Formation of a ternary complex between GM2 activator protein, GM2 ganglioside and hexosaminidase A.

The GM2 activator is a 17 kDa protein required for the hydrolysis of GM2 ganglioside by the lysosomal enzyme hexosaminidase A (HexA). The activator behaves as a substrate binding protein, solubilizing GM2 ganglioside monomers from micelles (in vitro) or membranes (in vivo). However, the activator also shows a high order of specificity for activation of lysosomal hydrolases and has been predicted to form a ternary complex with the heterodimeric enzyme (alphabeta) Hex A and GM2 ganglioside. We demonstrated a transient interaction between HexA and the GM2 activator. A chimeric protein containing the FLAG epitope sequence upstream of the GM2 activator was expressed in Escherichia coli and purified using the M1 immunoaffinity (anti-FLAG) column. Binding of the FLAG-GM2 activator (FLAG-AP) fusion protein to the M1 column led to the specific retardation of Hex A applied to the column. Other proteins were not retarded by the column nor did they compete with Hex A for binding to FLAG-AP. Hex A and GM2 ganglioside could be simultaneously bound to the column, but the binding of each ligand was independent of the other. The homodimeric (beta beta) isozyme Hex B did not bind to the immobilized activator. The alpha alpha homodimer, HexS, bound weakly, confirming that a hexosaminidase alpha subunit is required for interaction of enzyme and activator.

Elimination of the yeast RAD6 ubiquitin conjugase enhances base-pair transitions and G.C----T.A transversions as well as transposition of the Ty element: implications for the control of spontaneous mutation.

The RAD6 gene of the yeast Saccharomyces cerevisiae encodes an enzyme that conjugates ubiquitin to other proteins. Defects in RAD6 confer a mutator phenotype due, in part, to an increased rate of transposition of the yeast Ty element. To further delineate the role of protein ubiquitination in the control of spontaneous mutagenesis in yeast, we have characterized 202 mutations that arose spontaneously in the SUP4-o gene carried on a centromere vector in a RAD6 deletion strain. The resulting mutational spectrum was compared to that for 354 spontaneous SUP4-o mutations isolated in the isogenic wild-type parent. This comparison revealed that the rad6 mutator enhanced the rate of single base-pair substitution, as well as Ty insertion, but did not affect the rates of the other mutational classes detected. Relative to the wild-type parent, Ty inserted at considerably more SUP4-o positions in the rad6 strain with a significantly smaller fraction detected at a transposition hotspot. These findings suggest that, in addition to the rate of transposition, protein ubiquitination might influence the target site specificity of Ty insertion. The increase in the substitution rate accounted for approximately 90% of the rad6 mutator effect but only the two transitions and the G. C----T.A transversion were enhanced. Analysis of the distribution of these events within SUP4-o suggested that the site specificity of the substitutions was influenced by DNA sequence context. Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad6 mutator did not reduce the efficiency of correcting mismatches that could give rise to the transitions or transversion nor did it bias restoration of the mismatches to the incorrect base-pairs. These results are discussed in relation to possible mechanisms that might link ubiquitination of proteins to spontaneous mutation rates.