SDS-RESISTANT AGGREGATION OF MEMBRANE-PROTEINS - APPLICATION TO THE PURIFICATION OF THE VESICULAR MONOAMINE TRANSPORTER

The vesicular monoamine transporter, which catalyses a H+/monoamine an tiport in monoaminergic vesicle membrane, is a very hydrophobic intrin sic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially wh en samples were boiled in the classical Laemmli buffer before electrop horesis. This behaviour was analysed in some detail. The aggregation w as promoted by high temperatures, organic solvents and acidic pH, sugg esting that it resulted from the unfolding of structure remaining in S DS. The aggregates were very stable and could be dissociated only by s uspension in anhydrous trifluoroacetic acid. This SDS-resistant aggreg ation behaviour was shared by very few intrinsic proteins of the chrom affin granule membrane. Consequently, a purification procedure was bas ed on this property. A detergent extract of chromaffin granule membran es enriched in monoamine transporter was heated and the aggregates wer e isolated by size-exclusion HPLC in SDS. The aggregates, containing t he transporter, were dissociated in the presence of trifluoroacetic ac id and analysed on the same HPLC column. This strategy might be of gen eral interest for the purification of membrane proteins that exhibit S DS-resistant aggregation.