Activation of soluble guanylyl cyclase at the leading edge during Dictyostelium chemotaxis.
Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.
Veltman
Douwe M
DM
Department of Biochemistry, University of Groningen, 9747 AG Groningen, The Netherlands.
Roelofs
Jeroen
J
Engel
Ruchira
R
Visser
Antonie J W G
AJ
Van Haastert
Peter J M
PJ
D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D002633:Chemotaxis; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000096:biosynthesis; D003600:Cytosol / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008561:Membrane Fusion; D012995:Solubility; D013379:Substrate Specificity
D007527:Isoenzymes; D049452:Green Fluorescent Proteins; D008345:Manganese; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D008274:Magnesium