Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.

Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated. The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified [32P]polymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized. This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in vivo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity.

Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated.
The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified ["2Plpolymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized.
This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in uiuo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity. In 16.2 (15). This suggests that the native protein kinase, if globular, has a molecular weight of approximately 135,000 and so could be composed of one or more of the four major polypeptides present in the DEAE-Sephadex pool. The specific activity of the protein kinase after DEAE-Sephadex chromatography is comparable to similar protein kinases purified by others (11,24) but is only 10% that reported for a yeast protein kinase purified by Lerch et al. (25). This latter enzyme exists as a monomer of 42,000 daltons, whereas the enzyme described here appears to be larger.
The subcellular localization of this protein kinase remains to be determined but it has general enzymatic properties similar to those described by others for nuclear protein kinases (26-28   Following partial acid hydrolysis of [32P]polymerase I and analysis of the hydrolysate by high voltage paper electrophoresis, approximately 7% of the radioactivity migrated with phosphothreonine, 22% with phosphoserine, 70% with inorganic phosphate, and the remaining 1% remained at the origin. These data suggest that these amino acids are present in RNA polymerase I, but do not exclude the possibility that other amino acids might be phosphorylated. 32Pi is produced under these hydrolysis conditions by decomposition of the phosphate ester of phosphoserine or phosphothreonine (21). Since the rate of decomposition of phosphoserine is approximately 4 times that of phosphothreonine, the fraction of the radioactivity in phosphoserine and, to a lesser extent, phosphothreonine is underestimated.
N-Phosphoamino acids also decompose under acidic conditions (33). The incorporated 32P was acid-stable (6% was released as 32Pi in 15 min in 0.5 N HCl at 60") and baselabile (69% was released as 32Pi in 15 min in 0.5 N NaOH at SO") which suggests that most of the phosphate is attached to Densitometer tracing of autoradiograms of in uivo phosphorylated RNA polymerase I, purified as described in the text, obtained after resolution of polymerase polypeptides by electrophoresis in sodium dodecyl sulfate-lo% polyacrylamide gels. a, the pattern of subunit phosphorylation observed when polymerase I was isolated from yeast cells grown in phosphate-depleted medium. b, the pattern of subunit phosphorylation observed when polymerase was isolated from yeast cells grown in complete medium. The numbers above the peaks indicate the molecular weight x 10m3. serine and threonine residues and not to lysine, arginine, or histidine residues which are acid-labile and base-stable (33). Of the incorporated 32P, 85% was sensitive to potato acid phosphatase and 65% was sensitive to bacterial alkaline phosphatase. It is possible that a small fraction of the phosphate label could be incorporated by adenylylation or ADP ribosylation of enzyme polypeptides. We consider the latter unlikely since we have been unable to detect any NAD+:protein ADPribosyltransferase activity in isolated yeast nuclei. The 185,000-and 44,000-dalton polypeptides were more highly labeled than the others (Fig. 7). The 20,000-dalton polypeptide was only lightly labeled. The relative extent of phosphorylation of this subunit appears to be sensitive to the phosphate content of the culture medium since it is more highly labeled when the enzyme was isolated from cells grown in complete rather than phosphate-depleted medium (Fig. 7). RNA Polymerase II-The RNA polymerase II purified as described under "Experimental Procedures" was 90 to 95% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Figs. 8 and 9a, Fractions 13 to 15) and has the following polypeptide composition: 205,000, 145,000, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein from sucrose gradient of yeast RNA polymerase II described in Fig. 8. The enzyme subunits are designated by their molecular weights. The fraction numbers correspond to those of the gradient in Fig. 8. The gel concentration was 11%. a, stained polypeptides. The lane designated M is myosin; the lane designated S is yeast RNA polymerase II reference. b, autoradiogram of gel. 46,000, 33,500, 28,000, 24,000, 18,000, 14,500, and 12,500 daltons (the 12,500-dalton subunit is not resolved in Fig. 9a but is evident in Fig. 12). The polypeptide composition of this enzyme preparation is similar to those described by Hager et al. (34) and Buhler et al. (32) except that the molecular weight of the largest subunit in these previous preparations was approximately 175,000 (Fig. 9o, Column S). However, the apparent molecular weight of the largest subunit when polymerase II was isolated by this procedure and in a preparation recently described by Dezelee et al. (35) is similar to that of the large subunit of myosin, i.e. approximately 220,000 (36) (Fig. 9a, Columns M and S and Fraction 14). This subunit has an apparent molecular weight of 205,000 as determined by electrophoresis in a 6.5% sodium dodecyl sulfate-polyacrylamide gel which resolves high molecular weights polypeptides better than an 11% gel (data not shown). There is little of the 175,000dalton polypeptide evident in this preparation. This is consistent with an origin by proteolysis during isolation as proposed by Dezelee et al. (35). Thus, when proteolysis is efficiently prevented, the only form of polymerase II isolated is one possessing the 205,000-dalton subunit. There is no apparent difference in molecular weight in the other subunits in the polymerase II preparation (Fig. 9u, Column S and Fraction 14). These subunits do not appear to be as sensitive to proteolysis. Dezelee et al. (35) have found no significant differences in cY-amanitin sensitivity or template preference between the two forms. The contaminants in this polymerase II preparation are several polypeptides with molecular weights between 46,000 and 145,000 that are relatively tightly associated with the polymerase since they sediment with the enzyme in 0.5 M KCl.
When polymerase II was purified by this procedure from yeast grown in 32P,, analysis of the sucrose gradient showed a small peak of radioactivity sedimenting with the enzyme. The identity of the radioactive polypeptides was determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins in the gradient fractions by autoradiography. Most of the radioactivity which sedimented with polymerase migrated with the 24,000-dalton subunit (Fig. 9, Fractions 14  and 15). In addition, there were radioactive polypeptides with apparent molecular weights of 225,000, 165,000, and 140,000 present in minor amounts which do not migrate with any polymerase II subunits. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P1polymerase II in a 6.5% resolving gel and subsequent autoradiography also indicated that the three large phosphorylated polypeptides are not polymerase II subunits (data not shown). These large phosphorylated polypeptides do not sediment exactly with the enzyme which also suggests that they are contaminating polypeptides. Sucrose gradient centrifugation resolved polymerase II from a 33,500-dalton radioactive polypeptide. The enzyme subunit of this molecular weight is not labeled.
RNA Polymeruse III-This enzyme has generally been difficult to purify; the procedure described here has been used to purify polymerase III from variable quantities of cells. After aftinity chromatography on denatured DNA-cellulose, polymerase III was approximately 80% pure (data not shown) and sucrose gradient sedimentation removed most of the remaining protein contaminants (Figs. 10 and lla). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the protein in the active fractions into 10 polypeptides (160,000, 128,000, 82,000, 41,000, 37,000, 34,000, 28,000, 24,000, 20,000, and 14,500 daltons) (Fig. lla, Fractions 14  and ~0 L,ooo and II33,500 daltons (Fig. 12). The protei  After incubation for 10 min at 30", 0.090-ml aliquots were applied to Whatman 3MM filter discs which were washed as described for the protein kinase assay and counted as described elsewhere (15). In these reactions, the incorporation of :i2P into acid insoluble material reflects the amount of polymerase phosphorylation, whereas the incorporation of ["HIUMP into acid-insoluble material indicates the amount of RNA synthesis.
There was no incorporation of 12P into acid-insoluble material in the presence of protein kinase alone under these conditions.

Enzyme
Divalent metal ion Addition Moles of phos-"*PC;;;;po-YHIUMP in-l~~e~&P&-phorus incorpocorporated tivity rated per mole of polymerase  (9)(10)(11)46 The concentration of [Wliso-Abu was written as 1.1 mM instead of 0.11 mM. The correct line should read: The extraction buffer also contained 0.3 M ammonium sulfate. The sentence should read: salts listed below and 0.11 rnM [l-'%]iso-Abu (61 fmol/cpm) to 140 PLg The pellet was suspended with a glass rod and finally a glass We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections int,o any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.
Monohy-of inside-out red cell membrane vesicles. Interactions with droxy acids from novel lipoxygenases. potassium.
Pierre Borgeat, Mats Hamberg, and Bengt Samuelsson Rhoda Blostein and Lily Chu The two monohydroxy acids isolated after incubation of Page 3038, Table V arachidonic acid and homo-y-linolenic acid should both have the "D" configuration, i.e. 5-n-hydroxy-6,8,11,14-eicosatetrae-In the title, K,,, should be K,,,. noic acid and 8-n-9,11-14-eicosatrienoic acid, respectively. Oxidative ozonolysis of the methoxycarbonyl derivatives of The heading should read the methyl esters afforded mainly the methoxycarbonyl derivatives of dimethyl 2-L-hydroxyadipate and of dimethyl 2-Lhydroxyazelate as described on p. 7818. Since the carbome-Effects of Na,,, on K,,, inhibition at 37" thoxy group of these dioates that should be oriented upwards in the Fischer projection formulas (C-l) does not correspond Under 0.05 PM ATP concentration, the value for E-P at 5.0 to the carboxyl group of the parent unsaturated hydroxy mM Na should be 0.168 pmol/mg.
acids, it follows that the latter acids should have the "D" configuration at C-5 (5-n-hydroxy-6, 8,11,14-eicosatetraenoic The line should read acid) and at C-8 (8-n-hydroxy-9,11,14-eicosatrienoic acid). 5.0 mM Na 0.168 23.8 142 Vol. 252 (1977( ) 1990( -1997( Vol. 252 (1977  The concentration of [Wliso-Abu was written as 1.1 mM instead of 0.11 mM. The correct line should read: The extraction buffer also contained 0.3 M ammonium sulfate. The sentence should read: salts listed below and 0.11 rnM [l-'%]iso-Abu (61 fmol/cpm) to 140 PLg The pellet was suspended with a glass rod and finally a glass We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections int,o any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.