alkaline peroxisomal pH, may result from the effects of CO₂ and/or HCO. All our preceding experiments were conducted in the nominal absence of CO₂, and selective permeability of the peroxisomal membrane to HCO, coupled with the appropriate transmembrane potential, could generate luminal alkalosis. We therefore undertook measurements of pH_p in the presence of CO₂ and HCO. For reference, the cytosolic pH was also measured in parallel experiments. As illustrated in Fig. 6A, introduction of CO₂ elicited a significant acidification of the peroxisomal lumen. As in the case of concanamycin A, we suspected that the change in pH_p may have been secondary to changes in the pH of the cytosol because the plasmalemma is known to be more permeant to CO₂ than to HCO. Cytosolic hydration and dissociation of CO₂ produces an acidification, which was verified using cytosolic pHluorin (Fig. 6B). As before, the changes in pH_p and cytosolic pH were similar in course and magnitude.

View larger version (8K): [in this window] [in a new window]   Fig. 6.   Peroxisomal permeability to CO2 and HCO. Cells were transfected with either pHluorin-SKL (A and C) or pHluorin (B) to measure peroxisomal or cytosolic pH, respectively. The cells were initially bathed in Na+-rich, CO2/HCO-free buffer. Where specified, the solution was rapidly exchanged for an iso-osmolar medium containing HCO that was in equilibrium with 5% CO2. In C, the plasma membrane was selectively perforated with streptolysin O prior to the addition of CO2/HCO. The results are representative of four experiments.

To more directly compare the permeation rates of CO₂ and HCO across the peroxisomal membrane, the plasmalemma was permeabilized with streptolysin O. When CO₂ and HCO were suddenly introduced under these conditions, pH_p was not measurably altered (Fig. 6C). This implies that the permeabilities of the weak acid precursor (CO₂ that hydrates to H₂CO₃) and of the conjugated base (HCO) are not markedly different. Therefore, differential permeability to HCO is unlikely to account for the alkaline pH reported by Dansen et al. (9).

Concluding Remarks-- We were able to measure the peroxisomal pH in a continuous, non-invasive manner using the fluorescent protein pHluorin. Our data indicate that at steady state the pH_p in CHO cells is near neutral: 6.92 as determined by the null-point method and 7.12 as determined using nigericin. The modest discrepancy between these determinations may be attributable to differences in the K⁺ concentration of peroxisomes and the cytosol, which were assumed to be identical. Because the cytosolic pH of CHO cells is similarly neutral, no significant gradient of H⁺ exists across the peroxisomal membrane. This likely reflects the high H⁺ (equivalent) permeability of the peroxisomal membrane, suggested by the parallel behavior of pH_p during the course of imposed changes in cytosolic pH. The neutrality of pH_p is also consistent with our inability to detect functional V-ATPases on the peroxisomal membrane.

While internally consistent and highly reproducible, our data differ from earlier determinations in yeast and in mammalian fibroblasts. In the former, peroxisomes were inferred to be acidic by NMR (8) and other determinations (24), and separate reports detected V-ATPases in yeast peroxisomal preparations (20). However, the biochemical determinations of proton-pumping ATPases are the subject of controversy, because some authors believe that they resulted from contamination of incompletely purified peroxisomes by other organelles (21). Moreover, the NMR determinations are somewhat indirect and required the induction of extreme peroxisomal proliferation (8). It is possible, therefore, that the observed variance in pH reflects differences between resting and proliferating peroxisomes. Alternatively, species differences may account for the disparate pH values recorded.

Our data are more difficult to reconcile with those of Dansen et al. (9), who also used mammalian fibroblastic cells. In this case, the apparent discrepancy may be methodological in origin. Dansen et al. (9) used SNAFL coupled to a water-soluble hexapeptide that, remarkably, permeated freely across the plasmalemma yet was retained within peroxisomes even in the absence of ATP. Their probe displayed a small dynamic range (10% change in fluorescence range per pH unit) and was calibrated externally, which may have failed to take into account the unique conditions prevailing within the peroxisomal lumen. Independent determinations of pH in mammalian peroxisomes will be required to establish whether the SNAFL or the pHluorin method is more accurate.

It has been argued that the peroxisomal pH is either acidic, in the case of yeast, or alkaline, in fibroblasts, in order to maximize the activity of enzymes with corresponding pH optima. A similar argument could be made for a near-neutral pH. The breakdown of fatty acids in the peroxisome, one of the primary functions of peroxisomes, is achieved by a series of oxidases that generate H₂O₂, which is then catalytically decomposed by catalase. While catalase activity is independent of pH over the 4.7-10.5 range (25), the activity of fatty acyl-CoA synthetase is optimal between pH 8 and 9 (26, 27). Therefore the neutral pH would favor the activity of catalase relative to that of the oxidases and thus limit the concentration of potentially harmful H₂O₂. Conditions where oxidase activity exceeds that of catalase, e.g. following addition of peroxisomal proliferators, can result in carcinogenesis through the release of H₂O₂ into the cytosol and nucleus (28).

Alternatively, the neutral pH of peroxisomes may be a consequence of the relatively high permeability of their membrane to small solutes, required for active metabolic traffic and documented in detail earlier (10, 11). By allowing the rapid passage of H⁺ equivalents across their membranes, peroxisomes are effectively connected to the cytosol and utilize its buffering power and the plasmalemmal acid/base transport systems to indirectly maintain pH_p homeostasis.

	FOOTNOTES

^* 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.

^§ Supported by a CIHR Studentship.

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.

Published, JBC Papers in Press, October 18, 2001, DOI 10.1074/jbc.M109003200

² pHluorin-SKL was not expressed in fibroblasts at sufficiently high levels to allow detection, likely because the cDNA employs the Aequora codon usage. Therefore, these experiments were performed using another pH-sensitive fluorescent protein, eGFP-SKL, the sequence of which has been humanized.

	ABBREVIATIONS

The abbreviations used are: PTS, peroxisomal targeting signal; pH_p, peroxisomal pH; CHO, Chinese hamster ovary; BCECF, 2',7'bis(2-carboxyethyl)-5(6)-carboxyfluorescein; GFP, green fluorescent protein; V-ATPase, vacuolar-type proton-pumping ATPase; CCCP, carbonyl cyanide m-chlorophenylhydrazone; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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