C2006/F2402 '02 -- Key to Exam #1
In general, each answer is worth 2 pts. Any exceptions are noted and value of explanations is included below.
1. A. Mannitol can not cross the epithelium because of tight junctions.
B. Mannitol diffuses within the epithelium using connexin. Transport here is most similar to diffusion through a channel. Explanation : Mannitol is diffusing through gap junctions; the channels/pores at gap junctions consist of connexons that are made up of the protein connexin. (2 pts)
2. A. None of the kinesins require a signal peptide. Explanation: they are all cytoplasmic proteins, so they do not need an SP as they do not enter the ER. (2 pts).
B. All kinesins should bind to tubulin AND none should bind to actin. Explanation: Kinesins are motor molecules that bind to microtubules (MT) and move things along the MT. Since MT are made of tubulin, all kinesins must bind to tubulin. Kinesins do not interact with microfilaments (MF) which are made of actin. (2 pts).
C. A -Kinesin and dynein should move in the same direction. Explanation: Ordinary kinesin (+K) moves in the opposite direction to dynein, so -K and dynein should move in the same direction. Alternatively, + K moves toward the + ends of the MT; dynein moves toward the - ends, as does -K. (2 pts)
D. Coated vesicles could be bound to a -K; secretory and default vesicles should not. Explanation: Secretory and default vesicles are moving out from the Golgi toward the periphery of the cell. This should be mediated by a +K, which moves outwards, towards the + ends of the MT. (The MT generally are connected by their - ends to a MTOC near the middle of the cell and extend so their + ends are near the plasma membrane.) Coated vesicles can be moving inwards or outwards -- if they are moving inwards as a result of RME they would require a -K. (2pts).
E. APP should be attached by its carboxyl end to a +K . Explanation (4 pts total): Single pass proteins are usually inserted in the ER membrane with their amino ends on the lumen/extracellular side and their carboxyl ends in the cytoplasm. If APP inserted in vesicles binds to kinesin, it must be the cytoplasmic part of the protein that binds to kinesin. (2 pts). The vesicles are moving away from the cell body, toward the + ends of the MT, so the vesicles need to attach to a + kinesin. (2 pts).
3. A. Picture of protein in plasma membrane (not ER membrane) should have ends of protein and sides of membrane correctly labeled. (3 pts). This question was included to make it easier for you to answer the remaining questions, and easier for us to grade them. It is really the explanation to part B.
B-1. The SP should be in the middle of the protein chain.
(It is probably closer to the amino end than to the carboxyl end, but it is not
on the extreme amino end. You can't be sure how close it is to each end, because
you don't know how long the individual protein sections are.)
B-2. TMS (transmembrane sequence) #8 is a stop transfer
sequence.
B-3. There are 3 start transfer sequences (you don't count
the SP).
B-4. The section between TMS 3 & 4 is extracellular.
B-5. S-S bonds could be between TMS 3 & 4. (Only formed
in lumen of ER, and found in extracellular domains of proteins.)
C. If signal peptidase were lacking, protein would be the same. Explanation: The SP of this protein is not removed, so a lack of the enzyme for SP removal would make no difference. (2 pts) You know the SP is internal and is not removed because the amino end of the protein is in the cytoplasm, not outside the cell.
D. (This part labeled C-1 & C-2 on exam by mistake): Ab #2 is best for measuring presenilin in the plasma membrane and Ab #1 is best for measuring presenilin in the ER and Golgi membranes. Explanation (4 pts total): If antibody is added from outside the cells, it cannot enter the cells (under normal conditions) and can only react with the extracellular domains of the plasma membrane proteins. So it can bind to the section of presenilin between TMS 3 & 4 but not to the section between TMS 2 & 3. (2 pts). If the plasma membrane is permeabilized, then antibodies can enter the cytoplasm and interact with the protein domains that are exposed to the cytoplasm. When presenilin is in the ER and Golgi, the parts corresponding to the extracellular domains (for the plasma membrane) are inside the lumen of the ER or Golgi. The domains accessible to the antibody are the domains that stick out into the cytoplasm such as the ones between TMS 2 & 3. (2 pts). Credit was given for the explanations, but not for the answers, if you misunderstood the experimental set up but correctly stated which domains of presenilin should be accessible to an antibody in the cytoplasm and which should be accessible to an antibody on the outside of the cell.
E. (This part labeled D on exam by mistake). Either
defective kinesin (+1) or defective APP (+1) should decrease the amount of
presenilin at the synapse. Explanation (6 pts total): Presenilin is a
plasma membrane protein. To get to the plasma membrane, it must move down the
axon toward the synapse in the membrane of a vesicle (2 pts). In more detail:
Presenilin is inserted into the membrane of the ER as it is made. From then on,
the protein remains in a membrane. Vesicles bud off the ER carrying the protein
in the membrane (not the lumen) of the vesicle. The vesicles fuse with the cis
side of the Golgi, and new vesicles bud off the trans side of the Golgi. These
vesicles move down the axon and fuse with the plasma membrane at the synapse.
Throughout this entire process, the presenilin remains inserted in a membrane in
its original orientation.
How do the vesicles move down the axon? The vesicles that
carry the presenilin also carry APP in their membranes -- that's how the
vesicles attach to kinesin. If APP is missing, the vesicles can't be attached to
a motor, so they don't move down the MT.(2 pts). Kinesin acts as a motor
molecule to move the vesicles down the MTs to the synapse. If kinesin is
missing, there is no motor and nothing moves down the MTs. (2 pts).
4. A. If you omit the ATP or K+, the initial rate of glucose
uptake stays the same. (This did not need to be explained.) If you omit
the Na+, the rate decreases, and if you increase the glucose,
the rate increases if the co-transporter is not saturated. (2 pts each
correct answer.) Explanation (4 pts total): Glucose uptake at the apical
surface is (primarily) by co-transport with Na+. Glucose uptake is
dependent on the Na+ gradient -- Na+ flows down its
gradient and that provides the energy to drive glucose up its gradient, from the
lumen into the cells. If there is no Na+ gradient, glucose uptake by
co-transport into the cells will not occur. (2 pts). The uptake of glucose in
this way is dependent on a supply of Na+, but if the [Na+]
is kept constant, the rate is proportional to the amount of glucose present
until the carrier or pump becomes saturated with glucose (2 pts). This is
demonstrated by the hyperbolic curves of initial rate of X uptake vs [X] on
handout 3B -- note that you get a hyperbolic curve for protein-mediated
transport whether uptake is passive or active.
In this experiment, the ATP is added on the outside, and so
it cannot be involved at all in either transport of glucose or maintaining an
ion gradient.
K+ is needed for operation of the Na+/K+
pump, but once the ion gradient is established, you don't need the K+
to transport the glucose.
B. The initial rate of glucose uptake will be the same whether you have ATP available on the inside of the cell or not. Explanation (4 pts total): Transport here is directly dependent on the Na+ gradient, not on the ATP supply. In the short run, the Na+ gradient can power glucose uptake -- it will take some time for the gradient to run down. (2 pts) ATP was needed to set up the Na+ gradient in the first place, and ATP will be needed it you want to replenish the gradient, but ATP is not needed immediately for glucose uptake. (2 pts)
C. A uniport protein (2 pts), and a permease or channel protein (2 pts for either one or both) are needed in the membrane to explain these results. (The membrane must also include a symport protein for glucose/Na+ co-transport, but no co-transport is taking place in this experiment, so you didn't have to include the symport protein here.) Explanation (3 pts total): This is an example of passive transport -- glucose is flowing down its gradient. This transport requires a protein as glucose cannot diffuse across a membrane by itself. (2 pts). You don't have enough information to distinguish carrier-mediated facilitated diffusion from channel-mediated facilitated diffusion. (1 pt). As far as I know, there are no channels for glucose (and carriers are quite common), but from the data given, you can't rule out a channel.
D. You should get glucose export (2 pts), using a channel or a carrier (2 pts) -- which ever mechanism you chose in part C. Explanation (4 pts total): There is no glucose/Na+ co-transport possible here, because there is no Na+ gradient. In the long run, the pump could set up a gradient, but in the short run, no co-transport is possible. (2 pts.) The results in part C indicate that glucose can cross the apical membrane by passive transport, with glucose flowing down its gradient using a carrier or channel. In the absence of co-transport, that should happen here. Glucose should exit the cells, not enter them, because the [G] is higher inside the cells than outside. (2 pts). Note: In at least some epithelia, it is thought that the apical membrane really does have two transport systems for glucose -- one active, and one passive, as described here.