Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.
The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide. After receptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport. We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact live cells. Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy. After 1 h at 37 degrees C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including alpha-mannosidase II, beta-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex. Imaging of the emission of fluorescein-labeled VT1B was used to measure intra-Golgi pH (pHG), which was calibrated in situ with ionophores. In intact Vero cells, pHG averaged 6.45 +/- 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar-type ATPases, pHG remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H+ antiport. Similarly, pHG was unaffected by acute changes in cytosolic calcium. Furthermore, pHG recovered quickly toward the basal level after departures imposed with weak bases. These findings suggest that pHG is actively regulated, despite the presence of a sizable H+ "leak" pathway. The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.
Kim
J H
JH
Division of Gastroenterology and Nutrition, Hospital for Sick Children, Toronto, Ontario, Canada.
Lingwood
C A
CA
Williams
D B
DB
Furuya
W
W
Manolson
M F
MF
Grinstein
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015415:Biomarkers; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002522:Chlorocebus aethiops; D019793:Fluorescein; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005915:Globosides / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism*; D006056:Golgi Apparatus / Q000502:physiology* / Q000648:ultrastructure; D006367:HeLa Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D015388:Organelles / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry; D014709:Vero Cells
D015415:Biomarkers; D005452:Fluoresceins; D005915:Globosides; D006017:Glycolipids; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C068556:Shiga-like toxin receptor; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium; D019793:Fluorescein