GLYCOSYLATION OF A VESICULAR MONOAMINE TRANSPORTER - A MUTATION IN A CONSERVED PROLINE RESIDUE AFFECTS THE ACTIVITY, GLYCOSYLATION, AND LOCALIZATION OF THE TRANSPORTER

The role of N-glycosylation in the expression, ligand recognition, act ivity, and intracellular localization of a rat vesicular monoamine tra nsporter (rVMAT1) was investigated. The glycosylation inhibitor tunica mycin induced a dose-dependent decrease in the rVMAT1-mediated uptake of [H-3]serotonin. Part of this effect was due to a general toxic effe ct of the drug. Therefore, to assess the contribution of each of the g lycosylation sites to the transporter activity, the three putative N-g lycosylation sites were mutated individually, in combination, and in t ote (''triple'' mutant). Mutation of each glycosylation site caused a minor and additive decrease in activity, up to the triple mutant, whic h retained at least 50% of the wildtype activity. No significant diffe rences were found either in the time dependence of uptake or the appar ent affinity for ligands of the triple mutant compared with the wildty pe protein. It is interesting that in contrast to plasmamembrane neuro transmitter transporters, the unglycosylated form of rVMAT1 distribute d in the cell as the wildtype protein. Pro(43) is a highly conserved r esidue located at the beginning of the large loop in which all the pot ential glycosylation sites are found. A Pro(43)Leu mutant transporter was inactive. It is remarkable that despite the presence of glycosylat ion sites, the mutant transporter was not glycosylated. Moreover, the distribution pattern of the Pro43Leu mutant clearly differed from that of the wild type. In contrast, a Pro (43)Gly mutant displayed an acti vity practically identical to the wild-type protein. As this replaceme nt generated a protein with wild-type characteristics, we suggest that the conformation conferred by the amino acid at this position is esse ntial for activity.