Exam 1 (SNARE & TOM) Flashcards
The SNARE hypothesis is an attempt to explain how the various vesicles in the cytoplasm, which are trafficking between numerous compartments, TAREGT to and DOCK with appropriate targets. What additional function for the SNARE complex are the authors proposing to test in this paper? What is the overall strategy for testing their hypothesis?
They will test whether SNARE complex formation is sufficient to cause membrane fusion in addition to allowing vesicles to identify the proper target membrane for docking. The strategy is to create 2 artificial membranes in the form of proteoliposomes. The only protein incorporated into the first proteoliposome is the v-SNARE, and the only proteins incorporated into the second proteoliposome are the t-SNAREs. If these two membranes can fuse, this proves that the SNARE complex is sufficient to cause fusion.
What is the source of the SNARE proteins used in this experiments?
The cDNA for the v-SNARE VAMP2 is altered to encode 6 histidine residues at the C-terminus. The protein was then overexpressed in genetically engineered bacteria and then purified from bacterial cell lysates based on the binding property of the HIS (histidine) tag. The 6 histidine’s cause the protein to bind tightly and specifically to nickel. Agarose beads were coated with nickel and incubated with the bacterial lysate. Then the beads were washed, and the VAMP that was bound to them was eluted with an excess of imidazole which also binds nickel and replaces the VAMP on the coated beads.
The t-SNARE SNAP-25 cDNA was engineered to encode a glutathione S transferase (GST) tag, followed by a protease cleavage site and then the entire SNAP-25 protein. The GST tagged SNAP-25 was then overexpressed in bacteria along with the t-SNARE syntaxin. This required that two different plasmids coexist in the bacteria, and this was achieved by having one plasmid confer ampicillin resistance and the other confer kanamycin resistance. The two proteins will form a complex in bacteria and this complex was purified in a similar manner to the VAMP (above) except the beads were coated with glutathione which binds GST. The complex was eluted by releasing the complex from the beads with a protease that cleaves between the GST and the VAMP portions of the polypeptide.
How were the SNAREs incorporated into lipid vesicles? Do all the SNAREs get incorporated into the vesicles in the desired orientation? How do the authors determine what fraction of the SNAREs are in the right-side orientation (the authors don’t show the data).
Lipids dissolved in detergent are mixed with proteins dissolved in detergent. The detergent is rapidly diluted, causing the lipids to form a continuous bilayer (vesicle) into which the proteins are incorporated. All the detergent is removed by dialysis. The proteins can be incorporated in the vesicles right-side-out or wrong-side-out. In this type of liposome, you can expect to find the protein in the correct orientation only 50% of the time (random process). Thus, it somewhat unusual that the authors find that about 70% of their vesicles have the SNAREs in the proper orientation. In cells the orientation of a transmembrane protein is controlled so that a specific protein is always in one specific orientation. This is controlled when the protein is inserted into the membrane during translation-coupled transport across the endoplasmic membrane.
The authors determine the fraction of proteins oriented in a particular direction by monitoring the sensitivity of the proteins to proteases. Unfortunately, they don’t state what protease was used. Presumably, BoNT D could be used on the vSNARE, but a different protease would have to be used on the tSNARE since it is insensitive to BoNT D.
Botulinum neurotoxin D (BoNT D) is a potent inhibitor of synaptic transmission because of its effect on SNARE complex formation. How does BoNT D inhibit fusion? How was BoNT D used for the experiment in Figure 1B to provide evidence that v-SNAREs and t-SNAREs in liposomes are forming complexes?
BoNT D cleaves the N-terminus of VAMP (v-SNARE) prior to SNARE complex formation and fusion, thus preventing fusion.
BoNT D degrades uncomplexed v-SNAREs. Hence, they can measure the amount of radioactively labeled v-SNARE that resists degradation. However, the assay involves more than just adding BoNT D to a mixture of vesicles because v-SNARES that were outside-in in the vesicles are also resistant to BoNT D even though they are not complexed with t-SNAREs. To get at these outside-in v-SNAREs, they dissolved the membrane with Triton X-100. But this results in another problem. Because t-SNAREs were in excess, these were able to bind to the exposed outside-in v-SNARES before the BoNT D could attack the v-SNARES. To prevent this, the authors added an excess of a “truncated” version of the v-SNARE so this could sequester all the t-SNARES before the t-SNARE captured the outside-in v-SNARES.
How was fluorescence used to monitor fusion of vesicles?
Donor vesicles are prepared with fluorescent phospholipids containing either NDB or rhodamine. The rhodamine fluors quench the NBD fluorescence when the rhodamine is in close proximity to the NDB. Upon fusion to an unlabeled acceptor vesicle, the phospholipid bilayers mix, and the rhodamine and NBD are diluted. Thus, rhodamine is no longer in close proximity to NBD and NBD fluorescence can be detected. The level of NBD fluorescence is, thus, a measure of fusion.
The 4°C sample in Figure 2A is essentially the same as the “Acceptor” sample in Figure 2B. Why do the plots appear different?
The authors have reset the y-axis to emphasize what’s happening at 37°C when fusion is occurring. The fluorescence that occurs at 4°C reflects incomplete quenching and is set to zero. The fluorescence detected after Triton X-100 (which mixed the lipids completely and dequenched the fluorescence of NBD) is added is the maximum fluorescence possible so its set to 100%
The experiment in Fig. 3 is performed to investigate whether fusion has fully occurred. What is meant by hemifusion? How does dithionite allow the authors to determine if they are achieving complete fusion of membranes or hemifusion?
Hemifusion means that the outer leaflets of two bilayers have fused, but the inner leaflets have not. Dithionite will reduce the NBD in the outer leaflet to a non-fluorescent form, allowing the authors to measure unquenching / fusion in the inner leaflet
The authors notice an effect of preincubation of v-SNARE and t-SNARE vesicles at 4°C (Fig. 2A). What is happening at 4°C and what subsequently happens at 37°C? Or in other words, can you describe a model for the sequence of events leading to fusion?
During incubation at 4°C, v-SNARE vesicles and t-SNARE vesicles collide as a result of Brownian motion. The highly stable SNARE complex forms while the vesicles are at 4°C, causing the vesicles to stick together, but fusion does not occur until the temperature is changed to 37°C
Figures 4B, 4C, and 4D show that addition of BoNT D, soluble VAMP, or soluble t-SNARE before preincubation blocks fusion but not after preincubation. Why does adding the reagent before preincubation block fusion?
Incubation of the vesicles with BoNT D prior to preincubating the vesicles together allows BoNT D to destroy the VAMP thus inhibiting docking and fusion. Incubating the vesicles with soluble VAMP or soluble t-SNARE allows the soluble forms of the protein to binding their cognates in the vesicles and block binding to the cognates on the vesicles. From these results, we can infer that the addition of the soluble SNAREs or BoNT D after the 4°C preincubation must inactivate free SNAREs on the vesicles thus indicating that the free SNAREs are not involved in fusion event when the vesicle are shifted to 37°C. Thus, the complexes that formed at 4°C must be sufficient to mediate fusion.
Which of the following transport mechanisms transports proteins in an unfolded state: nuclear import, co-translational transport into the ER, vesicular transport between the ER and the Golgi, TOM/TIM-mediated import into the mitochondria?
Co-translational transport into the ER and TOM/TIM-mediated import into the mitochondria.
What chemical characteristic of the mitochondrial signal sequence matches well with a chemical feature identified on the inside of the Tom40 beta barrel?
The signal sequence is a positively charged, amphipathic alpha helix. The crosslinking obtained with 35SpSu9-DHFR and displayed in Figure 1B revealed that the peptide crosslinked to a region inside the Tom40 barrel that was rich in negatively charged amino acids. Thus, we can envision that the positively charged side of the signal sequence associates with the negatively charged area of the barrel through electrostatic interactions.
Would a hydropathy plot analysis be able to predict the transmembrane domains that constitute Tom40?
NO! The hydropathy plot analysis will detect continuous regions of hydrophobic amino acids. Because of the structure of the beta barrel, the amino acids may alternate between hydrophobic and hydrophilic resulting in a hydropathy score near zero.
What two regions of aminoacyl tRNA synthetase had to be altered in order for cells to couple the crosslinkable amino acid BPA to the desired tRNA? What else had to be altered in the cell in order to have the cell insert BPA into a specific site in Tom40?
The aminoacyl tRNA synthetase had to be modified so that it would recognize the unnatural amino acid, BPA, and the anticodon CUA, since the normal cadre of synthetases don’t recognize either of these. Other
changes were necessary to insert BPA into a specific site. The amber codon had to be inserted into the coding sequence of Tom40 at the position where BPA was to be inserted. Also, the modified tRNA containing the CUA anticodon had to be expressed.
Why was the sequence encoding Tom40 changed so that 10 histidine’s were attached to one end of the protein? How was this used to generate the data in Figure 1A? What was learned from Figure 1A?
The 10 histidine’s made it possible so the researches could purify Tom40 from a cells lysate by using Ni-NTA affinity chromatography. Proteins such as Tom22 that were covalently crosslinked to Tom40 copurify with the Tom40 when the Tom40 sticks to the affinity resin. The Tom40 is eluted from the column along
with any covalently attached proteins. The eluted mixture of proteins was then analyzed by western blotting with antibody against Tom22.
As far as I can tell, the beta barrel structure of the Tom40 complex that is presented in the paper is only a model. Based on which amino acids of Tom40 crosslinked to Tom22, the authors deduce which amino acids
were pointing on the outside surface of the barrel.
Figures 1A and 1B both display protein bands on gels. How does the detection of these bands differ? What was learned from the data in Figure 1B?
Figure 1A is a Western blot (immunoblot). The authors state that they detected proteins on the immune-blot using antibodies but they don’t describe how they detected the antibodies.
There are several ways this can be done and one of them is similar to the indirect immunofluorescence microscopy. A primary antibody is bound to the protein that resides on the immunoblot.
A fluorescently labeled secondary is then bound to the primary antibody and the fluorescence is detected machine. Alternatively, the secondary antibody may be coupled to an enzyme such as luciferase and a chemical reaction that produces light is done on the blot and this light is detected with film.The bands in figure 1B result from the 35S-radioactivity that can be detected by exposing the gel to film or a machine called a phosphoimager. Figure 1B reveals regions of Tom40 that are in close proximity to the protein passing through the barrel called pSu9-DHFR. pSu9-DHFR is radioactively labeled and Tom40 contains the crosslinker inserted at various locations. Its interesting to note that there are several places where crosslinking occurs at every other amino acid. For example, crosslinking occurs at amino acids 101 and 103 but not 102 and 104. This is consistent with the beta barrel structure where every other amino acid faces the inside of the barrel.
