FIGURE 4.

The highly conserved Pro³¹⁴ is crucial for TBZ binding and plays a role in the transport function of rVMAT2. A, replacement of Pro³¹⁴ by Leu or Thr confers resistance to TBZ in yeast cells. ADU1–7 cells were transformed with pAES426 (empty vector) or pAES426 harboring rVMAT2 and Pro³¹⁴ mutants. Growth was assayed and analyzed as described in the legend to Fig. 1A. B, time course of [³H]serotonin transport by Pro³¹⁴ mutants reconstituted in proteoliposomes. The uptake assay was performed as described in the legend to Fig. 1B. All data are mean ± S.E. of 2–3 experiments. C, conservation of Pro³¹⁴. The sequence of rVMAT2 was used to query the National Center for Biotechnology Information (NCBI) using the psi-BLAST tool as provided by the NCBI server with default parameters. Multiple sequence alignment and the conservation logo were created as described in the legend to Fig. 1C. Only a fraction of the alignment corresponding to residues 311–320 is shown. D, kinetic properties of Pro³¹⁴ mutants. Proteoliposomes were prepared from HEK293 cells expressing rVMAT2 or Pro³¹⁴ mutants. K_D for [³H]TBZOH binding, K_m, V_max for [³H]serotonin uptake, and IC₅₀ for reserpine and TBZ, were determined as described in the legend to Fig. 1D.