Neurexin Iβ and neuroligin are localized on opposite membranes in mature central synapses

Synaptogenesis requires formation of trans-synaptic complexes between neuronal cell-adhesion receptors. Heterophilic receptor pairs, such as neurexin Iβ and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre- and postsynaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the postsynaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Iβ and neuroligin in mature cerebrocortical synapses, we have developed a novel method for physical separation of junctional membranes and their direct analysis by western blotting. Using urea and DTT, we disrupted trans-synaptic protein links, without dissolving the lipid phase, and fractionated the pre- and postsynaptic membranes. The purity of these fractions was validated by electron microscopy and western blotting using multiple synaptic markers. A quantitative analysis has confirmed that neuroligin is localized strictly in the postsynaptic membrane. We have also demonstrated, that neurexin Iβ is largely (96%) presynaptic. Thus, neurexin Iβ and neuroligin normally form trans-synaptic complexes and can transduce bidirectional signals.