Structure-function analysis of the yeast NAD+-dependent tRNA 2'-phosphotransferase Tpt1

Abstract

Tpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2'-PO4 from the splice junction to NAD+ to form ADP-ribose 1''-2''cyclic phosphate and nicotinamide. To understand the structural requirements for Saccharomyces cerevisiae Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea. We thereby identified four residues-Arg23, His24, Arg71, and Arg138-as essential for Tpt1 function in vivo. Structure-activity relationships at these positions were clarified by introducing conservative substitutions. The activity of the Escherichia coli ortholog KptA in complementing tpt1Delta was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125. Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30 degrees C. These findings attest to the structural and functional conservation of Tpt1-like 2'-phosphotransferases and identify likely constituents of the active site.

FIGURE 1.

Mechanism and primary structure of NAD⁺-dependent 2′-phosphotransferase. (Left ) Two-step mechanism of tRNA 2′-phosphate removal catalyzed by S. cerevisiae Tpt1 and its E. coli ortholog KptA (Spinelli et al. 1999). See text for details. (Right) The amino acid sequence of S. cerevisiae (Sce) Tpt1 from residues 14–185 is aligned to the sequences of the homologous proteins of E. coli (Eco), Schizosaccharomyces pombe (Spo), Homo sapiens (Hsa), Drosophila melanogaster (Dme), Leishmania major (Lma), Trypanosoma cruzi (Tcr), Pyrococcus horikoshii (Pho), Nostoc punctiforme (Npu), Clostridium perfringens (Cpe), Aeropyrum pernix (Ape), and Archaeoglobus fulgidus (Afu). Positions of side chain identity or similarity in all six proteins are denoted by an arrowhead under the alignment. Residues of Tpt1 that were subjected to alanine scanning are indicated by shaded boxes. Amino acids found to be essential for Tpt1 function in vivo are indicated by vertical bars. Nonessential residues are indicated by +. The V8 protease-sensitive site of Tpt1 (Glu107–Ala108) is indicated by an arrow.

FIGURE 2.

Sedimentation and limited proteolysis of Tpt1. (A) Glycerol gradient sedimentation. An aliquot (80 μg) of the Ni-agarose preparation of His₁₀-Tpt1 was mixed with BSA (40 μg) and cytochrome c (50 μg). The protein mixture was applied to a 4.8-mL 15%–30% glycerol gradient containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.025% Triton X-100. The gradient was centrifuged in a Beckman SW50 rotor at 50,000 rpm for 16 h at 4°C. Fractions (~0.26 mL) were collected from the bottom of the tubes. Aliquots (12 μL) of odd-numbered fractions were analyzed by SDS-PAGE. (B) Proteolysis. Reaction mixtures (20 μL) containing 10 mM Tris-HCl (pH 8.0), 5 μg of Tpt1, and 1.6, 3.1, 6.2, 12.5, 25, 50, or 100 ng of V8 protease (from left to right) were incubated for 15 min at 22°C. V8 protease was omitted from the control mixture shown in lane –. The reactions were quenched by adding SDS and the digests were resolved by SDS-PAGE. The Coomassie Blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. Duplicate samples were resolved by SDS-PAGE and the gel contents were transferred electrophoretically to a polyvi-nylidene difluoride membrane. Polypeptides were visualized by staining the membrane with Coomassie Blue dye. Membrane slices containing intact His₁₀-Tpt1 and individual proteolytic products (denoted by arrow-heads on the right) were excised and subjected to automated Edman sequencing. The sequences are indicated in single-letter code (right).

FIGURE 3.

Limited proteolysis of purified KptA. Reaction mixtures (20 μL) containing 10 mM Tris-HCl (pH 8.0), 5 μg of KptA, and 1.6, 3.2, 6.2, or 12.5 ng of trypsin (from left to right) were incubated for 15 min at 22°C. Trypsin was omitted from the control mixture shown in lane –. The reactions were quenched by adding SDS and the digests were resolved by SDS-PAGE. The Coomassie Blue-stained gel is shown. A duplicate gel was electroblotted to PVDF; intact His₁₀-Tpt1 and individual tryptic products were subjected to automated Edman sequencing; their sequences are indicated in single-letter code (right).