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 gamma-phosphate hydrolysis within the hydrophilic interior of an eight-strand beta barrel (the "triphosphate tunnel"), which rests upon a globular protein core (the "pedestal"). We performed a structure-guided alanine scan of 17 residues located in the tunnel (Ser(373), Thr(375), Gln(405), His(411), Ser(429), Glu(488), Thr(490)), on the tunnel's outer surface (Ser(378), Ser(487), Thr(489), His(491)), at the tunnel-pedestal interface (Ile(304), Met(308)) and in the pedestal (Asp(315), Lys(317), Arg(321), Asp(425)). Alanine mutations at 14 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. Two of the mutations (R321A and D425A) elicited a thermosensitive (ts) yeast growth phenotype. The R321A and D425A proteins had full phosphohydrolase activity in vitro, but were profoundly thermolabile. Arg(321) and Asp(425) interact to form a salt bridge within the pedestal that tethers 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, whereas 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. Replacement of Ser(429) by alanine or valine elicited a cold-sensitive (cs) yeast growth phenotype. The S429A and S429V proteins were fully active when produced in bacteria at 37 degrees C, but were inactive when produced at 17 degrees C. Replacement of Ser(429) by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress the cs growth defect in yeast.
Bisaillon
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M
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
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S
D017766:Acid Anhydride Hydrolases / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D020134:Catalytic Domain; D002455:Cell Division; D017124:Conserved Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000596:Amino Acids; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; C023090:RNA triphosphatase; D000409:Alanine