Alexis Evans 10.16.2019The Synthesis and Posttranslational Fate of aBee Venom ProteinSource: Ibrahimi, I. 1987. Signal recognition particle causes a transient arrest in thebiosynthesis of prepromelittin and mediates its translocation across mammalianendoplasmic reticulum. J. Cell Biol. 104: 61–66.Corresponding chapter(s) in the textbook: Chapter 11Review the following terms before working on the
...[Show More]
Alexis Evans 10.16.2019
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(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 5 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,
[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
[Show Less]
