Question

Protein trafficking has been one of the biggest challenges to explore and elucidate in the past few decades. Yet we now know much about this multi-step process.

d. Explain how proteases were used with rough microsomes to demonstrate the presumed presence of an SRP receptor.

e. Explain the differences between co-translational translocation versus post-translational translocation of newly synthesized proteins. What is the role of Bip in the latter process?

f. Challenge: You are studying a particular protein (Protein Z) that is found in normal cells as a single pass integral dimer protein. As such, it has a topology similar to glycophorin. As a research clinician and as a well known expert of this protein, you are contacted by a physician who has a patient that presents with excess soluble (not membrane bound) Protein Z (referred to as Protein Zsol) with little present in the plasma membrane. What might be the basis for the mutation in Protein Zsol?

g. How is pH important to RER resident proteins?

Answer 1

d: The purifies microsomes were treated with protease in the presence of absence of a detergent. The labeled secretory proteins associated with microsomes are digested by proteases only if the microsomes is destroyed by the detergent

e:.  In eukaryotes, secretory proteins enter the ER by co-translational translocation while in yeast some secretory proteins enter the ER lumen in post-translational translocation. SRP and its receptor are involved in co-translational translocation but in the post-translational translocation they are not involved. Bip is a chaperone protein that keeps the protein unfolded and prevents slipping.

f:.  The excess amount of nascent polypeptide chains in the open translocon Type Icould cause this mutation in the protein because too much protein is made in the first step of the Type I single pass proteins.

g:pH-dependent sorting between the ER and ERGIC. Proteins travel between the ER and the ERGIC in anterograde and retrograde directions. The pH of the ER is 7.4 (Wu et al., 2001), whereas that of the ERGIC is more acidic. The scheme shows three different pHdependent sorting mechanisms of anterograde-directed and/or recycling proteins in the early secretory pathway. ERGIC-53 functions as a transport receptor for the secretory/lysosomal protein procathepsin Z (pro-catZ). Association between ERGIC-53 and pro-catZ occurs in the ER at neutral pH, and dissociation occurs in the ERGIC following protonation of the ligand-binding site in ERGIC-53 (Appenzeller-Herzog et al., 2004). ERGIC-53 is then recycled back to the ER; pro-catZ proceeds through the secretory pathway. The LDL receptor-related protein (LRP) forms a complex with the escort protein RAP (Bu et al., 1995), which inhibits receptor-ligand interactions in the early secretory pathway. Again, low pH in the ERGIC triggers the dissociation of the two proteins. Subsequently, RAP is recycled back to the ER, probably by the KDEL receptor (KDEL-R), and LRP travels through the Golgi to the cell surface. By contrast, binding of the KDEL receptor to escaped ER proteins containing a C-terminal KDEL signal is thought to require the acidified pH in post-ER compartments (Scheel and Pelham, 1996). Ligand binding in turn triggers the retrograde transport of KDEL-receptor–ligand complexes from ERGIC and cis-Golgi to the ER.