Question

An mRNA is translated into a protein in a cell-free extract in the absence of presence of microsomes or endoplasmic reticulum. Samples are treated as follows: (I) no treatment, (II) addition of protease, (IIl) addition of protease and detergent, and (IV) disruption of mircosomes and addition of endoH, an enzyme removing N-linked sugars that are added in the ER. Analysis by gel electrophoresis is shown in the figure. 7. MICROSOMESİ MICROSOMES ABSENT PRESENT TREATMENT protease a. Proteih synthesis at the ER requires the signal recognition particle (SRP) containing a deterent GTPase activity. Explain this statement and endo H why does this mechanism include a GTP hydrolyzing step? 0 b. Explain the experimental results in the absence of microsomes c. How would you argue that, in the presence of microsomes, the protein is transported across microsomal membranes? d. Considering that vesicles from the ER are moved towards the Golgi, explain the importance of vesicle coating (clatherin coat), the SNARE mechanism and the Rab system

Answer 1

A. Translocation of protein across ER membranes is a GTP dependent process. The signal receptor particle and signal receptor both contain subunits for GTP binding domain which are essential for binding .GTP hydrolysis activity is inhibited by GDP but not ATP.

B. In absence of microsomes, protein synthesis is inhibited

Example, Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting synthesis

C. ER has specialized regions called ER exit sites, whose membrane lakes bound ribosome.

D. Mostly vesicles are transport in coated form which are bud off later. Mostly they are clathrin coated vesicle ,which contain mainly clathrin and subunits triskelion( 3 large and 3 small polypeptide chains) other type of clathrin proteins are adaptin

Transported vesicles must be highly selective in recognising there target membrane this mechanism is carried out by proteins a. SNAREs they are mainly vSNARE and tSNARE unite and helps in binding target

b. Rabs : similar to SNAREs