Functional groups required for the stability of yeast RNA triphosphatase in vitro and in vivo

Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent T-phosphate hydrolysis within the hydrophilic in terior of an eight-strand beta barrel (the "triphosphate tunnel"), which re sts upon a globular protein core (the "pedestal"). We performed a structure -guided alanine scan of 17 residues located in the tunnel (Ser(373), Thr(37 5), Gln(405), His(411), ser(429), Glu(488), Thr(490)), on the tunnel's oute r surface (Ser(378), Ser(487), Thr(489), His(491)), at the tunnel-pedestal interface (IIe(304), Met(308)) and in the pedestal (Asp(315), Lys(317) Arg( 321), Asp(425)). Alanine mutations at 14 positions had no significant effec t on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 func tion in vivo. Two of the mutations (R321A and D425A) elicited a thermosensi tive (ts) yeast growth phenotype. The R321A and D425A proteins had full pho sphohydrolase activity in vitro, but were profoundly thermolabile. Arg(321) and Asp(425) interact to form a salt bridge within the pedestal that, teth ers two of the strands of the tunnel. Mutations R321Q and D411N resulted in ts defects in vivo and in vitro, as did the double-mutant R321A-D435A, whe reas the R321K protein was fully stable in vivo and in vitro. These results highlight the critical role of the buried salt bridge in Cet1 stability. R eplacement of Ser(429) by alanine or valine elicited a cold-sensitive (es) yeast growth phenotype. The S429A and S429V proteins were fully active when produced in bacteria at 37 degreesC,, but were inactive when produced at 1 7 degreesC. Replacement of Ser(429) by threonine partially suppressed the c old sensitivity of the Cet1 phosphohydrolase, but did not suppress the cs g rowth defect in yeast.