TRANSLOCATION OF CYTOSOLIC ANNEXIN-2 TO A TRITON-INSOLUBLE MEMBRANE SUBDOMAIN UPON NICOTINE STIMULATION OF CHROMATIN CULTURED-CELLS

To gain a better understanding of the function of annexin 2, we have i nvestigated the subcellular distribution of the monomeric and heterote trameric forms of annexin 2 and their relationship to the cytoskeleton upon stimulation of chromaffin cells, Quantitative immunoblotting has revealed that in resting cells a large amount of annexin 2 is monomer ic and cytosolic. Upon nicotine stimulation 80% of total annexin 2 bec omes associated with a Triton-X100-insoluble fraction where the monome ric and the heterotetrameric forms are found, The translocation of mon omeric annexin 2 is Ca2+-dependent and complete at 1 mu M free Ca2+. W e have shown that about 66% of the annexin 2 associated with the Trito n-X100-insoluble fraction is soluble in octylglucoside while the remna nts are insoluble in the detergent and remain likely associated with a ctin filaments and associated cytoskeleton proteins, The octylglucosid e-soluble fraction contains integral proteins from the plasma membrane and from granule membrane, but does not contain caveolin, Moreover, u pon nicotine stimulation, a redistribution of proteins was detected in this fraction, These dynamic processes appear concomitantly with the phosphorylation of annexin 2 in this compartment and with catecholamin e release, It is suggested that the soluble octylglucoside fraction ma y represent a special Lipidic membrane compartment where the NSF attac hment proteins and the cytosolic proteins like annexin 2 and rab3a may become concentrated upon stimulation of the cell, The presence of ann exin 2 is consistent with its proposed function on granule and target membrane proteins required for the close apposition of two distinct me mbranes and supports its functional role in the regulated exocytosis/e ndocytosis process. (C) 1997 Federation of European Biochemical Societ ies.