Direct measurement of trans-Golgi pH in living cells and regulation by second messengers.
In the endocytic compartment, an acidic pH plays a key role in receptor and ligand sorting, vesicular transport, and protein degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and following viral infection suggest a mildly acidic pH of > 6.0. We developed a liposome microinjection method to introduce fluorescent indicators into the aqueous compartment of trans-Golgi in living cells. In the presence of ATP and at 37 degrees C, 70-nm diameter liposomes delivered their fluid-phase contents selectively into the trans-Golgi compartment as assessed by colocalization with the trans-Golgi stain N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosine (C6-NBD-ceramide). Liposome fusion was ATP- and temperature-dependent and blocked by N-ethylmaleimide but not by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). trans-Golgi pH in skin fibroblasts was 6.17 +/- 0.02 (S.E., n = 174) as measured by ratio imaging confocal microscopy using fluorescein and rhodamine-based indicators and an in vivo calibration procedure. trans-Golgi pH increased to 6.8 +/- 0.1 by cAMP agonists and to 6.5 +/- 0.1 by protein kinase C activation. These results provide the first direct measurement of trans-Golgi pH in living cells and demonstrate pH regulation by second messengers.
Seksek
O
O
Department of Medicine, University of California, San Francisco 94143-0521.
Biwersi
J
J
Verkman
A S
AS
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology; D002460:Cell Line; D002518:Ceramides; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006706:Homeostasis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D008845:Microinjections; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D015290:Second Messenger Systems / Q000187:drug effects; D012867:Skin / Q000378:metabolism; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D002518:Ceramides; D005456:Fluorescent Dyes; D008081:Liposomes; D010982:Platelet-Derived Growth Factor; D013873:Thionucleotides; D000643:Ammonium Chloride; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D009327:4-Chloro-7-nitrobenzofurazan; D013755:Tetradecanoylphorbol Acetate