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 μm2/s. The equation for diffusion is t = x2/2D, 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 multi-subunit protein translocators in the mitochondrial inner membrane, are much less abundant than those of the TOM 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?
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?
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 trans-membrane segment to a hydrophilic segment? Sketch the arrangement of the modified protein in the ER membrane.
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.
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?
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