Protein Transport & Nuclear Import

By what mechanisms can proteins enter membrane-enclosed organelles and identify the organelles that use them?

What membrane-bound organelles can receive proteins directly from the cytosol?

What is the fate of proteins that lack a sorting signal?

What organelle serves as an entry point for proteins destined for all organelles or for the cell surface?

Describe the structure of the nuclear envelope.

What is the difference in conformation adopted by proteins during nuclear transport and that of proteins transported into mitochondria and chloroplasts?

Why is the nuclear localization sequence not cleaved off?

How do nuclear pores restrict the passage of larger molecules while allowing small, water-soluble molecules to pass freely between the nucleus and cytosol?

How do nuclear import receptors escort proteins bearing a nuclear localization signal from the cytosol into the nucleus?

How is the energy supplied by GTP used to drive nuclear transport?

Explain how the Ran protein and GTP catalyze the transport of materials into and out of the nucleus.

Explain how the gradient of RAN-GTP/RAN-GDP maintains directionality of import.

What would happen to a protein that bears both a nuclear localization signal and a nuclear export signal?

What would happen to a protein that bears both an ER sorting signal and a nuclear localization signal?

X = Proteins being imported/exported/phenomenon

1. What would happen to localization of a nuclear protein if an NLS/NES is mutated?

X = Proteins being imported/exported/phenomenon

1. What would happen to if a import/export receptor is mutated? Or a drug inhibiting import is used?

X = Proteins being imported/exported/phenomenon

1. What would happen to X if Ran-GTPase cycle was messed with.

Explain how mitochondrial proteins are recognized and transported into the mitochondrial matrix, and describe the role played by chaperone proteins inside the organelle.

What is the difference between free ribosomes and membrane-bound ribosomes?

What is the source of energy for the transport of proteins into the ER?

Compare a polyribosome and the rough ER.

What is the difference between post-translational and co-translational transmembrane transport into the ER?

How is post-translational used in the import of proteins into the ER?

How is co-translational transmembrane used in the import of proteins into the ER?

What are the important components for the import machinery into the ER?

What are the function of the important components of import machinery into the ER?

How is translation halted to allow threading of the protein into the translocation channel? How are proteins released into the ER lumen after the process is complete?

What are “stop transfer” and “start transfer” sequences and how do they lead to insertion of proteins into the ER membrane?

List the types of covalent modifications that take place in the ER and describe the functions these modifications serve.

How are proteins selected for glycosylation in the ER and how these sugars are attached to the protein?

How are proteins that are destined to function in the ER are kept in the ER or returned to the ER if they accidentally enter the Golgi apparatus?

How are misfolded or not fully assembled proteins retained in the ER?

What is the unfolded protein response? What are the consequences of triggering this?

What are the major stages of vesicular transport?

What is the difference between COPII, COPI, and clathrin transport routes?

How do coat proteins contribute to cargo selection and budding?

What is the mechanism by which vesicles dock and fuse with the correct target membranes? List the proteins involved in each.

What is the importance of maintaining specificity in both docking and fusion of vesicles at their target compartments and how Rabs, tethers, and SNAREs facilitate this process?

How are escaped ER resident proteins recaptured and sent back to ER?  What type of signal is used in this process?

Name or provide examples of proteins that would be transported regularly by both COPI- and COPII-coated vesicles?

What methods are used to determine whether a labelled protein bearing a particular signal sequence is transported into a preparation of isolated organelles?

Outline biochemistry and cell free assays based on sensitivity to cleavage by endoglycosidase.

Outline how temperature-sensitive mutants have been used to dissect the protein secretory pathway in yeast.

Outline microscopy and biochemistry-based assays for following the trafficking of proteins through the secretory pathway

Contrast the endocytic and exocytic pathways in terms of directionality, purpose, and participation of certain organelles and membranes.

Compare how proteins are sorted in the cis and trans Golgi networks.

How and where are additional oligosaccharides added to the glycoproteins within the Golgi stack? What are the advantages of glycosylation?

Contrast the constitutive and regulated exocytosis pathways, and describe the behavior of the proteins secreted by each pathway.

What are the three pathways to the lysosome?

What general kind of chemical reactions occur in the lysosome, and how and why is it maintained at a low pH?

Describe how cholesterol is transported into the cell cytosol by receptor-mediated endocytosis.

Outline the possible fates of receptor proteins and cargo molecules following endocytosis into endosomes.

What happens to the final products of macromolecule digestion by lysosomes?