Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

CHAPTER 12 END-OF-CHAPTER PROBLEMS
693
(A)
(B)
solution
gel
30 s
10 min
complex. They were initially identified using a genetic
trick. The yeast Ura3 gene, whose product is an enzyme
that is normally located in the cytosol where it is essential
for synthesis of uracil, was modified so that the protein
carried an import signal for the mitochondrial matrix.
A population of cells carrying the modified Ura3 gene in
place of the normal gene was then grown in the absence
of uracil. Most cells died, but the rare cells that grew were
shown to be defective for mitochondrial import. Explain
how this selection identifies cells with defects in components
required for import into the mitochondrial matrix.
Why don’t normal cells with the modified Ura3 gene grow
in the absence of uracil? Why do cells that are defective for
mitochondrial import grow in the absence of uracil?
MBP-mCherry
30 min
30 s
10 min
12–11 If the enzyme dihydrofolate reductase (DHFR),
which is normally located in the cytosol, is engineered to
carry a mitochondrial targeting sequence at its N-terminus,
it is efficiently imported into mitochondria. If the modified
DHFR is first incubated with methotrexate, which binds
tightly to the active site, the enzyme remains in the cytosol.
How do you suppose that the binding of methotrexate
interferes with mitochondrial import?
importin-MBP-GFP
30 min
Figure Q12–2 FG-repeat gel and influx of proteins into the nucleus
(Problem 12–9). (A) Cartoon of the meshwork (gel) formed by pairwise
interactions between hydrophobic FG repeats. For FG-repeats
separated by 17 amino acids, as is typical, the mesh formed by
extended amino acid side chains would correspond to about 4 nm on
a side, which would be large enough to account for the characteristic
passive diffusion of proteins through nuclear pores. (B) Diffusion of
MBP-mCherry and importin-MBP-GFP into a gel of FG-repeats. In each
group, the solution is shown at left and the gel at right. The bright areas
indicate regions that contain the fluorescent proteins.
Problems p12.201/12.04
A. FG-repeats only form gels in vitro at relatively high
concentration (50 mM). Is this concentration reasonable
for FG repeats in the NPC core? In yeast, there are about
5000 FG-repeats in each NPC. Given the dimensions of the
yeast nuclear pore (35 nm diameter and 30 nm length),
calculate the concentration of FG-repeats in the cylindrical
volume of the pore. Is this concentration comparable
to the one used in vitro?
B. A second question is whether the diffusion of
importin-MBP-GFP through the FG-repeat gel is fast
enough to account for the efficient flow of materials
between the nucleus and cytosol. From experiments of
the type shown in Figure Q12–2B, the diffusion coefficient
(D) of importin-MBP-GFP through the FG-repeat gel was
determined to be about 0.1 μm 2 /s. The equation for diffusion
is t = x 2 /2D, where t is time and x is distance. Calculate
the time it would take importin-MBP-GFP to diffuse
through a yeast nuclear pore (30 nm) if the pore consisted
of a gel of FG-repeats. Does this time seem fast enough for
the needs of a eukaryotic cell?
12–10 Components of the TIM complexes, the multisubunit
protein translocators in the mitochondrial inner
membrane, are much less abundant than those of the TOM
12–12 Why do mitochondria need a special translocator
to import proteins across the outer membrane, when the
membrane already has large pores formed by porins?
12–13 Examine the multipass transmembrane protein
shown in Figure Q12–3. What would you predict would
be the effect of converting the first hydrophobic transmembrane
segment to a hydrophilic segment? Sketch the
arrangement of the modified protein in the ER membrane.
NH 2
1 3 5
2 4 6
COOH
CYTOSOL
ER LUMEN
Figure Q12–3 Arrangement of a multipass transmembrane protein
in the ER membrane (Problem 12–13). Blue hexagons represent
covalently attached oligosaccharides. The positions of positively and
negatively charged amino acids flanking the second transmembrane
segment are shown. Problems p12.24/12.19
12–14 All new phospholipids are added to the cytosolic
leaflet of the ER membrane, yet the ER membrane has a
symmetrical distribution of different phospholipids in its
two leaflets. By contrast, the plasma membrane, which
receives all its membrane components ultimately from the
ER, has a very asymmetrical distribution of phospholipids
in the two leaflets of its lipid bilayer. How is the symmetry
generated in the ER membrane, and how is the asymmetry
generated and maintained in the plasma membrane?