Question

You are using reconstitution experiments to study the formation of COPIl-coated vesicles. Specific components that you have available are shown in the table below. The ER membraneis an artificial membrane that contains the Sec12 protein and V-SNAREs. GTP,S is an analog of GTP that is functionally identical in every way except that it cannot be hydrolyzed. You perform the following experiments. After ncubating the components indicated in experiment 1 you observe normal fomation of COPlI-coated vesides that are able to fuse at a target membrane. Experiment Component GTP S ER Membrane x:13 2 R31 A. Describe the steps that would occur ih the process of vesicular transport to the target membrane with the components in experiment 2. Explain the role(s) for each component in these steps. components in experiment 3. Explain the role(s) for each component in these steps. C. Describe the steps that would occur in the process of vesicular transport to the target membrane with the components in experiment 4. Explain the role(s) for each component in these steps. pt Ha он GTP GTPyS

Answer 1

Experiment Number 2

1. In this case, there occurs binding of the COP-II coat on the endoplasmic reticulum which is associated with formation of the secretory cargo and formation of transport vesicles.
2. Sar-1 is activated by means of GTP binding and recruits Sec-23-24 complex that acts as primary cargo-binding apparatus. Simultaneously, Sec-13-31 complex is activated which provides the structural outer layer for the formation of vesicle.
3. This process involved formation of the budding vesicle and sec-13 is involved in generation of the transport vesicle simultaneously. The Sar-1 is involved in membrane curvature and recruitment.
4. Rab proteins are associated with GTP activity. When GTP is bound, they get activated. These proteins are associated with the plasma membrane and play a vital role in membrane trafficking and tethering to the target membrane of the golgi complex. As GTP is used in this case, the mechanism can be easily regulated by means of inhibition carried out by hydrolysis of the GTP to form GDP.
5. Sar-1 GDP is absent and hence cannot be involved in recruitment of the effector involved in vesicle formation and formation of transport vesicle. So, this step would be inhibited except for assembly of the Rab proteins.

Experiment Number 3

1. In this case, the mechanism of COP-II will be the same involved in binding to the donor membrane. This will activate the Rab by means of regulatory enzymes that adds GTP moiety to Rab forming the active protein. This will also be associated with activation of the Sar-1 GTP from GDP which will lead to recruitment of sec-23-24 complex followed by sec-13-31 complex. Both the mechanisms occur simultaneously.
2. In case if Rab is not present, the vesicle formation process may be affected however major impacts can be seen in terms of membrane tethering and binding process. So, tethering factors will not get activated and hence transport and trafficking will be affected.

Experiment Number 3

1. To be specific in this case, the GTP-gamma-S is used instead of GTP which cannot be easily hydrolysed and hence the inhibition cannot happen. This is suitable for experimental purposes where continuous stimulation is needed.
2. So, basically the mechsnism is going to be same in which there will be simultaneous activation of Rab and Cop-II binding followed by recruitment of Sar-1 GTP which helps in addition of Sec protein complexes and transport vesicles will be forms after budding. This will be followed by activation of tethering proteins that will create docking site in the target membrane.