1. The following results were obtained in early studies on the translation of secretory proteins. Based on what we now know of this process, explain the reason why each result was observed.

2. Describe the source or sources of energy needed for unidirectional translocation across the membrane in (a) cotranslational translocation into the endoplasmic reticulum (ER); (b) post-translational translocation into the ER; (c) translocation into the mitochondrial matrix.

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3. Translocation into most organelles usually requires the activity of one or more cytosolic proteins. Describe the basic functions of three different cytosolic factors required for translocation into the ER, mitochondria, and peroxisomes, respectively.

4. Describe the typical principles used to identify topogenic sequences within proteins and how these principles can be used to develop computer algorithms. How does the identification of topogenic sequences lead to prediction of the membrane arrangement of a multipass protein? What is the importance of the arrangement of positive charges relative to the membrane orientation of a signal-anchor sequence?

5. An abundance of misfolded proteins in the ER can result in the activation of the unfolded-protein response (UPR) and ER-associated degradation (ERAD) pathways. UPR decreases the abundance of unfolded proteins by altering gene expression of what type of genes? What is one manner in which ERAD may identify misfolded proteins? Why is dislocation of these misfolded proteins to the cytoplasm necessary?

6. Temperature-sensitive yeast mutants have been isolated that block each of the enzymatic steps in the synthesis of the dolichol-linked oligosaccharide precursor for N-linked glycosylation. Propose an explanation for why mutations that block synthesis of the intermediate with the structure dolichol-PP-(GlcNAc)₂Man₅ completely prevent addition of N-linked oligosaccharide chains to secretory proteins, whereas mutations that block conversion of this intermediate into the completed precursor—dolichol-PP-(GlcNAc)₂Man₉Glc₃—allow the addition of N-linked oligosaccharide chains to secretory glycoproteins.

7. Name four different proteins that facilitate the modification or folding of secretory proteins within the lumen of the ER. Indicate which of these proteins covalently modifies substrate proteins and which brings about only conformational changes in substrate proteins.

8. Describe what would happen to the precursor of a mitochondrial matrix protein in the following types of mitochondrial mutants: (a) a mutation in the Tom22 signal receptor; (b) a mutation in the Tom70 signal receptor; (c) a mutation in the matrix Hsp70; and (d) a mutation in the matrix signal peptidase.

9. Describe the similarities and differences between the mechanism of import into the mitochondrial matrix and the chloroplast stroma.

10. Design a set of experiments using chimeric proteins, composed of a mitochondrial precursor protein fused to dihydrofolate reductase (DHFR), that could be used to determine how much of the precursor protein must protrude into the mitochondrial matrix in order for the matrix-targeting sequence to be cleaved by the matrix-processing protease.

11. Peroxisomes contain enzymes that use molecular oxygen to oxidize various substrates, but in the process, hydrogen peroxide—a compound that can damage DNA and proteins—is formed. What is the name of the enzyme responsible for the breakdown of hydrogen peroxide to water? What is the mechanism of the import of this protein into the peroxisome, and what other proteins are involved?

12. Suppose that you have identified a new mutant cell line that lacks functional peroxisomes. Describe how you could determine experimentally whether the mutant is primarily defective for insertion/assembly of peroxisomal membrane proteins or matrix proteins.

13. The nuclear import of proteins larger than 40 kDa requires the presence of what amino acid sequence? Describe the mechanism of nuclear import. How are nuclear transport receptors able to get through the nuclear pore complex?

14. Why is localization of Ran-GAP in the nucleus and Ran-GEF in the cytoplasm necessary for unidirectional transport of cargo proteins containing an NES?