Microbiology 7: Nuclear structure

Question 1

What are key features of a nuclear localisation signal (NLS)?

Answer 1

Must be exposed on the protein surface

Basic - Amino group with positive charge

Question 2

What would be a good approach to examine the role of the NLS in nuclear import of a specific protein?

Answer 2

Mutating one of more key residues of the NLS

Question 3

What experiment would you use to determine the effect of the NLS mutation on nuclear import of the protein?

Answer 3

Fluorescence microscopy

Question 4

Describe the function + structure of importin proteins

Answer 4

Soluble import receptors -> bind to the NLS

A range of importins bind to different NLS sequences

• Importin alpha binds classic NLS + importin beta
• Other face of the importin (importin beta) binds to FG repeats on the nuclear pore complex

Question 5

Function and properties of Ran protein / Ran cycle? Facilitating proteins?

Answer 5

Ran small GTPase
- in rho, ras family

GEF protein (Guanine Nucleotide exchange factor) facilitates exchange of GDP for GTP -> switches Ran on

GAP (GTPase activating protein) activates Ran GTPase activity -> switches Ran off

Question 6

How does Ran allow importins/exportins to sense they are inside or outside of the nucleus?

Answer 6

Ran-GAP exists in cytosol

Ran-GEF exists in nucleus bound to chromatin

Question 7

How do Ran complexes interact with importins? How do they affect nuclear import?

Answer 7

Ran-GTP binds to importins, changing their conformation to allow cargo release (predominates NUCLEUS)

Ran-GDP dissociates from importins, changing their conformation to cause cargo binding (predominates CYTOSOL)

Question 8

Differences of exportins to importins?

Answer 8

Transported proteins carry Nuclear export signal (NES)

• CRM1 protein binds to leucine rich NES

Major difference -> Ran-GTP promotes BINDING rather than cargo release (Ran opposite function)

Question 9

Examples of molecules undergoing nuclear import and export?

Answer 9

Import

• DNA/RNA polymerases
• Transcription Factors
• Histones, nuclear lamines (structural)
• Chromatin Modifying enzymes

Export

• mRNA and tRNA
• partly-assembled ribosomal subunits
• shuttle proteins
• proteins that must be excluded from nucleas at certain times e.g. cyclin B1

Question 10

Mechanisms of control of nuclear import/export

Answer 10

Masking NLS/NES

• binding inhibitory proteins
• post-translational modification

Tethering in cytoplasm/nucleoplasm

Question 11

Example of masking of NLS/NES?

Answer 11

Binding of IKappaB to NF-KappaB -> covers NLS

-> IKappaB degraded upon stimulation