Calcium-promoted resonance energy transfer between fluorescently labeled proteins during aggregation of chromaffin granule membranes.
Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor. Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact upon Ca2+-induced aggregation of the membranes. The four major findings were: (1) Raising the Ca2+ concentration to approx. 500 microM causes the proteins to aggregate in the plane of the membrane. This is demonstrated by Ca2+-induced increases in the fluorescence resonance energy transfer in double labeled membranes. This effect is not protein-concentration dependent and occurs at calcium concentrations too low for granule aggregation, implying intra-membrane protein clustering or patching. To our knowledge this is the first direct demonstration of the fluid mosaic nature of subcellular organelles. (2) If two sets of granules are labeled separately, Ca2+-induced aggregation brings at least 74% of the labeled proteins into close transmembrane proximity. This effect is also observed at 10-100-fold slower rates in the absence of calcium and can be greatly reduced by depleting the granule membrane of labeled peripheral proteins. It is enhanced if the granules are aggregated by Ca2+ or K+. We conclude that (some) peripheral proteins can transfer from one membrane surface to another. (3) Aggregation of separately labeled sets of membranes by Ca2+ also produces transmembrane energy transfer since: (a) the Km for Ca2+-induced quantum transfer is in the same range as the Km for aggregation; (b) the reaction is protein-concentration dependent; (c) reversal of aggregation also (partially) reverses donor quenching. (4) A kinetic analysis of the transmembrane effect shows it to be 5-10-fold slower than aggregation itself, supporting earlier suggestions (Haynes, D.H., Kolber, M. and Morris, S.J., (1979) J. Theor. Biol. 81, 713-743) that lipid and protein rearrangements are secondary to granule membrane aggregation.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002837:Chromaffin Granules / Q000187:drug effects / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D004735:Energy Transfer; D005452:Fluoresceins; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D005452:Fluoresceins; D008565:Membrane Proteins; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C037041:fluorescein 5-maleimide; D011188:Potassium; D002118:Calcium