title: pH regulation in glycosomes of procyclic formTrypanosoma brucei.
abstract: Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic formTrypanosoma bruceiWhen added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+exchangers are required for glycosomal pH regulation.
authors.affiliation: From the Departments of Chemistry and.
authors.forename: Sheng
authors.initials: S
authors.lastname: Lin
authors.affiliation: From the Departments of Chemistry and.|the Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602.
authors.forename: Charles
authors.initials: C
authors.lastname: Voyton
authors.affiliation: Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634 and.
authors.forename: Meredith T
authors.initials: MT
authors.lastname: Morris
authors.affiliation: the Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602.
authors.forename: P Christine
authors.initials: PC
authors.lastname: Ackroyd
authors.affiliation: Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634 and.
authors.forename: James C
authors.initials: JC
authors.lastname: Morris
authors.affiliation: the Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602 kenc@chem.byu.edu.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Christensen
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000584:Amiloride / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D003600:Cytosol / Q000737:chemistry; D003847:Deoxyglucose / Q000737:chemistry; D004072:Digitonin / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D018942:Macrolides / Q000737:chemistry; D008830:Microbodies / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D011188:Potassium / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D000072417:Protein Domains; D015800:Protozoan Proteins / Q000737:chemistry; D019810:Sodium Azide / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000737:chemistry*
chemical_list: D018942:Macrolides; D015800:Protozoan Proteins; C057620:bafilomycin A; D000584:Amiloride; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D011392:Proline; D003847:Deoxyglucose; D005947:Glucose; D004072:Digitonin; D011188:Potassium; C039614:ethylisopropylamiloride
keywords: Trypanosoma brucei; biosensor; fluorescence; glucose metabolism; microscopy; parasite metabolism; parasitology; proton pump; trypanosome