Determination of ATP Synthase Subunits Interacting with DAPIT: Experimental Analysis, Exams of Biochemistry

Download Determination of ATP Synthase Subunits Interacting with DAPIT: Experimental Analysis and more Exams Biochemistry in PDF only on Docsity! Page 1 of 2 Semester 2 Examinations 2011/2012 Exam Code(s) 4BS2; 4BO2 Exam(s) BI401 Biochemistry Module Code(s) BI442; BI436 Module(s) Research paper analysis Paper No. 1 Repeat Paper External Examiner(s) Prof. M. J. R. Stark Internal Examiner(s) Prof. C. Morrison Instructions: Please read the enclosed research paper, and answer the questions relating to this research paper. Answer the question from Section A, and two questions from Section B. Please use a separate answer book for each Section. Duration 3 hours No. of Pages 3 Discipline(s) Biochemistry Course Co-ordinator(s) Dr. L. Byrnes Requirements: MCQ Release to Library: Yes x No Handout Yes Statistical/ Log Tables Cambridge Tables Graph Paper Log Graph Paper Other Materials P.T.O. Page 2 of 2 BI442 and BI436 Research paper analysis Time allowed: Three Hours Please use a separate answer book for each Section. The percentage of the total marks for each question is shown in parentheses. Answer the following question. Section A 1. Provide a Title (10%) and an Abstract (40%). The Title should not be longer than 15 words and the Abstract must not exceed 250 words. The Abstract should succinctly and clearly describe the major findings reported in the manuscript, mentioning the aims of the study and the data used to draw the conclusions of the paper. Section B Answer two questions (25% each) 1. Stable shRNA expression was used to disrupt DAPIT expression. Suggest two alternative approaches and compare their advantages and disadvantages with shRNA. 2. How could it be determined experimentally which ATP Synthase subunits interact with DAPIT? 3. Describe two experiments to test whether DAPIT localises to mitochondria, including appropriate controls. 4. Loss of DAPIT led to a marked decline in cell proliferation. How might the authors test the hypothesis that this decline is due to increased levels of apoptosis in the absence of DAPIT. availableDAPIT limits the population ofATP synthase inmito- chondria. Free monomers of - and -subunits were also seen faintly and clearly, respectively. For each subunit, the amount of free subunit appeared to correlate with the amount of ATP synthase, suggesting that the free subunits are products of dis- assembly of ATP synthase. Because free monomers were found even in shMock cells, disassembly was not characteristic of DAPIT knockdown but occurred naturally in all cells. e rea- son why the amount off ree -subunit was much smaller than that off ree -subunit is not known. SDS-PAGE analysis showed that total amounts of - and -subunits in mitochon- dria of shDAPIT-2 and shDAPIT-4 cells matched their amounts in Clear Native PAGE (Fig. 2B). ese results show that DAPIT knockdown results in loss of population of mature ATP synthase in mitochondria. Transcription andMitochondrial Transport of - and -Sub- units Are Normal in DAPIT-knockdown Cells—To examine a possibility that shRNA directly suppressed transcription of genes of ATP synthase, levels of mRNAs of - and -subunits were measured by real-time PCR (Fig. 3). As seen, there was no significant difference in mRNA levels of - and -subunits between the DAPIT-knockdown cells and the shMock cell. Given that translation ofmRNAby ribosomeswas not impaired in the knockdown cells as shown for normal VDAC expression (Fig. 1), this result indicated that - and -subunits, and most likely other subunits as well, should be synthesized normally in the DAPIT-knockdown cells. Further, transport of - and -subunits intomitochondria in the DAPIT-knockdown cells was not impaired because accu- mulation of precursor - and -subunits with a mitochondria- targeting presequence was not found in SDS-PAGE of whole cell extracts of all tested cells (data not shown). erefore, it appears that - and -subunits are synthesized and transported into mitochondria normally. ATP Synthesis, Cell Growth, and Viability Are Impaired by DAPIT Knockdown—Mitochondrial ATP synthesis activity of the DAPIT-knockdown cells was measured (16). e proton motive force necessary for ATP synthesis was generated by suc- cinate oxidation. As expected from the decreased population of ATP synthase, ATP synthesis activity of shDAPIT-2 cells and FIGURE 1. Expression of DAPIT protein in whole cells ( A) and the isolated mitochondria of the mock-treated ( shMock ) and DAPIT-knockdown (shDAPIT -1, 2, 3, 4) HeLa cells ( B). Whole cell (20 g of protein) and mito- chondria (5 g of protein) were analyzed with SDS-PAGE, and DAPIT was stainedwithWestern blotting. VDACwas also stained as an internal standard of mitochondrial protein. Experiments were carried out at least four times, and representative results are shown. FIGURE2.ATP synthase in themitochondria in shMock andDAPIT-knock- down cells. Mitochondria (5 g of protein) were analyzed with Clear Native PAGE (A) or SDS-PAGE (B). The protein bands of -subunit, -subunit, and DAPIT were stainedwithWestern blotting. -Subunit was stained strongly to show the presence o ree subunit. The positions of ATP synthase, free -sub- unit, and free -subunit in A are indicated. Experiments were carried out at least ve times, and representative results are shown. FIGURE 3. Expression of mRNAs of -subunit and -subunit in DAPIT- knockdowncells relative to that in shMockcells. TheamountofmRNAwas measured by real-time quantitative PCR. Details are described under “Exper- imental Procedures.” 3 that of shDAPIT-4 cells were 90 and 60% of that of the shMock cells, respectively (Fig. 4A). e respiratory electron transfer chains in these cells were functioning to produce pro- ton motive force as demonstrated by accumulation of a mem- brane-potential probing dye, TMRE, in mitochondria that was dissipated by CCCP (Fig. 4B). ATP synthesis was completely inhibited by oligomycin in all cases, ensuring that we observed ATP synthesis by mitochondrial ATP synthase but not by adenylate kinase. e measured mitochondrial ATP synthesis activity did not agree with the population of ATP synthase in mitochondria; in the case of shDAPIT-4, 25% of ATP syn- thase was responsible for 60% ATP synthesis. is apparent disagreement can be explained by the presence of 2-fold more population of ATP synthase in mitochondria than needed for the maximum ATP synthesis activity as seen in -subunit knockdown cells (16). Cell proliferation of shDAPIT-2 and shDAPIT-4 after a 2-day culture was 70 and 50% of that of shMock cells (Fig. 5A). As the population of ATP synthase in the cell decreases, ATP production should becomemore dependent on glycolytic path- way, and viability of cells should be more susceptible to depri- vation of glucose. Indeed, this was observed for the -subunit knockdown cells (sh -3), in which population of ATP synthase was 5% of the mock cell (16). When glucose in the culture medium of the sh -3 cells was substituted with galactose that was unavailable for glycolysis, 80% of the cells died in 2 days (Fig. 5B). Under the same conditions, 40% of shDAPIT-4 cells died in galactose whereas only 10% of the shMock cells died in galactose (Fig. 5B). DISCUSSION DAPIT is not essential for function and structure of mature ATP synthase. It is easily lost from ATP synthase during puri- fication (12) or by exposure to relatively strong detergent (13) without losing the structural integrity and the core function of ATP synthase (13). is work, however, reveals that DAPIT is essential to maintain the population of ATP synthase in mito- chondria in living cells. It appears that the transcription and translation are not impaired inDAPIT-knockdown cells (Fig. 1, 3), and therefore, assembly or degradation of ATP synthase should be affected by DAPIT. Given that DAPIT-less ATP syn- thase is structurally stable (13), it is likely that DAPIT plays a role in assembly of ATP synthase even if a potential role in protection from degradation is not excluded. e absence of structurally indispensable subunit should cause the failure of the assembly of ATP synthase as observed for knockdown cells of -subunit (21) and -subunit (16). However, because DAPIT is dispensable for mature ATP synthase, it might play a chaper- FIGURE 4. Mitochondrial activities of ATP synthesis ( A) and membrane potential generation ( B) of shMock and DAPIT-knockdown cells. A, ATP synthesis activities were measured with luciferase (16). Filled symbolsrepre- sent the data in the presence of oligomycin (10 g/ml) of the corresponding open symbols.B, CCCP, uorescent images of cells incubated for 10minwith TMRE that accumulates in mitochondria in response to establishment of membrane potential. CCCP, uorescent images of the same eld after addi- tion of an uncoupler CCCP that dissipates membrane potential and proton gradient. Details are described under “Experimental Procedures.” FIGURE 5. Cell proliferation in the glucose-containing medium ( A) and viability in the glucose-free medium ( B) of shMock and DAPIT-knock- down cells. A, proliferation of cells in 0.1% glucose is shown as an increase of cell numbers after a 2-day culture (-fold). B, glucose in the medium was replacedwith0.1%galactose and the culture continuedanother 2days. Then, numbers o iving cells and dead cells were counted. Viability was expressed as percent o iving cells per total cells. Details are described under “Experi- mental Procedures.” 4 one-like role in the assembly of ATP synthase.e assembly of mitochondrial ATP synthase proceeds through several candi- date intermediate subcomplexes such as F1, c-ring-F1, c-ring-a, and peripheral stalk (22) and, different from prokaryotic ATP synthase, the assembly depends on many factors and chaper- ones (23, 24). DAPI as a putative transmembrane segment and should be associatedwith the F0 portion inATP synthase. It is, therefore, tempting to assume that DAPIT assists subunit assembly of the F0 portion.Without assembled F0 portion, F1 or even its subcomplex cannot exist stably as demonstrated in Clear Native PAGE (Fig. 2A), where no protein band other than free and the ATP synthase-integrated subunit was seen. Our finding provides a plausible explanation for physiologi- cal consequence caused from failure of the controlled DAPIT production. It was reported that the mRNA level of DAPIT in skeletal muscle in rat was elevated after active stretching of muscles, apparently to meet the increasing demand for ATP by increasing population of ATP synthase. e DAPIT gene was located within the locus whose deletion caused diabetes-asso- ciated phenotype of a diabetic rat (25) and mRNA level of DAPIT was lowered when diabetes was induced by streptozo- tocin (15).e poor DAPIT production should result in scarce population of ATP synthase and low level of ATP production, which in turn suppresses mitochondrial respiration first and glucose consumption next. Low level of cellular glucose con- sumption suppresses glucose uptake from blood and weakens the effect ofi nsulin. e factor controlling DAPIT expression and actual roles of DAPIT in mitochondrial ATP production and in glucose metabolism are worth studying especially in the insulin-sensitive cells. REFERENCES 1. Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621–628 2. Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299–302 3. Boyer, P. D. (1997) Annu. Rev. Biochem. 66, 717–749 4. Yoshida,M.,Muneyuki, E., andHisabori, T. (2001)Nat. Rev.Mol. Cell Biol. 2, 669–677 5. Weber, J., and Senior, A. E. (2003) FEBS Lett. 545, 61–70 6. Nakamoto, R. K., Baylis Scanlon, J. A., and Al-Shawi, M. K. (2008) Arch. Biochem. Biophys. 476, 43–50 7. Junge, W., Sielaff, H., and Engelbrecht, S. (2009) Nature 459, 364–370 8. von Ballmoos, C., Wiedenmann, A., and Dimroth, P. (2009) Annu. Rev. Biochem. 78, 649–672 9. Kagawa, Y. (2010) Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86, 667–693 10. Düser, M. G., Zarrabi, N., Cipriano, D. J., Ernst, S., Glick, G. D., Dunn, S. D., and Börsch, M. (2009) EMBO J. 28, 2689–2696 11. Walker, J. E., and Dickson, V. K. (2006) Biochim. Biophys. Acta 1757, 286–296 12. Chen, R., Runswick, M. J., Carroll, J., Fearnley, I. M., and Walker, J. E. (2007) FEBS Lett. 581, 3145–3148 13. Meyer, B., Wittig, I., Trifilieff, E., Karas, M., and Schägger, H. (2007) Mol. Cell. Proteomics 6, 1690–1699 14. Carroll, J., Fearnley, I. M., and Walker, J. E. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16170–16175 15. Päivärinne, H., and Kainulainen, H. (2001) Acta Diabetol. 38, 83–86 16. Fujikawa, M., and Yoshida, M. (2010) Biochem. Biophys. Res. Commun. 401, 538–543 17. Bookout, A. L., and Mangelsdorf, D. J. (2003) Nucl. Recept. Signal. 1, e012 18. Ishiyama,M.,Miyazono, Y., Sasamoto, K., Ohkura, Y., andUeno, K. (1997) Talanta 44, 1299–12305 19. Pallotti, F., and Lenaz, G. (2007)Methods Cell Biol. 80, 3–44 20. Wittig, I., Karas, M., and Schägger, H. (2007) Mol. Cell. Proteomics 6, 1215–1225 21. Havlícková, V., Kaplanová, V., Nùsková, H., Drahota, Z., and Houstek, J. (2010) Biochim. Biophys. Acta 1797, 1124–1129 22. Wittig, I., and Schägger, H. (2008) Biochim. Biophys. Acta. 1777, 592–598 23. Rak, M., Zeng, X., Brière, J. J., and Tzagoloff, A. (2009) Biochim. Biophys. Acta 1793, 108–116 24. Ludlam, A., Brunzelle, J., Pribyl, T., Xu, X., Gatti, D. L., and Ackerman, S. H. (2009) J. Biol. Chem. 284, 17138–17146 25. Granhall, C., Park, H. B., Fakhrai-Rad, H., and Luthman, H. (2006)Genet- ics 174, 1565–1572 5