ANALYSIS OF THE TRANSMEMBRANE TOPOLOGY OF THE GLYCINE TRANSPORTER GLYT1

A theoretical 12-transmembrane segment model based on the hydrophobic moment has been proposed for the transmembrane topology of the glycine transporter GLYT1 and all other members of the sodium- and chloride-d ependent transporter family, We tested this model by introducing N-gly cosylation sites along the GLYT1 sequence as reporter for an extracell ular localization and by an in vitro transcription/translation assay t hat allows the analysis of the topogenic properties of different segme nts of the protein, The data reported herein are compatible with the e xistence of 12 transmembrane segments, but support a rearrangement of the first third of the protein, Contrary to prediction, hydrophobic do main 1 seems not to span the membrane, and the loop connecting hydroph obic domains 2 and 3, formerly believed to be intracellular, appears t o be extracellularly located, In agreement with the theoretical model, we provide evidence for the extracellular localization of loops betwe en hydrophobic segments 5 and 6, 7 and 8, 9 and 10, and 11 and 12.