Identifying sites of protein synthesis in Chlamydomonas using erythromycin and cyclohexamide as protein synthesis inhibitors. October 16, 2009 Introduction: In living cells, prokaryotic or eukaryotic, the synthesis (construction) of proteins is accomplished by similar machinery. Amino acids, ribosomes, messenger RNA (mRNA), and transfer RNA (tRNA), are all necessary for the building of functional proteins in a cell. Ribosomes are the site of protein synthesis in a cell, and there are two different types, depending on the type of cell. Only the 70S ribosomes are found in prokaryotes (bacteria, archaea).
Eukaryotes, (cells that contain a membrane-bound nucleus), have both 70S and 80S ribosomes. The 70S ribosomes are present in mitochondria and chloroplasts of eukaryotic cells (Willey, et al, 20083). The theory of endosymbiosis is based on the thought that mitochondria and chloroplasts were, at one time, prokaryotic organisms that were engulfed into a eukaryotic cell and formed an equally beneficial relationship. Since mitochondria and chloroplasts contain their own DNA and self-replicate, their genetic codes are passed along with the rest of the cell when reproduction occurs.
The two types of ribosomes made in a eukaryotic cell will respond differently when given certain antibiotics (Nelson, et al 2009). This experiment focused on two: erythromycin and cyclohexamide. When given erythromycin, tRNA is inhibited from transferring from one site of ribosomal RNA (rRNA) to another site during protein synthesis. However, this inhibition only occurs in the 70S ribosome (DrugBank, 2011). In contrast, when a cell is given cyclohexamide, the 80S ribosomes are blocked from completing the process of elongation (the addition of triplet codons that are complimentary to an mRNA molecule).
This occurs in the last step of elongation, during the translocation phase (Willey, et al, 20081). Translocation is the movement of the tRNA molecule from one part of the ribosome to another as it adds its complimentary amino acid opposite the mRNA strand. (Willey, et al, 20082). As a result of the inhibition actions, a cell given erythromycin will only produce proteins using 80S ribosomes (cytoplasmic proteins). Conversely, when given cyclohexamide, proteins synthesis of cytoplasmic proteins decreases (Dzyubinskaya, 2006).
In order to determine the number of proteins made by Chlamydomonas when treated with these antibiotics, the cells were treated with 35S, a radioactive isotope of sulfur. The algal cells used the sulfur in the synthesis of new proteins. It is then possible to measure the amount of radioactivity in the new proteins after the antibiotic treatment with a scintillation counter. As Chlamydomonas contains only a single chloroplast, which accounts for a significant amount of its volume, affecting protein synthesis in either type of ribosome will constitute a noticeable decrease in production.
This experiment sets out to determine where the resultant proteins would have remained chloroplast and mitochondrial proteins, in the cytoplasm of the cell, or in the cell membrane. Materials and Methods: Into each of three microfuge tubes, one milliliter (1mL) of Chlamydomonas culture was inserted and spun at top speed (14,000 rcf) for two minutes. When completed, the supernatant was removed and discarded. Each of the three tubes was then resuspended with 50 microliters (µL) of erythromycin (100µg/mL), 50µL of cyclohexamide (10µg/mL), or 50µL of TRIS buffer (as the control).
Each of the three tubes was then mixed with 50µL of the radioactive sulfur isotope 35S and incubated at room temperature for 30 minutes. The samples were then centrifuged at top speed for five minutes, and the supernatant discarded. The remaining pellet was frozen at -20°C for 2 minutes, then thawed (room temperature for 2 minutes) 3 times to burst the cells. The cells were again resuspended, using 100µL of TRIS buffer and mixed. The tubes were respun in the centrifuge at top speed for five minutes.
The supernatant containing the solute proteins was then transferred into three new centrifuge tubes. To the pellet, (the membranous proteins), 100µL of Sodium Dodecyl Sulfate (SDS) lysis buffer was added and mixed to resuspend cell membrane proteins, then 400µL of cold acetone was added and the tubes incubated in a -20°C freezer for 15 minutes. To the supernatant, (the soluble proteins), 400µL of cold acetone was directly added, and those microfuge tubes were also placed in a -20° freezer for 15 minutes.
After freezing, all six tubes were spun in the centrifuge at top speed for five minutes and the supernatant from each tube discarded. Each tube was then given 50µL of SDS lysis buffer and mixed. Ten (10) µL of sample from each tube were placed into corresponding scintillation vials and sent to the scintillation counter for analysis. Results: Figure 1. 0 shows the results from the scintillation counter for each sample. The scintillation counter reads the amount of radioactivity present in the sample, so the higher the reading, the higher the number of proteins present that contain the 35S isotope.
It also gives a clear initial indication of the impact of the antibiotic treatment on the cell’s protein synthesis activity. As the graph shows, both antibiotics had a marked impact on the synthesis of proteins in cellular solute and in the cells’ membranes. The graph includes the averages for all four experimental groups as well as the data for the author’s group. Figure 1. 0: Graphic representation of average protein counts in control, after erythromycin treatment, and after cycloheximide treatment for all experimental groups (red) and author’s group (blue).
The (S) indicates proteins in solution, while the (M) indicates proteins in membranes. Averaged over all four experimental groups, erythromycin showed a 42. 5% decrease the synthesis of proteins in cell membranes when compare to the control count. It decreased the proteins in solution by 34%. The cycloheximide decreased synthesis of proteins in the membranes by 57. 6% when compared to the control, and a 53. 6% decrease in soluble proteins. Cyclohexamide treatment resulted in a greater overall decrease in protein synthesis throughout all cellular components (See Table 2. ). Erythromycin decreased protein synthesis in all cellular components by 38. 25%, while cycloheximide treatment decreased synthesis by 56. 75% when compared to the control. The percent decrease in proteins, when averaged over all four experimental groups, is slightly lower than the percent decrease calculated for the author’s group. While erythromycin and cycloheximide both decreased protein synthesis in soluble and membrane proteins, our percentages were higher than the average. Erythromycin showed a 63. % decrease in soluble proteins, and a 37. 5% decrease in membrane proteins (from control). Cycloheximide decreased protein synthesis in soluble proteins by 53. 6%, while proteins in membranes were decreased by 85. 3% (from control). These numbers also resulted in higher than average overall protein synthesis reductions for the two inhibitors. Erythromycin treatment resulted in a 56% reduction in overall protein synthesis, while cycloheximide resulted in an overall 72. 6% decrease in protein synthesis in our group’s experiment. Membrane Proteins| Solute Proteins| Overall Proteins| Average % Decrease After Erythromycin| 42. 5%| 34. 0%| 38. 3%| Average % Decrease After Cyclohexamide| 57. 6%| 53. 6%| 56. 8%| Table 1. 0: Average decrease in protein counts after antibiotic treatment (compared to control counts). Figures averaged from all groups. Discussion: While both antibiotics caused a significant decrease in the production of proteins within the cell, it was the cyclohexamide that had the greatest impact on both soluble proteins and membrane proteins.
With its blocking effect of blocking elongation in 80S ribosomes, it can be deduced that the majority of proteins made within the cell are synthesized with 80S ribosomes. Erythromycin had a smaller percentage decrease of protein production, but when the data from Group 4 is examined, erythromycin actually had a larger percentage decrease in membrane proteins than the sample treated with cycloheximide. In groups 1 and 2, cycloheximide had a greater percentage decrease, but the percent differences were 7. 5% and 15. 2%, respectively.
The greatest difference in the membrane proteins were found in group 3, which showed a 47. 8% difference between treatment with cycloheximide and erythromycin. When evaluating the scintillation counts for the solute proteins, the data remains very similar. While cycloheximide still caused a greater overall reduction in solute proteins for every group tested, two groups had results that did not show a considerable difference in protein synthesis after treatment. Group 1 showed a 10. 4% difference in protein production between cycloheximide and erythromycin, while group 3 had a very minor 3. % difference between the two antibiotics. From this data, we are forced to conclude that in a eukaryotic cell, the greatest numbers of proteins are synthesized by 80S ribosomes. As Table 2. 0 shows: cycloximide’s effect on 80S ribosomes caused a greater decrease in solute and membrane proteins, leading to a 56. 8% overall reduction in protein synthesis. This experiment also showed that within the cell, if there is a decrease in protein synthesis, there is a greater reduction of cell membrane proteins than there is for the solute proteins.
So, whether the proteins are synthesized by 70S or 80S ribosomes, the proteins have a greater chance of ending up as membrane proteins than they do for remaining in the cytoplasm of the cell. As stated in the introduction, the 70S ribosomes are found in the mitochondria and chloroplasts. As we have shown in this experiment, when erythromycin affects the synthesis of proteins from 70S ribosomes, it causes a larger decrease in the number of membrane proteins than it does for proteins that remain in the cytoplasm of the cell.
These proteins can be found not only in the cell membrane as well as the membranes of mitochondria and chloroplasts. This decrease was not as significant as the decrease of protein synthesis caused by cycloheximide; however, cycloheximide also caused a more significant reduction in membrane proteins than in solute proteins. My interpretation of this is that when protein synthesis is decreased in any way, the membrane is the most affected part of the cell.
This can lead to problems for the cell as far as transporting materials across the cell membrane, receptor proteins being absent or diminished in numbers, and will affect the building of protein antibodies that reside on the cell membrane (Freeman, 2008). Bibliography: * DrugBank – Open Data Drug and Drug Target Database. (10/06/2011). URL: http://www. drugbank. ca/drugs/DB00199 * Dzyubinskaya, E. V. ; D. B. Kiselevsky; L. E. Bakeeva, and V. D. Samuilov. (2006). Programmed cell death in plants: Effect of protein synthesis inhibitors and structural changes in pea guard cells.
Biochemistry (Moscow). 71 (4), 395-405. * Freeman, Scott. (2008). Biological Science. Pearson Education, Inc. San Francisco, CA. pps. 109-116 * Nelson, Mark L. ; Mark C. Grier; Susan E. Barbaro; and Mohamed Y. Ismail. (2009). Polyfunctional antibiotics affecting bacterial membrane dynamics. Anti-Infective Agents in Medical Chemistry. 8, 3-16. * Willey, Joanne M, Linda M. Sherwood, Christopher J. Woolverton. (2008). Prescott, Harley, and Klein’s Microbiology. McGraw-Hill. New York, NY. 1. Willey, et al. Pps. 284-285 2. Willey, et al. Pps. 625-626 3. Willey, et al. Pps. 272-280