Testing the 3Q:1R "rule": mutational analysis of the ionic "zero" layer in the yeast exocytic SNARE complex reveals no requirement for arginine.

The crystal structure of the synaptic SNARE complex reveals a parallel four-helix coiled-coil arrangement; buried in the hydrophobic core of the complex is an unusual ionic layer composed of three glutamines and one arginine, each provided by a separate alpha-helix. The presence of glutamine or arginine residues in this position is highly conserved across the t- and v-SNARE families, and it was recently suggested that a 3Q:1R ratio is likely to be a general feature common to all SNARE complexes. In this study, we have used genetic and biochemical assays to test this prediction with the yeast exocytic SNARE complex. We have determined that the relative position of Qs and Rs within the layer is not critical for biological activity and that Q-to-R substitutions in the layer reduce complex stability and result in lethal or conditional lethal growth defects. Surprisingly, SNARE complexes composed of four glutamines are fully functional for assembly in vitro and exocytic function in vivo. We conclude that the 3Q:1R layer composition is not required within the yeast exocytic SNARE complex because complexes containing four Q residues in the ionic layer appear by all criteria to be functionally equivalent. The unexpected flexibility of this layer suggests that there is no strict requirement for the 3Q:1R combination and that the SNARE complexes at other stages of transport may be composed entirely of Q-SNAREs or other noncanonical combinations.