A fluorescent probe of polyamine transport accumulates into intracellular acidic vesicles via a two-step mechanism.
Mammalian polyamine carriers have not yet been molecularly identified. The fluoroprobe Spd-C2-BODIPY faithfully reports polyamine transport and accumulates almost exclusively in polyamine-sequestering vesicles (PSVs). Polyamines might thus be imported first by a plasma membrane carrier and then sequestered into pre-existing PSVs (model A), or be directly captured by polyamine receptors undergoing endocytosis (model B). Spd-C2-BODIPY uptake was unaffected in receptor-mediated endocytosis-deficient Chinese hamster ovary cell mutants. PSVs strongly colocalized with acidic vesicles of the late endocytic compartment and the trans Golgi. Virtually perfect colocalization between PSVs and acidic vesicles was found in Chinese hamster ovary cell mutants that are blocked either in the late endosome/lysosome fusion process or in the maturation of multivesicular bodies. Prior inhibition of the V-ATPase dramatically decreased total Spd-C2-BODIPY accumulation while increasing cytosolic fluorescence. Conversely, cells pre-loaded with the probe slowly released it from PSVs upon V-ATPase inhibition. The present data thus support model A, and indicate that polyamine accumulation is primarily driven by the activity of a vesicular H+:polyamine carrier.
Soulet
Denis
D
Oncological and Molecular Endocrinology Research Center, CHUL Research Center, Sainte Foy, Quebec G1V 4G2, Canada.
Gagnon
Bruno
B
Rivest
Serge
S
Audette
Marie
M
Poulin
Richard
R
D000818:Animals; D001692:Biological Transport; D001896:Boron Compounds / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006056:Golgi Apparatus / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008956:Models, Chemical; D008985:Monensin / Q000378:metabolism; D009154:Mutation; D011073:Polyamines / Q000737:chemistry*; D011522:Protons; D013095:Spermidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D018942:Macrolides; D011073:Polyamines; D011522:Protons; C494777:Spd-C2-BODIPY; C040929:bafilomycin A1; D008985:Monensin; D025262:Vacuolar Proton-Translocating ATPases; D013095:Spermidine