13–4 In a nondividing cell such as a liver cell, why must the flow of membrane between compartments be balanced, so that the retrieval pathways match the outward flow? Would you expect the same balanced flow in a gut epithelial cell, which is actively dividing?
13–5 Enveloped viruses, which have a membrane coat, gain access to the cytosol by fusing with a cell membrane. Why do you suppose that these viruses encode their own special fusion protein, rather than making use of a cell's SNAREs?
13–6 For fusion of a vesicle with its target membrane to occur, the membranes have to be brought to within 1.5 nm so that the two bilayers can join (Figure Q13–1). Assuming that the relevant portions of the two membranes at the fusion site are circular regions 1.5 nm in diameter, calculate the number of water molecules that would remain between the membranes. (Water is 55.5 M and the volume of a cylinder is πr2h.) Given that an average phospholipid 1.5 nm
Figure Q13–1 Close approach of a vesicle and its target membrane in preparation for fusion (problem 13–6).
occupies a membrane surface area of 0.2 nm2, how many (A) strain A strain B phospholipids would be present in each of the opposing monolayers at the fusion site? Are there sufficient water molecules to bind to the hydrophilic head groups of this number of phospholipids? (It is estimated that 10–12 water molecules are normally associated with each phospholipid head group at the exposed surface of a membrane.) 13–7 SNAREs exist as complementary partners that carry out membrane fusions between appropriate ves icles and their target membranes. In this way, a vesicle with a particular variety of v-SNARE will fuse only with a membrane that carries the complementary t-SNARE. In some instances, however, fusions of identical membranes (homotypic fusions) are known to occur. For example, when a yeast cell forms a bud, vesicles derived from the mother cell’s vacuole move into the bud where they fuse with one another to form a new vacuole. These vesicles carry both v-SNAREs and t-SNAREs. Are both types of
SNAREs essential for this homotypic fusion event?
To test this point, you have developed an ingenious assay for fusion of vacuolar vesicles. You prepare vesicles from two different mutant strains of yeast: strain B has a defective gene for vacuolar alkaline phosphatase (Pase); strain A is defective for the protease that converts the pre cursor of alkaline phosphatase (pro-Pase) into its active form (Pase) (Figure Q13–2A). Neither strain has active alkaline phosphatase, but when extracts of the strains are mixed, vesicle fusion generates active alkaline phosphatase, which can be easily measured (Figure Q13–2).
Now you delete the genes for the vacuolar v-SNARE, t-SNARE, or both in each of the two yeast strains. You prepare vacuolar vesicles from each and test them for their ability to fuse, as measured by the alkaline phosphatase assay (Figure Q13–2B).
What do these data say about the requirements for v-SNAREs and t-SNAREs in the fusion of vacuolar vesicles? Does it matter which kind of SNARE is on which vesicle?
13–8 If you were to remove the ER retrieval signal from protein disulfide isomerase (PDI), which is normally a soluble resident of the ER lumen, where would you expect the modified PDI to be located?