A screen for dominant negative mutants of SEC18 reveals a role for the AAAprotein consensus sequence in ATP hydrolysis

An evolutionarily ancient mechanism is used for intracellular membrane fusi on events ranging from endoplasmic reticulum-Golgi traffic in yeast to syna ptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), sol uble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their mol ecular mechanisms of action remain unclear. To illuminate important structu re-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a G AL1-regulated SEC18 yeast expression plasmid. Several dominant negative all eles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulati on of a membranous tubular network, growth suppression, increased cell dens ity) is due to a single A-G substitution in SEC18 resulting in a missense m utation in Sec18p (Tkr(394)-->Pro). Thr(394) is conserved in most AAA prote ins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18 -109p indicates that the mutation does not prevent hexamerization or intera ction with yeast alpha-SNAP (Sec17p), but instead results in undetectable A TPase activity that cannot be stimulated by Sec17p. This suggests a role fo r the AAA protein consensus sequence in regulating ATP hydrolysis. Furtherm ore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functio nal domains in their mammalian counterparts.