Question: The Synthesis And Posttranslational Fate Of A Bee Venom Protein Source: Ibrahimi, I. 1987. Signal Recognition Particle Causes A Transient Arrest In The Biosynthesis Of Prepromelittin And Mediates Its Translocation Across Mammalian Endoplasmic Reticulum. J. Cell Biol. 104:61-66. Corresponding Chapter() In The Textbook: Chapter 11 Review The Following …

Question: The Synthesis And Posttranslational Fate Of A Bee Venom Protein Source: Ibrahimi, I. 1987. Signal Recognition Particle Causes A Transient Arrest In The Biosynthesis Of Prepromelittin And Mediates Its Translocation Across Mammalian Endoplasmic Reticulum. J. Cell Biol. 104:61-66. Corresponding Chapter() In The Textbook: Chapter 11 Review The Following …

The Synthesis and Posttranslational Fate of a Bee Venom Protein Source: Ibrahimi, I. 1987. Signal recognition particle causes
FIGURE RM RM-E * الا 1 2 3 4 kD -21.5 - 14.3 -6.5 PPM- PM QUESTIONS 1. Explain how the bands pPM and PM were resolved and sug
Biosynthesis and Characterization of pPM and PM Promelittin (PM) is the predominant secretory protein of queen bee venom glan
123.56 HD -215 100-SRP (0) - SRP ( -143 Preprenelitin arbitrary with Preligichan arbitrary units 50 pPM- PM- -6.5 Figure Synt

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The Synthesis and Posttranslational Fate of a Bee Venom Protein Source: Ibrahimi, I. 1987. Signal recognition particle causes a transient arrest in the biosynthesis of prepromelittin and mediates its translocation across mammalian endoplasmic reticulum. J. Cell Biol. 104:61-66. Corresponding chapter() in the textbook: Chapter 11 Review the following terms before working on the problem: secretion, protein synthesis, posttranslational protein processing, in vitro translation, mRNA, [‘H]proline/[S]methionine labeling, microsomal fraction, endoplasmic reticulum, signal recognition particle (SRP), SRP receptor, proteases, SDS-polyacrylamide gel electrophoresis, autoradiography. Read the paper and answer the questions below that refers to the data described in Figure 1 of the paper. Be prepared to discuss the other experiments described in class. EXPERIMENT Promelittin (PM) is a small secretory protein, a predominant component of queen bee venom. Its synthesis and posttranslational metabolism was studied in an in vitro translation system containing total bee venom gland mRNA, wheat germ lysate, [‘H]proline, [“S]methionine, and other molecules required for protein synthesis. Supplements were added to this system as follows: Sample 1: rough microsomal fraction (RM) Sample 2: no additional components Sample 3: cycloheximide (a protein synthesis inhibitor) was added at the end of translation, followed by the addition of RM fraction and another 30 minute incubation Sample 4: rough microsomes (RM-E) treated with N-ethylmaleimide (an inhibitor of SRP receptor) were added at the beginning of translation Sample 5: same as sample 4, except this sample was digested with proteinase K after translation The samples were subjected to SDS polyacrylamide gel electrophoresis followed by autoradiography FIGURE RM RM-E * الا 1 2 3 4 kD -21.5 – 14.3 -6.5 PPM- PM QUESTIONS 1. Explain how the bands pPM and PM were resolved and suggest a possible relationship between them 2. What conclusion can be drawn from comparing samples 1 and 2? 3. What conclusion can be drawn from comparing samples 1 and 3? 4. What conclusion can be drawn from comparing samples 1 and 4? 5. What conclusion can be drawn from comparing samples 4 and 5? Biosynthesis and Characterization of pPM and PM Promelittin (PM) is the predominant secretory protein of queen bee venom gland. In vitro translation of total mRNA from these glands results in a product that is larger than PM, and this has been shown to be PPM (Kaschnitz and Kreil, 1978). It contains a typical amino terminal signal sequence comprising 21 amino acid residues. Subcellular fractions from rat liver were shown to convert PPM to PM (Suchanek and Kreil, 1977). Since antibodies to melittin could not be produced, other criteria were used to establish the identity of the in vitro translation products. PM can be selectively separated from pPM by extraction with n-butanol (Mollay et al., 1982; Mollay et al., 1976). The identity of the separated products was previously established by sequence analysis (Kaschnitz and Kreil, 1978). The two components could also be separated by electrophoresis using SDS polyacrylamide gels containing 22% acrylamide and 6 M urea. A single ma- jor band that comigrated with standard PPM was obtained 123.56 HD -215 100-SRP (0) – SRP ( -143 Preprenelitin arbitrary with Preligichan arbitrary units 50 pPM- PM- -6.5 Figure Synthesis and characterization of PM and PM. The ORNA from queen bee venom gland was translated in whesterm lysate in the absence or presence of RM. The translation mixtures were supplemented with no components (lane 1) or RM (lane >> portion of the translation shown in lane 2 was extracted with butanol and the labeled proteins in the butanol phase (lase 3). the aqueous phase (lane), and the interphase (lane 5) are shown Lane 6shows tritated pPM standard. The numbers on the right side indicate molecular mass standards in kilodaltons, values 20 40 10 incubation time Iming Figure 2. Time coure of pPM synthesis in the whence and pres- ence of SRP: Translations in wheat rem lysate were done in the absence and presence of SRP using limiting concentrations of MRNA. At the indicated time intervals, samples were withown and subjected to SDS PAGE and autoradiography. The hands on the Autoradiogram were quantitated by densitometric scanning. The for pPM were plotted against incubation time. Wees in the presence of SRP were corrected for specific variation as de scribed in Results. In parallel experiment using prelight chain mRNA, an early and alle ample were taken and analyzed as de scribed above. in the synthesis of prelight chain. The arrest in the biosynthe sis of pPM was only transient, as indicated by the slowdown in the rate of chain formation in the presence of SRP. Rabbit globin mRNA was included in the translations as a cytoplas- mic protein marker whose synthesis is not affected by SRP For each time point the amount of PPM or prelight chain formed in the presence of SRP was determined by densitom- etry and corrected by multiplying by the ratio of globin in the absence of SRP over globin in the presence of SRP. This ratio was always very close to 1. SRP Causes a Specific and Concentration-dependent Arrest in the Biosynthesis of pPM Fig. 2 shows that after 15 min of incubation in the presence of SRP, the synthesis of pPM was 80% inhibited by SRP. while after 90 min the inhibition was only 18%. Fig. 3 shows that this inhibition is specific and dependent on the concen tration of SRP. The incubation was carried out, as in Fig. 2. for 15 min in the presence of decreasing amounts of SRP. It is clear that the synthesis of globin was not essentially affected by SRP while that of pPM was arrested by SRP in a concentration-dependent manner. when total translation products were displayed after SDS PAGE (Fig. 1, lanes / and 6). Addition of RM from mam malian ER to the translation mixture in either wheat germ or reticulocyte lysate generated a lower molecular mass pro tein, probably due to the cleavage of the signal sequence from pPM (Fig. I lane 2). The two proteins could be sepa- rated from each other by extraction of the translation mixture with w-butanol. As expected, PM partitioned in the aqueous phase and pPM in the interphase (Fig. 1, lanes 3-5). The two products had the expected molecular size based on their mo bility in SDS PAGE. Their identity was confirmed by amino acid sequence analysis (results not shown). SRP Causes a Transient Arrest in the Biosynthesis of PPM In a tightly synchronized translation assay that favored the formation of monosomes, the synthesis of pPM, in contrast to that of IgG K prelight chain, was not affected by SRP (results not shown). Because the size of PPM is 70 amino acids, which is the expected size of the SRP arrested peptide the above results could be interpreted by assuming that chain termination takes place before SRP is able to bind to the na- scent chain. An alternative possibility is that the two events coincide or fall close together, in which case the arrested peptide and the completed peptide would co-migrate as a re- sult of being identical or different by only a few amino acids. To discriminate between these possibilities, I studied the effect of SRP on the rate of synthesis of pPM in a nonsyn chronized translation system. Sublimiting concentrations of mRNA were used for translations in the wheat germ system in the presence and absence of SRP to enhance polysome for mation and allow continuous chain synthesis. The logic of this experiment was that when mRNA is limiting, a transient arrest by SRP will delay the recycling of mRNA, and hence lower the overall rate of PPM synthesis. Samples were with drwn at time intervals, precipitated with TCA, and sub- jected to SDS PAGE. A parallel experiment was done on light chain. Fig. 2 shows that SRP caused a permanent arrest LOW US on August, 2018 SRP Slows Down the Release of PPM from Peptidyl RNA Because SRP slows down the rate of pPM synthesis, the pos- sibility arose that PPM might stay in association with the ribosomes as a peptidyl tRNA for a longer period in the pres- ence of SRP than in its absence. This possibility was tested by precipitating peptidyl tRNA from translation assays in the absence and presence of SRP. Translation assays were done in wheat germ lysates using excess mRNA. After 5 min of incubation, further initiation was blocked by adding 7-meth- ylguanosine phosphate. Aliquots of 10 ul were withdrawn at the time intervals shown in Table I and put on ice. One-half of each aliquot was precipitated with CTABt and prepared The wround plamic Resin 63