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(s) in the textbook: Chapter 11
Review the following terms before working on the problem: secretion, protein synthesis, posttranslational protein processing, in vitro translation, mRNA, [3H]proline/[35S]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.
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, [3H]proline, [35S]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.
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?
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