Characterization of 14-3-3 proteins in adrenal chromaffin cells and demonstration of isoform-specific phospholipid binding.
Isoform-specific antisera were used to examine which 14-3-3 isoforms were present in bovine adrenal chromaffin cells. The eta, tau and sigma isoforms were not detectable, and the epsilon isoform was present at only low levels. 14-3-3 isoforms were readily detected with antisera against the beta, gamma and zeta isoforms. The latter isoforms were found to leak from digitonin-permeabilized chromaffin cells, as expected for cytosolic proteins, but a proportion of each isoform was retained. In subcellular fractionation studies isoforms recognized by the beta and zeta antisera were found in the cytosol and Triton-insoluble cytoskeletal fractions, while the gamma isoform was found in cytosol and also in microsomal and chromaffin granule membrane fractions. The gamma 14-3-3 protein associated with granule membranes was partially removed by a high-salt/carbonate wash, and the membranes could bind further gamma from cytosol or from a purified brain 14-3-3 protein mixture. The binding of gamma 14-3-3 was not Ca(2+)-dependent, nor was it affected by phorbol ester, GTP analogues or cyclic AMP. Using pure phospholipid vesicles it was found that gamma and also epsilon 14-3-3 proteins bound directly to phospholipids. Little binding of brain beta, eta or zeta to phospholipid vesicles was detected. Brain 14-3-3 proteins were also able to aggregate phospholipid vesicles. Recombinant 14-3-3 isoforms (tau and the Xenopus protein) were able to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized chromaffin cells. The Xenopus proteins lacks part of the extreme N-terminus, indicating that this domain is not essential for function in exocytosis.
Roth
D
D
Physiological Laboratory, University of Liverpool, U.K.
Morgan
A
A
Martin
H
H
Jones
D
D
Martens
G J
GJ
Aitken
A
A
Burgoyne
R D
RD
D048948:14-3-3 Proteins; D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism; D005089:Exocytosis; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012756:Sheep; D013347:Subcellular Fractions / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase; D014981:Xenopus
D048948:14-3-3 Proteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D002118:Calcium