C2006/F2402 ’02 -- Key to Review Questions for Exam #1

1. A. Part of the rough ER; B. Transmembrane or extracellular.  
: These proteins are being followed from the Golgi. To reach the Golgi, they must be made on ribosomes attached to the ER and must enter the lumen of the ER as they are made. All these proteins must have signal peptides to enter the ER, but the proteins could be transmembrane proteins that lodge in the ER membrane or soluble proteins that enter the ER lumen completely. In either case, vesicles budding off the ER could ferry the proteins (either in the lumen or membrane of the vesicle) to the Golgi.

2. A. In the lumen of the intestine; B. a signal peptide on the amino end; C. 0 transmembrane sequences in the mature protein.  
Explanation: A. Since the protein is soluble, it will be released to the outside of the cell when the vesicle fuses with the plasma membrane. As the apical surface faces the lumen = inside of the intestine, the protein will be released into the lumen. (Note: Any enclosed space can be called a lumen – for example, the lumen of the intestine or the lumen of the ER.)
    B & C. Any protein that reaches the Golgi must have a signal peptide to enter the ER (see above). If the protein is entirely within the lumen, then the signal peptide must be removed and there must be no other transmembrane segments – otherwise, the protein would remain attached to the membrane. An internal signal peptide is not likely to be removed – it is expected to remain as an anchor to hold the protein in the membrane.

3. A. On the carboxyl end; B. In the cytoplasm; C. no transmembrane sequences or SP. 
: If the amino end of protein P is replaced with GFP, the recombinant protein doesn’t reach the Golgi. The most likely explanation is that the amino end of protein P contains the signal peptide, and GFP has no signal peptide of its own. (We known from question 2 that the SP of protein P is on the amino terminal end, and the data here fit with that.) So if the SP is replaced with GFP, the ribosomes making the recombinant protein never get attached to the ER. There is no SP to be recognized by the SRP, so the ribosomes are never carried to the ER, and the protein is completed in the cytoplasm. On the other hand, if GFP replaces the carboxyl end of normal protein P, the fusion protein still has an SP that is recognized by the SRP. So the ribosomes making the recombinant protein get attached to the ER, the protein enters the ER, the SP is removed and the recombinant protein moves on to the Golgi and enters vesicles targeted to the apical surface (just like for normal protein P). If GFP had any transmembrane seqments, the fusion protein would end up in the membrane of the carrier vesicles, not entirely in the lumen. If GFP had an SP, then the SP might also act as a transmembrane protein when the GFP is on the carboxyl end. 

4. A.  Facing the lumen. .(This question was included so we could know how to grade the rest of the question if you reversed the apical and basolateral surfaces.)
    B.  (1) Apical protein: Most obvious possibility: Glucose/Na+ co-transporter or symporter – uses Na
+ gradient to provide energy to carry glucose from the lumen into the cell against a glucose gradient. Needed to utilize glucose that you eat. An example of secondary active transport.
         (2) Basolateral Protein: Two obvious possibilities (not the only ones): Na+/K+ pump, glucose transporter (GLUT).
    Pump moves Na+ out of the cell so there is always a downhill Na+ gradient from the inside of the lumen (outside of apical surface) to the inside of the cell. The Na+ gradient is needed for transport of glucose and other nutrients into the cell from the lumen. (More details on pump below.)
GLUT is a permease or carrier for facilitated diffusion – allows movement of glucose across the membrane down its gradient. Needed to get glucose out of the cell and into the interstitial fluid for transport to rest of body.
    Note: GLUT does not facilitate transport into the blood directly. That occurs in a separate step. Glucose diffuses out of the interstitial fluid into the capillaries through spaces between the endothelial cells lining the capillaries. No carrier protein is required for entry into (or exit from) capillaries.

   C. K+ should enter the cell using an antiporter.  The protein is called the Na+/K+ pump. It moves Na+ out  and K+ in. The pump uses splitting of ATP to provide energy to move the ions; this is an example of primary active transport. 

    D-1. Secondary active transport; facilitated diffusion. No explanation needed.

    D-2. If the reverse Na+ gradient is set up,  and all processes then work normally, Na+ should enter across neither surface; glucose should exit across both surfaces (in the short run); glucose should exit across the apical membrane only (in the longer run). 
   What happens to Na+? It will flow out across both membranes as follows: Once the reverse gradient is set up, if nothing else is altered, Na+ will continue to be pumped out the BL surface by the Na+/K+ pump. Na+ will flow out the apical surface, down its gradient, using the Na+/glucose co-transporter. The Na+ will carry glucose with it – the transporter will operate in the reverse of its usual direction -- it will carry glucose and Na+ "out" instead of "in." This is possible because it is the binding of Na
+ that makes the protein bind glucose and the loss of Na+ that makes the protein release glucose. In normal cells, the Na+ concentration is always higher on the outside, so Na+ and glucose flow in only one direction -- into the cell.  However, if Na+ is higher on the inside, Na+ will bind on the inside and fall off on the outside of the cell , carrying glucose with it to the outside of the cell.
    What happens to glucose?
It will be transported out across the apical surface with Na+ as explained above. In the short run, it will continue to flow out, down its gradient, across the BL surface using the GLUT carrier protein. Once enough glucose leaves the cell, across both membranes, the level of glucose inside the cell will reach a level lower than the level in the interstitial fluid. Then glucose will flow into the cell across the BL membrane, using the GLUT carrier protein. GLUT acts as a permease, promoting passage of glucose down its gradient – transport can occur in either direction. When you have eaten recently, the glucose level inside the cell is relatively high and the gradient favors transport out of the cell. However if the glucose level in the cell is very low, as in this case, or because you have not eaten for a long time, then the gradient will favor flow of glucose into the cell.

5. Light Microscopy Explanation: They are looking at fluorescence, which results from the emission of visible light. Therefore they must be using light microscopy. The items they are looking at are very small, and below the resolution of the light microscope, but you can see them because you are looking at emission from a point source of light, not transmission of light from outside the sample. You can also deduce that they must be using light microscopy, not any form of electron microscopy, because they are looking at a process in living cells.

6. A. None of these; B. RME; C. Both.  
: Endocytic traffic = vesicles involved in endocytosis. Small molecules such as glucose and free ions do not generally enter cells by endocytosis. Macromolecules or large complexes, such as proteins or LDL (lipid-protein complex) enter by RME = receptor mediated endocytosis. (Iron enters by RME, but it does so complexed to the protein transferrin.)
    One possible transmembrane protein needed – a receptor to bind the ligand.
   A possible peripheral protein – clathrin; needed to help form coated pits and internalize the receptor + ligand in a coated vesicle. A coat protein such as clathrin is required to allow membrane to curve and form vesicle.

7. Gap junction; down its gradient.  
: Gap junctions are made up of proteins that form channel-like structures (made of connexons) connecting neighboring cells. Ions and small molecules such as glucose can pass through the hydrophylic spaces inside the tubes by simple diffusion. No active transport is involved. The other types of junctions mentioned serve to join cells together but do not provide any structure that allows transport between the cells. (Glucose is bigger than water or an ion, but is considered a small molecule relative to macromolecules such as proteins, polysaccharides, etc.)

8. A. MT, dynein . Explanation: Kinesin and dynein work in partnership with structures made of tubulin, that is, microtubules. Therefore the process involves MTs. Kinesin and dynein are "motor molecules" that attach to fibers, vesicles, or particles and move the attached materials along MTs. The energy to move the motor molecules and their attached cargo along the MTs is derived from hydrolyzing ATP. Kinesin and dynein carry their cargoes in opposite directions relative to the MTs, so if kinesin moves things "out" then dynein will move them "in." 
    B. No.
  Kinesin is a soluble protein found in the cytoplasm, not inside the endomembrane system.  Only proteins (or their domains) that extend into the lumen of the ER & Golgi can be modified by attachment of carbohydrates. This is because the enzymes for N- & O-glycosylation of proteins are inside the ER & Golgi.   Proteins like kinesin that remain in the soluble cytoplasm are not exposed to the enzymes of glycosylation. (It is not correct that only extracellular proteins are glycosylated – the proteins of the endomembrane system itself, such as lysosomal enzymes & the enzymes in the ER & Golgi, are also glycosylated.)

9. A Hormone; B. & C. Both; D at low pH (inside endosomes); E. Coated vesicles.  

F. For receptor: plasma membrane --> coated vesicles --> endosomes --> exocytotic vesicles --> back to plasma membrane .

    For hormone: plasma membrane --> coated vesicles --> endosomes --> lysosomes.

Explanation (not required): In this case, the hormone and its receptor must separate in the endosomes, which have a lower pH than coated vesicles and the outside of the cell. Then the hormone must be transported to lysosomes, where it is degraded, while the receptor is recycled back to the plasma membrane.