Download Problem Set #6

1. We are beginning to see an important role for small GTPases in controlling different
processes in cell biology. For example, we discussed an important role for Ran in controlling
nuclear import and export. We also discussed small GTPases such as Arf and Sar in membrane
trafficking. Write a brief paragraph comparing and contrasting Ran versus Arf or Sar. Finding
the similarities shouldn’t be too difficult, but I also want to hear your opinion as to how the two
systems differ.
2. Cognate v-SNAREs and t-SNAREs bind to each other and fuse two membranes together.
NSF is an ATPase that uses the energy of nucleotide hydrolysis to break apart SNARE
complexes. Why then is NSF required for membrane fusion in cells?
3. In order to probe retention of proteins in the ER, you transfected cells with three different
constructs, each with a test protein behind an inducible promoter. You then induce expression
and measure the rate at which the factors are secreted. The graph below compares the rates at
which a test substrate (SS-EGFP-EGFP-EGFP) is secreted from a cell to the rates of BiP and BiP
lacking its C-terminal KDEL tag. The triple EGFP protein is roughly the same molecular weight
as BiP. What does this experiment tell us about the mechanism of ER retention operating in
cells?
4. GFP is normally a cytosolic protein but can enter the secretory pathway if you attach a signal
peptide to its N-terminus. Cathepsin is a protease that is normally secreted. You can attach a red
fluorescent protein (RFP) to cathepsin and it will still be secreted from the cell with normal
kinetics. How could you use these two contstructions to determine whether cathepsin exits the
cell by bulk flow versus a receptor mediated process? Keep it simple. A few sentences should
suffice.
5. Shibire is a mutation in Drosophila that results in paralysis when flies are incubated at a
restrictive temperature. Anything that paralyzes flies is cause for celebration, so you decide to
investigate this further. Electron microscopy of fly brains at the restrictive temperature reveals
the following:
Figure 1. EM of presnynaptic nerve terminals in Shibire flies at the restrictive temperature.
The images in Figure 1show nerve terminals. The vesicles are most likely synaptic vesicles that
contain neurotransmitter for synaptic transmission, and they are readily detected under all
conditions. The structures with the collar however are only seen in the mutant at the restrictive
temperature. Based upon the EM, what cellular process do you think is disrupted by the Shibire
mutation and a brief statement as to why you think that. Finally, briefly describe an experiment
that would help test your hypothesis.
6. Cloning of the Shibire gene plus biochemical analysis reveal that it is a large GTPase that is
also known as dynamin. You express and purify recombinant dynamin and prepare it two
different salt concentrations then view it in the electron microscope and obtain the following
images. (the magnification in figure 2 is much higher than that in figure 1)
Figure 2. i) shows pure dynamin under low salt conditions. t) shows dynamin in the presence of
higher salt concentrations. 2i and 2t are at approximately the same magnification.
6a) How did dynamin get so huge in figure 2t?
6b) Based upon these images generated from in vitro work, state an interesting hypothesis with
respect to the images in Figure 1.
6c) You are interested in studying how GTP might control the morphological changes shown in
figure 2, but your advisor says that the bill ($) for using the electron microscopy facility is
getting too high. Now she will only pay fo more EM if you provide her with strong evidence
that the sample will be worth looking at in the microscope. Design a simple experiment to test
changes in dynamin as a function of GTP that would only cost a couple of dollars.
Unfortunately, you do not have access to a dynamic, quasi-elastic light scattering instrument.
7. Your experiment in 6B worked, so know you have a blank check (a colloquial phrase
meaning you are free to proceed at any time) to use the EM facility, and your advisor bought you
a light scattering instrument!
Figure 3 is an EM of dynamin. In (A) Dynamin + lipid vesicles in the absence of GTP. In (B),
GTP was added to the sample used in A for five minutes before fixing the sample. In addition to
these images, light scattering experiments show that the size of particles in the sample decreases
rapidly upon addition of GTP.
Figure 3. EM of dynamin + vesicles +/- GTP.
Based upon these results, what might be the function of GTP hydrolysis by dynamin and how
might this explain the phenotype seen in figure 1?
8. Cells are constantly endocytosing material using clathrin which requires that clathrin must
continuously be assembling on membranes in some regions of the cell while disassembling to
allow vesicle fusion in other regions of the cell. This is clearly a non-equilibirum behavior
requiring an energy source. Clathrin does not hydrolyze nucleotide. Where do you think the
energy powering clathrin turnover dynamics comes from?
9. We considered two alternative hypotheses regarding disassembly of coats on vesicles. One
stated that uncoating is tightly coupled to vesicle formation and therefore occurs soon after
scission. The second proposes that uncoating occurs “later” or at least later than we might have
thought. You knock the expression of a key component of the membrane tether complex TRAPP
down using RNAi. Recall TRAPP is thought to tether vesicels from the ER to the cis golgi. If
the “uncoating is late” hypothesis is true, what should you see in cells?
9b. How might tethers help control uncoating of vesicles?
10. Specific membrane fusion requires the SNAREs. It is thought that each vesicle contains a
specific v-SNARE while the target membrane contains the cognate t-SNAREs that bind
specifically to incoming vesicles with the correct v-SNARE . Now consider how resident
enzymes of the ER are retained in the ER. Do you see a major problem? In other words, why
would membrane trafficking appear to require more specificity than that afforded by the
SNAREs alone? (if you’re stuck o this one, try drawing a cartoon of membrane traffic between
ER and cis golgi).
11. The first SNAREs were identified in a wonderfully simple pull-down, affinity assay which
revealed that four proteins associated with NSF(NEM sensitive factor) in an ATP dependent
manner. Four proteins and yet Sollner and Rothman had the audacity to proclaim that the
SNAREs could explain membrane fusion specificity throughout the entire cell. What is it about
the SNAREs that led them to propose such a bold, groundbreaking model?