E. (114 points) The “Beer” toxin is composed of two subunits, “Ping” and “Pong”. The toxin binds to the “Springbreak” receptor on the plasma membrane of pancreatic β cells and enters the cell via clathrin-mediated endocytosis. Once the toxin-receptor complex reaches the early endosome, “Ping”, “Pong”, and “Springbreak” dissociate from one another. “Ping” exits the early endosomes and enters the cytosol whereas “Springbreak” is targeted to the lysosomes for degradation. Although “Ping” alone can interact with “Springbreak”, its cytosolic targeting requires the presence of “Pong”.
(a) (6 points) “Pong” exists as a hexamer with each monomer containing a 25 a.a.-long amphipathic sequence at its N-terminus. This sequence is sensitive and resistant to the protease treatment in the presence of liposomes under neutral and acidic pH conditions, respectively. Based on these data, what is the most likely mechanism by which “Pong” facilitates the cytosolic targeting of “Ping”?
(b) (8 points) You have performed a western blot analysis of “Ping” using the cell lysates prepared 5 min and 1 hr after adding “Beer” to the cell medium (result shown below). Can you speculate how “Beer” kills infected cells?
(c) (6 points) What would be the most likely fate of “Pong” after it enters the early endosomes? Please CIRCLE THE CORRECT ANSWER and EXPLAIN YOUR ANSWER IN SHORT. (“Pong” would stay in the early endosome membrane, stay in the early endosome lumen, return to the plasma membrane, return to the extracellular space, be sorted to the internal vesicles of the MVB, be sorted to the lumen of the MVB but remained outside of internal vesicles, none of the above)
(d) (8 points) “Springbreak” contains a single Asn residue on its extracellular domain. If this Asn residue is mutated to an Ala, “Beer” can no longer enter the cell. You conducted a pulse-chase analysis of wild type and mutated “Springbreak” and the results are shown below. Please provide an explanation for this observation.
(e) (6 points) Your biochemical study shows that “Springbreak” alone can enter a clathrin-coated vesicle, yet you cannot find any short amino acid sequences known to mediate clathrin-dependent internalization. Can you explain it?
(f) (6 points) “Springbreak” has three transmembrane domains with an O-glycan located between the second and third transmembrane domains. Please draw the topology of “Springbreak” if it resides on the internal vesicles of MVB.
(g) (6 points) (True/False). Both kinesin (a plus-end motor) and dynein (a minus-end motor) are directly involved in the transport of “Springbreak” from the ER to the plasma membrane. Circle the answer and explain it in brief.
(h) (8 points) Under normal conditions, “Beer” can enter the β cells only when the blood glucose level is elevated. However, increasing the extracellular potassium concentration can by-pass the glucose requirement. What would be the most likely physiological function of “Springbreak” in the β cells?
(i) (30 points) (1) cytosol (2) ER (3) mitochondria (4) nucleus (5) Golgi (6) plasma membrane (7) extracellular (8) COPII vesicles (9) VTC (10) Golgi-derived transport carriers destined to the plasma membrane. Please predict the location of “Springbreak” (or its fusions) under the following conditions
( ) The wild type “Springbreak” is replaced by a fusion in which a NLS is fused to its cytoplasmic side
( ) The wild type “Springbreak”is replaced by a fusion in which a mitochondrial targeting sequence is fused to its N-
( ) The wild type “Springbreak” is replaced by a fusion in which KDEL receptor is fused to its C-terminus
( ) In cells carrying a mutation in Sec23 which results in a loss of GAP activity.
( ) In cells expressing a high level of an exogenous ER signal peptide
( ) In cells collected from an I-cell patient
( ) In cells with a temperature-sensitive mutant of Sar1 grown at a high temperature.
( ) In cells with a temperature-sensitive mutant of SNAP25 (a surface SNARE) grown at a high temperature.
( ) In cells with a temperature-sensitive mutant of Sec12 (the Sar1 GEF) grown at a high temperature.
( ) In cells with a temperature-sensitive mutant of NSF grown at a high temperature.
(j) (15 points) (1) extracellular (2) plasma membrane (3) clathrin-coated pits (4) clathrin-coated vesicles (5) internalization vesicles with no clathrin (6) early/recycling endosomes, (7) MVB (8) late endosomes (9) lysosomes (10) cytosol. Please predict the location of “Ping” after you add “Beer” into the cell medium under the following conditions.
( ) The cells are pre-treated with bafilomycin A1 which inhibits the endosomal and lysosomal proton-ATPases
( ) The wild type “Springbreak” is replaced by a mutant missing all the cytoplasmic domains (assuming such mutation does
not affect the function of the extracellular domain of “Springbreak” and the mutated protein can still move to the plasma membrane normally)
( ) The cells express a dominant-negative mutant of Dynamin GTPase.
( ) The cells lack the cytoplasmic Hsc70 chaperone.
( ) Instead of adding “Beer”, only subunit “Ping” is added to the cell medium.
(k) (15 points) “Springbreak” has a half-life of 6 hrs. Would the cells become (1) more, (2) equally, or (3) less sensitive to “Beer” toxin under the following conditions? Explain your answes briefly.
( ) in the presence of an inhibitor of the COPI coat for 24 hrs
( ) in the presence of an inhibitor of the ESCRT complex
( ) in the presence of an inhibitor of the acid hydrolses for 24 hrs