Synthesis and targeting of hexokinase to mitochondria in hepatoma cells

ARCHIVES OFBIOCHEMISTRYAND BIOPHYSICS

Vol. 27’4,No. 1, October, pp. 94-99,1989

Synthesis and Targeting of Hexokinase

to Mitochondria

in Hepatoma Cells

FIROZ KABIR AND B. DEAN NELSON’ Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-10691 Stockholm, Sweden Received February 14,1989, and in revised form May 5,1989

The synthesis and turnover of hexokinase has been measured in Zajdela hepatoma ascites cells labeled for short periods with [35S]methionine. Digitonin fractionation of the labeled cells into a soluble and a membrane fraction showed that only a small part of the newly labeled hexokinase is transferred to mitochondrial binding sites. The soluble enzyme disappears, however, with a half-life of less than 2 h. Glucose had no effect on the stability of the soluble enzyme in intact cells. Our experiments suggest that Zajdela cell hexokinase is synthesized in excess of binding sites and that the excess enzyme is IlOt stable. o 1989AcademicPWS, 1~. Transformed (l-4) and growth-stimulated (5-10) cells exhibit elevated glycolysis and hexokinase activity. Although the exact role played by hexokinase in regulating glycolysis is not known, it is generally considered to be one of the three regulatory enzymes in this pathway and one of the glycolytic enzymes present in the lowest amount in both normal (11-13) and transformed (14,15) cells. Thus, increases in hexokinase could, under some conditions, regulate glucose phosphorylation and glycolysis (2,16). It is thus important to understand how the cellular content of hexokinase is regulated. We recently showed (1’7) that the mRNA for hexokinase is increased at least lo-fold in hepatoma compared to normal hepatocytes. In the present study we show that most newly synthesized hexokinase in hepatoma is not transferred to mitochondria, but, rather, breaks down. These findings are consistent with a mechanism in which the increased level of hepatoma hexokinase is regulated by activated gene expression and by the availability of mitochondrial binding sites which stabilize hexokinase against breakdown (l&19).

i To whom correspondence should be addressed. 0003-9861/89 $3.00

Copyright Q 1989by AcademicPress.Inc. All rights of reproductionin any form reserved.

MATERIALS

AND METHODS

Isolation of ce.Us.Zajdela hepatoma ascites cells were propagated in the peritoneal cavity of male Sprague-Dawley rats. Cells were isolated 5-7 days after inoculation and washed as previously described (20, 21), with the exception that the last wash was performed in a medium containing 5’7 mM NaCl, 4.3 mM KCl, 5 m&f MgClz. 6Hz0,O.g mM KHzPOd, 0.6 mM NazS04, 25 mM Hepea, 24 mM Tes, 30 mM Tricine, pH 7.4. Washed cells were suspended in the same medium. Labeling and,fractionation of cells. Isolated cells (30 mg protein/ml) were incubated at 30°C for 5 min with shaking under a Oz/Nz atmosphere in the above medium supplemented with 4.5% (v/v) bovine serum, heparin (7 units/ml), penicillin, and streptomycin (both 60 pg/ml), and an amino acid mixture lacking methionine (21). [%]methionine (sp act > 0.5 mCi/ ml, Amersham) was added to a final concentration of 0.5 mCi/ml, and the cells were incubated for the times indicated in the figure legends. Chases were initiated by addition of unlabeled methionine to a final concentration of 2 mM. Labeled cells were fractionated with digitonin (22), and the fractions were immunoabsorbed as pre-

2 Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; Tes, 2-{[2-hydroxy-l,l-bis( hydroxymethyl)ethyl]amino}ethanesulfonic acid; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)-ethyllglycine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 94

HEXOKINASE

SYNTHESIS

viously described (21), except that the immunoabsorption buffer contained 1 mM each of N-tosyl-Lphenylalanylchoromethane, N-tosyl-L-lysylchloromethane, and phenylmethylsulfonyl fluoride, and 200 units/ml of trasylol to prevent proteolytic breakdown of hexokinase (18, 19, 23, 24). A second immunoabsorption with the same antiserum did not recover additional radiolabeled material. Antibodies were raised in rabbits against rat brain hexokinase purified by the method of Chou and Wilson (25). The specificity of the antibodies were checked by Western blotting. Preimmune sera did not react with brain or tumor hexokinase. Binding of tumor hexokinase to rat liver mitochondtiu. Hepatoma cells (10 mg protein/ml) were fractionated with digitonin as above, but in the absence of added M%+. The soluble fraction containing soluble hexokinase was fortified with 5 mM MgCls, and a 0.5ml aliquot was incubated for 15 min at 30°C with 10 mg of freshly isolated, hexokinase-free, rat liver mitochondria. Mitochondria were removed from the incubation mixture by centrifugation in an Eppendorf centrifuge, and the soluble and mitochondrial fractions were assayed for hexokinase activity and processed for immunoprecipitation. Miscellaneous methods. SDS-PAGE was run on 10% polyacrylamide slab gels in the buffer system of Laemmli (26). Densitometric analysis of the fluorographs (2’7) were done on an Ultro Scan laser densitometer (LKB Products, Bromma, Sweden). Protein was measured by the biuret method. Hexokinase activity was assayed spectrophotometrically following the reduction of NADP+ (28). RESULTS

Labeling of isolated hepatoma cells with [35S]methionine was nearly linear for 40 min (Fig. 1A). Labeling of hexokinase (100 kDa) in these cells was also increased over a period of 40-50 min (Fig. 1B). On the basis of these results, subsequent chase experiments were performed on cells labeled for 30 min. In order to study the transfer of hexokinase to binding sites on mitochondria (23), cells were labeled with r5S]methionine for 30 min and then chased for various time periods after addition of 2 mM unlabeled methionine (Fig. 2A). The cells were fractionated into soluble and particulate fractions using the digitonin method previously described (22). Under these conditions, approximately 80% of the total hexokinase activity is associated with the mitochondria in the pellet fractions (22).

IN HEPATOMA

50

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A

i

40 -

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,o x s g r -c 8

30 -

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I

I

,

I

IO

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tame

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HK

FIG. 1. Labeling of hexokinase (HK) in isolated Zajdela hepatoma cells. Isolated cells were labeled continuously with [?S]methionine for a maximum of 60 min. At each time point, samples were removed and the incorporation into total proteins (A) and into immunoabsorbed hexokinase (B) were measured as described under Materials and Methods. A fluorograph of immunoprecipitated hexokinase is shown in (B).

Figure 2B shows that only 10-15s of the newly labeled hexokinase is present in the pellet fraction of cells labeled for 30 min. This result is surprising in view of our previous finding in Zejdela hepatoma that several inner membrane polypeptides are completely imported and processed by mitochondria within a few minutes (21). Figures 2A and 2B also show that the disappearance of hexokinase from the supernatant is not associated with a concomitant increase in the mitochondrial fraction. The most reasonable interpretation of this finding is that a large part of the newly synthesized hexokinase does not reach the mitochondria, but, rather, breaks down.

96

KABIR AND NELSON A P 0

10

S

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FIG. 2. The subcellular distribution of newly synthesized hexokinase following short-term chase. Cells were labeled for 30 min with Ij5Slmethionine as described under Materials and Methods. At time zero, 2 mM unlabeled methionine was added and the incubation was continued for the indicated time periods. At the end of the chase, 0.5 ml cells was removed and fractionated with digitonin into soluble (S) and membrane-containing pellet (P) fractions. These fractions were immediately precipitated with cold trichloroacetic acid and immunoabsorbed with antibodies against rat brain hexokinase. (A) Fluorographs of immunoabsorbed hexokinase resolved by SDS-PAGE, (B) desitometric analysis of the Flourographs. Densitometric analysis were done using an LKB Ultro Scan laser densitometer. Density values were obtained by weighing paper representing areas under the curves.

Glucose is known to stabilize hexokinase against inactivation (29,30). In view of the data in Fig. 2 suggesting destabilization of hexokinase, probably via proteolysis, we decided to investigate possible effects of glucose on hexokinase stability. To this end, cells were isolated, washed, and preincubated for 5 min in media lacking glucose. Given the rate of glucose oxidation by Zajdela cells (31), 5 min was judged to be

sufficient to eliminate any glucose present in the serum added to the incubation media. Cells were then labeled with y5S]methionine for 30 min in the absence of glucose and then chased with unlabeled methionine in the presence or absence of glucose (Fig. 3A). In agreement with the results shown in Fig. 2B, only 10-S% of the newly labeled hexokinase is present in the mitochondrial

HEXOKINASE

SYNTHESIS

IN HEPATOMA

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97

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FIG. 3. Turnover of hexokinase in hepatoma cells in the presence and absence of glucose. Hepatoma cells were isolated, washed, preincubated for 5 min, and then labeled for 30 min with PSJmethionine, all in the absence of glucose. Unlabeled methionine (2 mM) was added to the labeled cells and incubation was continued for the indicated times either in the presence of glucose (20 mbi) or in its absence. (A) Fluorographs of immunoahsorbed hexokinase from cells chased for 0 min (lane l), 15 min (lanes 2 and 3), 30 min (lanes 4 and 5), and 45 min (lanes 6 and 7) either in the absence of glucose (lanes 2,4, and 6) or in the presence of glucose (lanes 3,5, and 7). (B) Densitometric analysis of the fluorographs.

fraction after a 30-min labeling period. Glucose, however, had no significant effect on the labeling of hexokinase or on its disappearance from the cytosol (Fig. 3). A slight increase in mitochondrial hexokinase was noted during the chase (Fig. 3B), but, as shown in Fig. 2B, this increase is not commensurate with that disappearing from the cytosol. Thus, glucose appears to have little if any immediate effect on either labeling or stability of hexokinase in these hepatoma cells. To test if the hexokinase in the soluble fraction is a bindable form of the enzyme

(23, 24), the soluble fraction from digitonin-treated cells was used as a source of hexokinase for binding to isolated rat liver mitochondria in vitro. Table I shows that 5540% of the hexokinase activity in the soluble fraction binds to hexokinase-free liver mitochondria. For reasons not yet understood, the radioactive hexokinase that could be immunoprecipitated from the liver mitochondria and the tumor soluble fraction after the incubation was too small to measure. However, it can be concluded from this experiment that the slow transfer of hexokinase to mitochondria is not

98

KABIR AND NELSON TABLE I IN VITROBINDING OF TUMOR HEXOKINASE TO RAT LIVER MITOCHONDRIA Hexokinase activity (nmol/min) Experiment 1 2

Added

Bound

Free

Recovered

% Bound

28.2 36.0

16.0 18.5

12.0 16.0

28.0 34.5

58 54

Note. Hepatoma cells were fractionated with digitonin as described under Materials and Methods. The soluble fraction containing hexokinase was fortified with 5 mM MgClz and then incubated for 15 min at 30°C with hexokinase-free rat liver mitochondria (10 mg protein). Mitochondria were removed by centrifugation, and hexokinase was assayed in the pellet and the supernatant.

due to the lack of the bindable form of the soluble enzyme. DISCUSSION

The present study shows that newly synthesized hexokinase is lost from the soluble fraction of the hepatoma cell but does not appear in the mitochondrial fraction. The rapid rate of clearance of hexokinase agrees with the short half-life (1 h) of destabilized hexokinase in Ehrlich cells reported by Warms and Rose (18). Our findings, like those of Warms and Rose (18), are best explained by the suceptability of soluble hexokinase to protease attack. Binding to mitochondria stabilizes hexokinase (18,19), which probably accounts for the long half-life of 28 h measured for skeletal muscle hexokinase II (32). Glucose stabilizes hexokinase against inactivation in vitro (29,30). We find, however, that glucose has no significant effect on the stability of newly synthesized hexokinase in viva. A similar lack of effect of glucose was also reported for Ehrlich cells in which hexokinase was destabilized by various methods (18). Whether this is due to the lack of an in viva effect of glucose on hexokinase stability or to a more trivial explanation, i.e., that glucose is metabolized so rapidly that protective concentrations are never reached, remains open. We previously showed that metabolic alterations in hepatoma cells do not influence hexokinase binding (22). It therefore seems probable that newly synthesized hexokinase in hepatoma cells does not bind

to mitochondria because the binding sites are already occupied. The excess, soluble hexokinase is unstable. Thus, since the amount of translatable hexokinase mRNA is at least lo-fold greater in hepatoma cells than that in liver (1’7), cellular concentrations of hexokinase in hepatoma appear to be controlled both by elevated expression of the gene (1’7) and by available binding sites on mitochondria. ACKNOWLEDGMENT This study was supported by the Swedish Cancer Society. REFERENCES 1. KNOX, E., JAMDAR, S. C., AND DAVID, P. A. (1970) Cancer Res. 30,2240-2244. 2. BUSTAMANTE, E., MORRIS, H. P., AND PEDERSEN, P. L. (1981) J. Bid Chem, 256,8699-8704. 3. WEINHOUSE, S. (1966) Gann Monogr. 1,99-115. 4. PEDERSEN, P. L. (1978) Prog. Exp. Tumor Res. 22,

190-274. 5. Roos, D., AND Roos, J. A. (1970) Biochim Biophys. Acta 222,565-582. 6. CIJLVENOR, J. G., AND WEIDEMANN, M. J. (1976)

B&him.

Biophgs. Actu 437,354-363.

7. WANG, T., MARQUARDT, C., AND FOKER, J. (1976)

Nature (Lxmdon) 261,702-705. 8. KRAAIJENHAGEN, R. J., RIJKSEN, G., AND STAAL, G. E. J. (1980) Biochim Biophys. Ada631,402411. 9. BRAND, K. (1985) Biochem. J. 228.353-361. 10. HUME, D. A., RADIK, J. L., FERBER, E., AND WEIDEMANN, M. J. (1978) Biochcm J. 174,703-

709. 11. MAGNANI, M., STOCCHI, V., PIATTI, E., DALLAPICCOLA, B., AND FORNAINI, G. (1983) Blood 61,

915-919.

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12. TEJWANI, G. A., CHAUHAN, S., DURUIBE, V., AND VASWANI, K. K. (1985) Arch. Biochem Biophys. 239,462-466. 13. YOUNG, P., CAWTHORNE, M. A., LEVY, A. L., AND WILSON, K. (1984) FEBSLetf. 176,16-20. 14. WEINHOUSE, S. (1972) Cancer Res. 32,2007-2016. 15. HERZFELD, A., AND GREENGARD, 0. (1972) Cancer

Res. 32,1826-1832. 16. JOHNSON, J. H., ZIMNIAK, A., AND RACKER, E. (1982) Biochemists 21,2984-2989. 17. JOHANSSON, T., BERREZ, J. M., AND NELSON, B. D. (1986) Biochem. Biophys. Res. Commun 133, 608-613. 18. ROSE, I. A., AND WARMS, J. V. B. (1982) Arch. Biothem. Biophys. 213,625-634. 19. MAGNANI, M., STOCCHI, V., DACHA, M., AND FORNAINI, G. (1984) Mel CeU.B&hem, 61,83-92. 20. KUZELA, S., KOLAROV, J., AND KREMPASKY, U. (1973) Neoplasma 21,623-630. 21. KOLAROV, J., AND NELSON, B. D. (1984) Eur. J B&hem 144,387-392.

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22. NELSON, B. D., KABIR, F., AND MUCHIRI, P. (1984) B&hem. J. 219,159-X4. 23. ROSE, I. A., AND WARMS, J. V. B. (1967) J. Biol.

Chem 242,1635-X45. 24. POLAKIS, P. G., AND WILSON, J. E. (1985) Arch

Biochem. Biophys. 236,328-337. 25. CHOU, A. C., AND WILSON, J. E. (1972) Arch. Bie them. Biophys. 151,48-55. 26. LAEMMLI, U. H. (1970) Nature &n&m) 227,680685. 27. CHAMBERLAIN, J. P. (1979) Anal. Biochem. 98, 132-135. 28. NELSON, B. D., AND KABIR, F. (1985) B&him. Bip phys. Acta 841,195-200. 29. MURAKAMI, K., AND ROSE, I. A. (1974) Arch Biothem Biophys. 165,519-523. 30. SCHIMKE, R. T., AND GROSSBARD, L. (1968) Ann. N. Y Acd Sci. 151,332-350. 31. NELSON, B. D., AND KABIR, F. (1986) Biochimie 68, 407-415. 32. FRANK, S. K., AND FROMM, H. J. (1986) Arch Bie

them. Biophys. 249,61-69.