Identification of functionally interacting SNAREs by using complementary substitutions in the conserved '0' layer

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes form bundles of four parallel a-helices. The central '0' layer of interacting amino acid side chains is highly conserved and contains one arginine and three glutamines, leading to the classification of SNAREs into R, Qa, Qb, and Qc-SNAREs. Replacing one of the glutamines with arginine in the yeast exocytotic SNARE complex is either lethal or causes a conditional growth defect that is compensated by replacing the R-SNARE arginine with glutamine. Using the yeast SNARE complex mediating traffic from the encloplasmic reticulum to the Golgi apparatus, we now show that functionally interacting SNAREs can be mapped by systematically exchanging glutamines and arginines in the '0' layer. The Q -> R replacement in the Qb-SNARE Bos1p has the strongest effect and can be alleviated by an Q -> R replacement in the R-SNARE Sec22p. Four Q residues in the central layer caused growth defects above 30 degrees C that were rescued by Q -> R substitutions in the Qa and Qc SNAREs Sed5p and Bet1p, respectively. The sec22(Q)/sed5(R) mutant is temperature sensitive and is rescued by a compensating R -> Q replacement in the R-SNARE Ykt6p. This rescue is attributed to the involvement of Sed5p and Ykt6p in a different SNARE complex that functions in intra-Golgi trafficking.