Journal: Cell

Article Title: The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity.

doi: 10.1016/s0092-8674(00)80975-7

Figure Lengend Snippet: Figure 6. mtEXO Activity Cofractionates with Suv3p andSuv3p-6his through Chromatography on a Nickel–NTA Histidine Affinity Column C-terminal hexahistidine extension of Suv3p (Suv3p- Mitochondrial lysates from strain 2-11 expressing either Suv3p (C) 6his), cloned into an expression vector (Ycplac22) under or Suv3p-6his (H) were passed over identical Affigel blue columns as the control of the galactose-inducible promoter GAL1. described in Experimental Procedures. Affigel blue column fractions As a control, the wild-type suv3 gene was cloned into containing peak levels of either Suv3p or Suv3p-6his were loaded the same vector. Constructs containing either suv3 or onto identical columns that each contained 1.5 ml of nickel–NTA the suv3-6his allele were placed into Ycplac22 (which resin (Qiagen). Columns were washed with column buffer and then developed with an imidazole step gradient as indicated. A sample contains TRP1 as a selectable marker) and transformed of the input, flowthrough (FT) and the imidazole-eluted material were into the Dsuv3 Dnuc1 strain, 2-11. Trp1 transformants analyzed for Suv3p and assayed for mtEXO activity as described were selected and maintained in selective galactose in Experimental Procedures. The top panel shows a Western blot medium. Western blot analysis confirmed that Suv3p analysis using Suv3p-specific antiserum on an aliquot of each frac- and Suv3p-6his were expressed only in Dsuv3 cells tion. The bottom two panels show PEI–cellulose TLC plates, spotted transformed withthe suv3 and suv3-6his contructs when with 3 ml of the mtEXO assay reaction mix for each fraction, devel- those cells were grown on galactose medium (data not oped in 1 M formic acid, 0.5M LiCl. mtEXO activity was assayed as described in Experimental Procedures in the presence (middle) or shown). Additional control experiments showed that absence (bottom) of 1 mM ATP. TLC plates were visualized by Suv3p-6his is functional in vivo, since a r1 mitochondrial autoradiography and scanned with a Molecular Dynamics phos- genome was stable in galactose-grown Dsuv3 cells phorimager to quantitate the amount of UMP product. transformed with the suv3-6his allele, but not when those cells were grown on glucose medium. Next, we determined whether the suv3-6his allele suv3 or suv3-6his allele were grown on YNBCG medium could restore NTP-dependent mtEXO activity to the toinduce expressionof Suv3p or Suv3p-6hisand mtEXO Dsuv3 cells. Mitochondrial lysates were prepared as be- activity.Mitochondrial lysates from these cells were pre- fore from suv3 and suv3-6his transformants of strain pared, partially purified by Affigel blue chromatography, 2-11 grown on YNBC glucose or galactose medium, and and then applied in buffer B to the nickel column (see those lysates were analyzed for the presence of NTP- Experimental Procedures). After washing the nickel col- dependent mtEXO activity. As shown in Figure 5 (lanes umn with buffer B, we eluted bound proteins with a 1–2 and 5–6), little or no activity was seen in samples step gradient of increasing concentrations of imidazole. prepared from cells grown on glucose medium. By con- Aliquots from this fractionation were then analyzed for trast, mitochondrial lysates prepared from either trans- Suv3p and Suv3p-6his by Western blotting and for formant grown on galactose medium contained robust mtEXO activity by an assay of its characteristic NTP- mtEXO activity that was dependent upon the presence dependent RNase activity. of ATP (Figure 5, lanes 3–4 and 7–8). These data show As shown by the Western blot analysis in Figure 6, that Suv3p-6his can substitute for Suv3p in restoring both the unmodified and histidine-tagged derivative of mtEXO activity to Dsuv3 cells. Suv3p bound to the nickelresin; little of either form of the protein was found in the flowthrough or wash fractions mtEXO Activity Cofractionates (lanes 3–6). However, Suv3p and Suv3p-6his behaved with Suv3p-6his very differently upon elution of the column with buffer Since Suv3p-6his restored NTP-dependent exoribo- containing imidazole. Greater than 95% of Suv3p eluted nuclease activity to Dsuv3 cells, we next investigated from the column at 1 mM imidazole (Figure 6, lane 8) whether mtEXO activity cofractionates with this deriva- with the remainder eluting at 5 mM imidazole (lane 10); tive protein when chromatographed on a histidine affin- no detectable Suv3p-6his eluted under those conditions ity resin (nickel–NTA; Qiagen). Cells of strain 2-11 trans- formed with Ycplac22 constructs containing either the (lanes 7 and 9). Elution of Suv3p-6his required higher

Article Snippet: The crude mitochondrial formamide sample buffer, and separated on 5% polyacrylamide– lysate fraction was adjusted to 0.15 M KCl, 10 mM Tris (pH 8.0), 5 urea gels. mM MgCl2, 0.25 mM EDTA (pH 8.0), 0.5% Triton X-100, 10 mM b-mecaptoethanol, 10% glycerol, and 15% (v/v) ethylene glycol and diluted 1:4.Production of Antisera against Suv3p The pMALsuv3 plasmid was expressed in E. coli according to the This crude lysate was loaded onto a column containing 10 ml of Affigel blue resin (Bio-Rad) preequilibrated with buffer A (0.15 Mprotocol provided by New England Biolabs for the pMAL-c vector system.

Techniques: Activity Assay, Chromatography, Affinity Column, Expressing, Clone Assay, Plasmid Preparation, Control, Construct, Marker, Transformation Assay, Western Blot, Functional Assay, In Vivo, Autoradiography, Purification, Nickel Column, Fractionation