FOLDING A WD REPEAT PROPELLER - ROLE OF HIGHLY CONSERVED ASPARTIC-ACID RESIDUES IN THE G-PROTEIN BETA-SUBUNIT AND SEC13

The beta subunit of the heterotrimeric G proteins that transduce signa ls across the plasma membrane is made up of an amino-terminal alpha-he lical segment followed by seven repeating units called mo (Trp-Asp) re peats that occur in about 140 different proteins. The seven WD repeats in G beta, the only WD repeat protein whose crystal structure is know n, form seven antiparallel beta sheets making up the blades of a toroi dal propeller structure (Wall, M. A., Coleman, D. E., Lee, E., Iniguez -Lluhi, J. A., Posner, B. A., Gilman, A. Gr., and Sprang, S. R. (1995) Cell 83, 1047-1058; Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369-374). It is likely that a ll proteins with WD repeats form a propeller structure. Alignment of t he sequence of 918 unique WD repeats reveals that 85% of the repeats h ave an aspartic acid (D) residue (not the D of WD) in the turn connect ing beta strands b and c of each putative propeller blade. We mutated each of these conserved Asp residues to Gly individually and in pairs in G beta and in Sec13, a yeast WD repeat protein involved in vesicula r traffic, and then analyzed the ability of the mutant proteins to fol d in vitro and in COS-7 cells. lit vitro, most single mutant G beta su bunits fold into G beta gamma dimers more slowly than wild type to a d egree that varies with the blade. In contrast, all single mutants form normal amounts of G beta gamma in COS-7 cells, although some dimers s how subtle local distortions of structure. Most double mutants assembl e poorly in both systems. We conclude that the conserved Asp residues are not equivalent and not all are essential for the folding of the pr opeller structure. Some may affect the folding pathway or the affinity for chaperonins. Mutations of the conserved Asp in Sec13 affect foldi ng equally in vitro and in COS-7 cells. The repeats that most affected folding were not at the same position in Sec13 and G beta. Our findin g, both in G beta and in Sec13, that no mutation of the conserved Asp entirely prevents folding suggests that there is no obligatory folding order for each repeat and that the folding order is probably not the same for different WD repeat proteins, or even necessarily constant fo r the same protein.