ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 258, No. 1, October, pp. 259-264, 198’7
LIM, MANFRED Department
in the Yeast pyc Mutant’
ROHDE,2 C. PHILLIP
MORRIS, AND JOHN C. WALLACE3
of Adelaide, Adelaide,
Pyruvate carboxylase deficiency was previously reported to be the biochemical lesion in a yeast mutant, designated WC, which cannot utilize ethanol, acetate, pyruvate, aspartate, or oxaloacetate as the sole carbon source [C. Wills and T. Melham (1985) Arch. Biochem. Biophys. 236,782791; C. Wills et al. (1986) Arch. B&hem. Biophys. 246,3063201. We present evidence here that the level of pyruvate carboxylase activity as well as the native and subunit molecular weights of this enzyme are identical in the mutant and the wild type. In addition we have used immunocytochemical labeling to demonstrate the exclusively cytosolic localization of this enzyme in both the mutant and wild-type yeast. 0 1987AcademicPress,Inc.
Pyruvate carboxylase (PC)4 [EC 188.8.131.52 be a formidable task. A far more efficient is an important anaplerotic enzyme with a approach would be to infer the amino acid wide species distribution (1). With the ex- sequence from the nucleotide sequences of clones of the gene encoding this enzyme. ception of Pseudomonas citronellolis, this enzyme in all organisms studied so far is Isolation of such clones of the yeast PC a tetramer composed of four apparently gene would be greatly facilitated by genetic identical subunits (iV& - 120,000) arranged complementation of PC deficient strains in a tetrahedron-like structure (2). Alwith plasmids harboring the PC gene. though there is already a small amount of Therefore a most welcome development sequence data available for several pyruwas the reported isolation (5) of a mutant vate carboxylases of vertebrate origin (3, from Saccharomyces cerevisiae designated 4), the determination of the entire primary pyc which could not grow on minimal medium with ethanol, lactate, aspartate, or structure of this - 1000 amino acid polypeptide by protein sequencing would indeed oxaloacetate as sole carbon sources but which was able to grow on ethanol or pyruvate if the medium was supplemented i This research was supported by Grant D284/15411 with aspartate. In this report, however, we from the Australian Research Grants Scheme. present evidence that despite having an a Present address: Institut fur Mikrobiologie der identical growth pattern to that previously Georg-August, Univcrsitat au Giittingen, Grisereported, the levels of PC and its cellular bachstrasse 8, D-3400 Giittingen, Federal Republic of Germany. distribution in this pyc mutant are idens To whom correspondence should be addressed. tical to the wild type. 4 Abbreviations used: PC, pyruvate carboxylase; NBT. Nitro Blue tetrazolium; BCIP, 5-bromo-4-chloro3-indolyl phosphate; GAR-AP, goat anti-rabbit immunoglobulin-alkaline phosphatase conjugate; IgG, immunoglobulin G; PBS, phosphate-buffered saline; GARG, goat anti-rabbit immunoglobulin-gold conjugate; SDS-PAGE, sodium dodeeyl sulfate-polyaerylamide gel eleetrophoresis.
Matwiak. Nitrocellulose (0.2 pm pore size) was obtained from Sartorius (Giittingen, West Germany); Nitro Blue tetrazolium (NBT), 5-bromo-4-chloro-3indolyl phosphate (BCIP), and avidin-alkaline pbosphatase conjugate were purchased from BRESA
0003-9861/87 $3.00 Copyright 0 1987 by AcademicPress.Inc. All
(Adelaide, Australia); goat anti-rabbit immunoglobulin-alkaline pbosphatase conjugate (GAR-AP) from Sigma Chemical Company (St. Louis, MO); LR gold resin from Sigma Pharmaceuticals Ltd. (Victoria, Australia); and benzoyl peroxide from Polaron Equipment Ltd. (Hertfordshire, England). Goat and rabbit polyclonal antibodies were raised and purified according to established procedures (6). The pyc mutant was obtained as a gift from C. Wills (5). Yeast culture. Yeast media were prepared as described by Wills and Melham (5). The control strain GPO01 used in these studies was ura3- but wild type in all other respects. Accordingly, where appropriate, the test media included 2% (w/v) uracil. Detection of pyruvate carboxylase in cell extracts. Yeast pyruvate carboxylase activity was measured at pH 8.0 in the presence of 250 PM acetyl-CoA by either the isotopic incorporation assay or the enzyme-linked spectrophotometric assay (7). The yeast cell pellets pH 7.2, 10 were suspended in 100 mM Tris-acetate, mM MgClz at 0.5 g/ml and lysed by passage through a French pressure cell at 83 MPa. Purification of yeast PC and Superose 6 gel filtration were as previously described (8). Western analysis Electrophoresis was conducted as described by Laemmli (9) and protein bands transfered to nitrocellulose according to Svboda et al (10). One of the nitrocellulose blots was incubated with 1 rig/ml avidin-alkaline phosphatase in Buffer 1 (100 mM Tris-Cl, pH 7.0, 100 mM NaCl, 2 mM MgCla, 0.5% (v/v) Tween 20) for 1 h at room temperature, washed twice with Buffer 2 (100 mM Tris, pH 9.5,lOO mM NaCl, 5 mM MgCla), and developed in 0.3 mg/ml NBT and 0.2 mg/ml BCIP in Buffer 2 for 5 min in subdued lighting. The same procedure was followed with the other blot except that 1 pgg/ml rabbit anti-yeast PC antibody replaced the avidin conjugate and a second incubation with GAR-AP (l/1000 dilution in Buffer 1) preceded the color development process. Fixation and embeclding of protoplasts. Yeast protoplasts were prepared as described by Cryer et al (11) and suspended in 1.5% (w/v) agar; after solidification the agar was cut into small cubes. Fixation was performed by immersion for 60 min in a solution containing 0.5% (v/v) formaldehyde and 0.3% (v/v) glutaraldehyde in 0.1 M potassium phosphate buffer, pH 7.0, held on ice. Dehydration was achieved by equilibration with a series of graded ethanol solutions (10, 25, 50, 70, 90, 100%) on ice. Samples were then embedded in LR gold resin (a) in 50% (w/v) LR gold resin and 50% ethanol for 60 minutes; or (b) in a 70% LR gold resin at 4°C followed by an incubation in the embedding mixture consisting of LR gold resin and 1.5% (w/v) benzoyl peroxide paste (60% in dibutyl phthalate) for 8 h or overnight at 4°C. Fresh embedding mixture was added prior to polymerization for 36-48 h at room temperature.
ET AL. Immunocytochemical labeling. Colloidal gold particles were prepared as described by Slot and Geuze (12) and covered with goat anti-rabbit IgG according to de Mey et al (13). Thin sections were mounted on Formvar-covered 300-mesh nickel grids. The grids were then incubated for 3 h at room temperature on a drop of various dilutions of rabbit anti-yeast PC polyclonal antibodies. Sections were rinsed in phosphate-buffered saline (PBS) and incubated with goat anti-rabbit immunoglobulin gold (GARG) for 1 h at room temperature. Finally, the sections were rinsed with PBS and water prior to poststaining with aqueous 4% (w/v) uranyl acetate for 4 min. The specificity of labeling was demonstrated by use of the following controls: (a) incubation of the sections with GARG only; (b) incubation of the sections with non-specific (rabbit anti-bacterial carbon monoxide dehydrogenase) IgG antibodies followed by GARG. Electron microscopy Electron micrographs were taken with a Philips EM 301 electron microscope at calibrated magnifications and at an acceleration voltage of 80 kV. RESULTS
Determination of Pyruvate Carboxylase Activity in pyc Mutant The cell yields from 500-ml cultures of the control and pyc yeasts in rich medium were 10.9 and 10.5 g, respectively. Samples of the control strain grew on minimal + glucose, minimal + ethanol, and minimal + pyruvate whereas the WC cells grew only on minimal + glucose. This indicated that revertants had not been generated during the culture in rich medium. Crude extracts obtained from the cells after passage through a French pressure cell were assayed for PC (7) by three different procedures: (a) measuring the incorporation of [14C02] into oxaloacetate in the presence and absence of pyruvate; (b) using the enzyme-linked spectrophotometric assay in the presence and absence of pyruvate; (c) using the enzyme-linked spectrophotometric assay in the presence and absence of avidin (500 pg/ml). Since several NADH-utilizing systems present in the crude extracts can interfere with the latter method, the use of avidin allowed calculation of biotin-dependent activity. The results from three separate experiments shown in Table 1 reveal no significant difference between the levels of activ-
PYRUVATE TABLE PYRUVATE
CARBOXYLASE OF WILD-TYPE
ACTIVITY IN CELL EXTRACTS AND pyc YEAST
Avidin-sensitive units* (spec. assay) Pyruvate-dependent units” (spec. assay) Pyruvate-dependent unitsd (isotopic assay)
9.8 f 0.5
10.8 f 0.5
4.3 * 0.4
4.6 f 0.4
4.5 f 0.5
a Enzyme units (rmol/min at 30°C) determined in triplicate in three different crude cell extracts made from 5-g pellets of either wild-type or WC mutant yeast. Values given are the mean of three measurements -t the standard error of the mean. * Measured using the enzyme-linked spectrophotometric assay (7) in the presence and absence of avidin. c Measured using the enzyme-linked spectrophotometric assay in the presence and absence of pyruvate. d Measured using the 14Cincorporation assay (7) in the presence and absence of pyruvate.
ity found in the WC mutant and the control cell extracts. Further purification of the cell extracts resulted in similar yields of PC from the control and pyc strains. The elution time of activity upon Superose 6 gel filtration in both cases (data not shown) was the same as that of purified PC, indicating that the native form of the enzyme in the mutant was of the same size as that of the wild type. Detection of Pyruvate Carboxylase in pyc Mutant by SDS-PAGE and Western Blotting When crude extracts of the WC mutant and wild-type yeast were electrophoresed in the presence of SDS the resulting patterns of Coomassie-stained protein bands (Fig. 1) were, with the exception of several unidentified bands of M, N 74,000 and 3: 98,000, essentially identical. In particular, both the pyc mutant and wild-type ex-
tracts revealed protein bands having the same mobility as purified yeast PC. The identity of these bands as pyruvate carboxylase was confirmed by probing Western blots of replicate gels with antibodies raised against yeast PC (Fig. 1). Similarly, when another Western blot of a replicate gel was probed with avidin-alkaline phosphatase conjugate, both the pyc mutant and wild-type cell extracts revealed a biotin-containing protein band having the same mobility as purified yeast PC. In addition, both extracts also revealed a biotincontaining band of M, N 205,000 which is most probably attributable to the presence of acetyl-CoA carboxylase [EC 184.108.40.206. A third biotin-containing protein band of M, N 47,000 was also detected in both extracts.
FIG. 1. Western analysis of pyc cell extracts. Crude extracts from the pyc mutant (pyc), the control strain (con), and purified yeast pyruvate carboxylase (YPC) were electrophoresed and (a) stained with Coomassie blue; (b) transferred to nitrocellulose and probed with anti-YPC antibody followed by goat anti-rabbit alkaline phosphatase; and (c) transferred to nitrocellulose and probed with avidin-alkaline phosphatase. Molecular weight markers (M) were carbonic anhydrase (BK), ovalbumin (45K), bovine serum albumin (66K), phosphorylase b (97.4K), B-galactosidase (116K), and myosin (205K). Arrows at 74K (con) and 98K (pyc) indicate prominent bands in those tracks, respectively, which appear to be largely absent from the other track.
FIG. 2. Localization of yeast pyruvate carboxylase using the antibody-gold method. Ultrathin sections of yeast protoplasm were treated with anti-yeast PC antibody (A, C, D, 100 pg/ml; B and E, 64 pg/ml) followed by GARG (l/25 dilution) and poststained with uranyl acetate. (A and B) Wild-type strain; (C, D, E., and F) pyc strain. In F the section was incubated with nonspecific antibody (115 pg/ml) followed by GARG (l/25 dilution). The scale bar represents 250 nm. M = mitochondria, N = nucleus, V = vacuole, G = gold particle.
It is apparently not a breakdown product of pyruvate carboxylase since it was not detected by the polyclonal anti-yeast PC antibodies. Its identity is unknown at this stage. Intracellular Localization Carboxylase in Yeast
The majority of evidence favors an intramitochondrial localization of PC in vertebrate tissues (l), whereas in yeast it has been reported on the basis of cell fractionation studies to be cytosolic (14), as it is in Aspergillus nidulans (15) and Rhixopus arrhixus (16). However, due to the inherent imprecision of cell fractionation techniques, it is not possible to be certain whether small amounts of PC activity associated with yeast mitochondrial fractions represent the occurrence of this enzyme in the mitochondria or its presence merely as an artifact of the fractionation procedure. Since it had been claimed (5) that yeast PC was mitochondrial, and that its absence from the mitochondria of the pyc mutant accounted for that mutant’s growth characteristics, we have reinvestigated the localization of this enzyme using a completely alternative approach not previously applied to PC, viz., immunogold labeling. When this technique was applied to protoplasts made from mutant and control cells it can be seen (Fig. 2) that the mitochondria, nucleus, and vacuole of the protoplasts are devoid of any labeling. A comparison of Figs. 2A, B, C, D, and E shows that the pyc strain exhibits approximately the same amount of PC as the control, and in both cases the enzyme is clearly cytosolic. The few gold particles observed at the periphery of some of the mitochondria can be attributed to the fact that the gold particles are separated from the enzyme by an immunoglobulin bridge of about 20 nm. No labeling is detectable when nonspecific antibodies are used (Fig. 2F). DISCUSSION
The growth patterns of the pyc mutant on minimal medium supplemented by var-
ious carbon sources differed from that of the wild-type yeast in a way consistent with a lack of PC activity (5). However, our results clearly demonstrate a normal level of activity of this enzyme in the pyc mutant. Furthermore, the results from Western analysis and gel filtration indicate that the size of denatured PC subunits as well as native complexes was unaltered in the pyc mutant. We suggest that the main reason for the previously reported absence of PC (5, 17) is that the enzyme activity was probably limited by the low levels of substrate (1 I.LM HCO;) and activator (4 j&M acetyl-CoA) used. Under these conditions negligible activity was detected even in the wild-type control strain whereas under optimum assay conditions (10 mM HCO, and 250 PM acetyl-CoA) (8) we have detected PC activity at levels similar to those previously reported (1,8, 14) for yeast. The interpretation of the physiological effect of the pyc mutant published previously (5) depended on an intramitochondrial location for PC in S. cerevisiae. However, we have found that the localization of PC in the pyc mutant is identical to that of the wild type and is exclusively cytosolic. In conclusion, our results show that the biochemical defect in the pyc mutant is not a deficiency in pyruvate carboxylase. However, we suggest that the aberrant growth characteristics of this mutant may be consistent with a lesion in one of the mitochondrial metabolite transport systems.
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