THE EFFECT OF N-LINKED GLYCOSYLATION ON ACTIVITY OF THE NA-DEPENDENT AND CL--DEPENDENT SEROTONIN TRANSPORTER EXPRESSED USING RECOMBINANT BACULOVIRUS IN INSECT CELLS()

The rat Na+- and Cl--dependent serotonin transporter was expressed in Sf9 insect cells using the baculovirus system. Expression of the serot onin transporter caused the Sf9 cells to accumulate [H-3]serotonin (K- m 78 nM) and to bind the specific transport inhibitor [I-125]RTI55 (2 beta-carbomethoxy-3 beta-(4- [I-125]iodophenyl)tropane) (K-d 0.22 nM). Ligand binding assays on isolated membranes showed 500,000 copies of the serotonin transporter/cell (9 pmol/mg of membrane protein). Immuno reactive bands of apparent M(r) 54,000 (unglycosylated) and 60,000 (gl ycosylated) were observed in Western blots of membrane proteins from i nfected cells. The 54-kDa band was significantly smaller than the expe cted M(r) of 72,500 predicted from the cDNA sequence. The 54-kDa band was shown to represent the intact serotonin transporter by expressing a recombinant serotonin transporter that contained c-Myc and FLAG epit ope tags engineered at the N and C termini, respectively. Both tags we re present on a membrane protein that migrated slightly slower than th e previously observed 54-kDa band, consistent with the extra mass adde d by the tags. The tags did not affect the K-d for [I-125]RTI55 bindin g. The effect of N-linked glycosylation on ligand binding and the leve l of expression were studied. The expression of the serotonin transpor ter in tunicamycin-treated Sf9 cells resulted in low levels of ligand binding activity (0.2 pmol/mg) but unchanged K-d. Similarly, mutated s erotonin transporters that contained reduced numbers of N-linked glyco sylation sites had unchanged K-d for [I-125]RTI55 binding whether ther e were 2, 1, or 0 N-linked glycosylation sites present on the serotoni n transporter. In contrast, B-max was dramatically reduced; levels of expression of the unglycosylated serotonin transporter (0.4 pmol/mg) w ere 20-fold lower compared with levels of the fully glycosylated serot onin transporter. The K-m for [H-3]serotonin uptake was also unchanged . These data indicate that glycosylation is required for optimal stabi lity of the serotonin transporter in the membrane but not for serotoni n transport or ligand binding per se.