Download C2006/F2402 `07

C2006/F2402 ’08
Review Questions for Exam #1 – Exam #1 of ‘07
1. There is a protein named emerin that is 254 amino acids long. The protein has 1 hydrophobic region 11 amino acids
away from its carboxyl end. All the rest is hydrophilic. Emerin has many sites where phosphorylation is possible, and only
one where glycosylation is possible. When cells are fractionated, emerin is in the membrane fraction. (See note at *
below.)
A. If emerin is in the part of the membrane fraction containing the plasma membrane, then:
A-1. You expect emerin to be (a peripheral protein on the cytoplasmic side) (a peripheral protein on the extracellular
side) (a single pass protein) (a multipass protein) (can’t predict).
A-2. The amino end of emerin is probably:
(in the cytoplasm) (in the membrane) (extracellular) (cytoplasmic or extracellular) (any of these – can’t predict).
A-3. If the protein is glycosylated, the sugars are probably attached to amino acids that are (near the amino end)
(near the carboxyl end) (near one end or the other) (near both ends) (not necessarily near either end).
A-4. The largest section of emerin that is in the cytoplasm should contain approximately _____________ amino acids.
Draw a labeled picture showing the major domains and features of emerin, including how it is oriented relative to the
bilayer of the membrane and the cytoplasm. Explain briefly how you got your number in A-4.
*Note (not on original exam): ‘Fractionated’ does not mean ‘freeze fractured.’ The two terms are unrelated. ‘Fractionated’
means the cells were broken open and the parts were separated into various fractions by biochemical methods.
B. Defects in emerin can cause the muscle wasting syndrome called EDMD or Emery-Dreifuss muscular dystrophy.
(That’s how the protein got the name ‘emerin.’) Defects in lamin A can also cause EDMD. Defects in other proteins do not
cause EDMD. Given this information,
B-1. The most likely cell localization for mature emerin is (the plasma membrane) (the ER or Golgi membrane)
(the inner nuclear membrane) (the outer nuclear membrane) (either nuclear membrane) (any cell membrane) (any of these).
B-2. In a normal person, emerin is probably bound to:
(MF) (MT) (IF) (IF or MF) (MF or MT) (any of these) (none of these). Explain your answers briefly.
C. Suppose you freeze fracture the membrane that normally contains emerin. In a person with EDMD, there are no
particles observable on either face of the membrane. In a normal person, would you expect to see particles?
Note (included on original exam): Only relatively large protein domains containing many amino acids form particles large
enough to see in freeze-fractured membranes.
You would expect to find particles: (primarily on the P face) (primarily on the E face)
(about equally distributed on both faces of the membrane) (on neither face) (can’t predict from information given).
Explain your answer briefly.
2. Many epithelial cells have a Na+ channel in their apical membrane. The protein that makes up the channel is called ENaC
for epithelial sodium channel. The channel is found in the epithelial cells surrounding the colon (part of the large intestine);
it is NOT found in the epithelium surrounding the small intestines and upper GI tract.
A. Na+ is absorbed in the colon. (Absorption = transport of Na+ from the lumen of the colon to the interstitial fluid.)
Assume the epithelial cells surrounding the colon have no other Na + transport protein (other than ENaC) in their apical
membrane.
A-1. Na+ absorption from the colon to the interstitial fluid should require transport of Na+ through:
(gap junctions) (adherens junctions) (desmosomes) (tight junctions) (the cytoplasm of epithelial cells)
(the endo-membrane system of epithelial cells) (none of these).
A-2. Na+ transport from the lumen to the interstitial fluid should require (primary active transport)
(secondary active transport) (channel mediated facilitated diffusion) (carrier mediated facilitated diffusion)
(simple diffusion without a protein) (transcytosis) (none of these).
For each part of A-1 & A-2, circle one or more correct answers, and explain after A-3.
A-3. Suppose the concentration of Na+ in the colon varies considerably under normal circumstances. The rate of Na +
absorption will probably be (proportional to the [Na+] at lower levels of Na+) (independent of the [Na+])
(proportional to the [Na+] at high levels of Na+) (proportional to the [Na+] at all levels of Na+).
Explain your answers to all parts of A briefly.
Question 2, cont.
B. Suppose you have an analog of ATP that binds like ATP but can not be hydrolyzed. You inject the analog into colon
epithelial cells. Assume the starting Na+ & ATP concentrations in the cells and/or the medium are normal, and you add a
large excess of the analog. (Ratio of [analog] / [ATP] is high.) After you add the analog, you measure Na+ transport.
B-1. The rate of Na+ leaving the lumen will (slow down immediately) (slow down after a while) (stay the same)
(slow down only when the cell dies) (increase immediately) (increase after a while) AND
B-2. The rate of Na+ reaching the interstitial fluid will ________________________________. (same choices).
Explain your answers briefly.
C. In the colon, the epithelial cells have a GLUT protein on the basolateral side. The cells do not have any other
glucose transport proteins.
C-1. In a normal human, you would expect this protein to transport glucose (into the cells) (out of the cells) (both
ways) (one way or the other, but can’t predict which) (any of these), AND
C-2. If you add the ATP analog described above to the cells, the rate of glucose transport should:
(increase) (decrease) (stay the same) (change direction) (stay the same at zero). Ignore any gross effects on overall energy
metabolism, and stick to effects on transport using GLUT. Explain both answers briefly.
3. You have an antibody to human ENaC. You break open cells, solubilize the membranes, and use the antibody to
precipitate the ENaC protein. You then analyze the proteins in the precipitate, and you find that it contains both ENaC and
spectrin A.
A. The simplest interpretation of these results is that:
A-1. (Antibody to ENaC binds to spectrin A) (ENaC binds to spectrin A) (both of these occur) (something else is going
on -- neither of these types of binding can explain this result).
A-2. In cells of the colon, ENaC is probably connected to (an actin cytoskeletal network) (a tubulin cytoskeletal
network) (either one) (neither) (any of these -- can’t tell from information given). Explain briefly.
B. You can alter epithelial cells (by genetic engineering) so that the cells produce protein segments fused to GFP. See
last page for a description of such an experiment. Suppose you can precipitate either of the fusion proteins (see last page)
with antibodies to ENaC. Given the results reported on the last page:
(1). You will expect to find spectrin A in the precipitate if you add antibodies to fusion protein (1) (2) (either one) (neither).
(2). Normal ENaC is probably anchored to the cytoskeleton by its (amino end) (carboxyl end) (both) (neither).
(3). In colon epithelial cells, spectrin A is probably found (throughout the cytoplasm) (in the ECM)
(on the apical side of the plasma membrane) (on the basolateral side of the plasma membrane)
(all around the plasma membrane) (can’t predict).
Explain how your answers follow from the results.
C. To detect the proteins in the precipitate you have four different types of antibodies. You have unlabeled antibodies
to ENaC and to spectrin A. You also have labeled secondary antibodies. The labeled antibodies are either goat anti-rabbit
(for detecting ENaC) or goat anti-mouse (for detecting spectrin A). I am not making this up – it comes from an actual
paper.
C-1. Two different labeled antibodies are needed here because the two unlabeled antibodies are
(directed against different proteins) (made in different species) (both) (neither). No explanation required.
C-2. Suppose you actually use the antibodies described above to analyze the precipitate. (See top of page for how you got
the precipitate.) You dissolve the precipitate, and separate the proteins by SDS gel electrophoresis. Then you use the
antibodies to visualize where the proteins are in the gel (really a blot of the gel). Suppose you add the unlabeled antibody to
ENaC to the gel, wash off unattached antibody, and add the labeled goat anti-mouse antibody.
(1). How many labeled bands will you see on the gel? (0) (1) (2) (>2) (can’t predict).
(2). Suppose you repeat the experiment, but use unlabeled antibody to spectrin A instead. All other conditions are the same.
How many labeled bands will you see on the gel? (0) (1) (2) (>2) (can’t predict).
Explain your answers to C-2.
4. The epithelial sodium channel, ENaC, is found in the epithelia of the kidney, as well as in the colon. In the kidney, ENaC
is targeted to lysosomes by addition of a chemical group (we’ll call it Q) to the side chains of certain lysines in ENaC. The
enzyme Nedd4 catalyzes the addition of Q. In normal kidney epithelium, under ordinary conditions, ENaC is sent to
lysosomes at a steady rate, but the amount of channel protein on the surface remains constant. When the need arises,
regulatory factors change the amount of ENaC on the surface (& the rate of Na + transport) by altering the rates of
production, removal, etc.
A-1. Inhibition of Nedd4 could be used for: (up regulation of Na+ transport) (down regulation of Na+ transport)
(both) (neither).
A-2. Endocytosis of ENaC is probably triggered by (binding of ENaC to its receptor) (binding of ENaC to its ligand)
(covalent modification of ENaC) (binding of ENaC to its receptor or ligand) (any of these).
A-3. If Nedd4 activity increases, you would expect to find that the ENaC in the cell:
(increases) (decreases) (relocates, but total stays the same) (stays the same in the same location).
A-4. In a normal cell, the amount of ENaC on the surface probably remains constant because:
(ENaC is recycled by exocytosis) (new ENaC is constantly synthesized) (ENaC protein is anchored to the cytoskeleton)
(Lateral diffusion of ENaC is blocked by tight junctions) (any of these).
Explain how the amount of ENaC on the surface remains constant, including the role (if any) of Nedd4.
B. Suppose the kidney cells have receptors for LDL, transferrin, & EGF. You isolate several kinds of vesicles from the
cells, and suppose the vesicles carry ENaC.
B-1. If you isolate uncoated endocytic vesicles that carry ENaC, the vesicles could also contain
(LDL receptors) (transferrin receptors) (EGF receptors) (none of these) (all of these), AND
B-2. If you isolate vesicles that have budded off from endosomes and carry ENaC, the vesicles could also contain
____________________________________________________________. (Same choices).
Pick one or more choices for each part, and explain briefly in a sentence or two.
C. In people with the genetic condition called Liddle’s syndrome, there is too much Na+ transport. Each individual channel
transports Na+ normally, but there is more overall transport than normal. (This causes high blood pressure.) People with
Liddle’s syndrome have ENaC that is missing the last 45-60 amino acids.
C-1. The most likely cause of the symptoms in Liddle’s syndrome is: (too many channels) (not enough total channels)
(channels that fail to reach the apical surface) (something else – number of channels in apical membrane wouldn’t affect
Na+ transport).
C-2. The primary cause of Liddle’s syndrome is probably: (a high rate of ENaC synthesis) (a low rate of ENaC synthesis)
(improper folding of ENaC) (a high rate of ENaC endocytosis) (a slow rate of ENaC endocytosis)
(an unusual rate of synthesis or rate of endocytosis) (any of these ), AND
C-3. The section with the last 45-60 amino acids probably contains a site for (binding of ENaC ligand)
(binding of ENaC receptor) (enzymatic modification of ENaC) (binding of ENaC ligand or receptor) (transport of Na+).
C-4. What result would you expect from changing the activity of Nedd4? (It should improve the symptoms.) (It should
make the symptoms worse.) (It should have no effect on the symptoms.) (Effect on symptoms depends on whether you
increase or decrease the activity of Nedd4.) (Can’t predict). Explain all your answers, and how they fit together.
________________________________________________________
Experiment for Problem 3B:
You have cells that produce one of the following 2 fusion proteins. Each protein contains a 50 amino acid section of ENaC
fused to GFP. Both fusion proteins are soluble, and are made in the cytoplasm. Normal ENaC has both ends in the
cytoplasm.
Fusion protein 1 = First 50 AA of ENaC fused to GFP. (Only first 50 AA of ENaC are included.)
Fusion protein 2 = Last 50 AA of ENaC fused to GFP. (Only last 50 AA ENaC are included.)
In cells making fusion protein 1, there is diffuse fluorescence all over the cytoplasm.
In cells making fusion protein 2, you ‘light up’ the apical portion of the cells. (That is you detect intense fluorescence in the
apical portion.)