Identification of human vesicle monoamine transporter (VMAT2) lumenal cysteines that form an intramolecular disulfide bond.
The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. To obtain structural information regarding this large membrane protein by analysis of disulfide bonds and other intramolecular cross-links, we engineered a strategic thrombin cleavage site into deglycosylated, HA-tagged human VMAT2. Insertion of this protease site did not disrupt ligand binding or serotonin transport. Thrombin cleavage at an engineered site in the predicted cytoplasmic loop between transmembrane (TM) domains 6 and 7 (loop 6/7) was rapid and quantitative in the absence of any detergent. The loop 6/7 thrombin site allowed assessment of an intramolecular disulfide bond between the N- and C-terminal halves of the transporter. Consistent with this hypothesis, after quantitative loop 6/7 thrombin cleavage, in the absence of reducing agent, VMAT2 migrated on SDS-polyacrylamide gels as a full-length transporter. Addition of dithiothreitol resulted in complete conversion from full-length to thrombin-cleaved size, demonstrating a DTT-reversible covalent bond. The identity of the disulfide-bound cysteine pair was suggested by the observation that replacement of Cys 126 or Cys 333 with serine both reduced [(3)H]serotonin transport. Replacement of either Cys 126 or Cys 333 was found to eliminate the DTT-reversible intramolecular covalent bond. We conclude that human VMAT2 Cys 126 in loop 1/2 and Cys 333 in loop 7/8 form a disulfide bond which contributes to efficient monoamine transport.
Thiriot
David S
DS
Department of Pharmacology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, Wisconsin 53706-1532, USA.
Sievert
Michael K
MK
Ruoho
Arnold E
AE
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides; D017433:Protein Structure, Secondary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D013917:Thrombin / Q000378:metabolism; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D004220:Disulfides; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; C493452:SLC18A2 protein, human; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D013917:Thrombin; D003545:Cysteine