NPC Transport

Question 1

Nucleocytoplasmic transport requires

Answer 1

Energy
Protein receptors
Unique targeting signals

Question 2

Nuclear Localization Signals

Answer 2

Amino acid sequence that is both necessary and sufficient for cytoplasm to nuclear targeting

Recognized by nuclear receptor proteins

Question 3

Classic NLS

Answer 3

Most common
Consists of a short stretch of positively charged (basic) amino acid residues

KKQRKKx

X= S/T/Y (can be phosphorylated)

Question 4

Bipartite NLS

Answer 4

Composed of 2 short stretches of basic amino acids and a 7-19 amino acid long “spacer” sequence
-2 sequences with a linker

Question 5

Transport Receptors

Answer 5

Mobile protein responsible for moving “ferrying” protein “cargo” across the nuclear envelope

Question 6

Karyoferins

Answer 6

Large family of receptor proteins (transport receptors) responsible for moving macromolecules either into nucleus (importins) or out of nucleus (exportins)

Question 7

ARC1

Answer 7

Shuttle between cytoplasm and nucleus

Posses a classic NLS (aa 261-266)

Question 8

Mutation of ARC 1 NLS

Answer 8

Mutation of residues 261-266 results in in myc-tagged ARC1 being mislocalized exclusively to the cytoplasm

Myc- short polypeptide derived from the c-myc oncogene protein
- Acts as a tag for immunodetection

Question 9

Addition of ARC1 NLS

Answer 9

Fusion of residues 261-266 in ARC1 to the cytoplasmic protein CAT results in the fusion protein (CAT-NLS) being redirected to the nucleus

Question 10

Ran-GAP1

Answer 10

Hydrolyzes GTP to GDP on Ran-GTP in cytoplasm

Question 11

RCC1

Answer 11

Converts Ran GDP to Ran-GTP in nucleus

Question 12

NES

Answer 12

LxxLxxL

X= any amino acid

Question 13

Nucelocytoplasmic Transport of ARC1:

Answer 13

Before pollination- localized in nucleus

During self pollination

• NLS is disrupted due to phosphorylation of adjacent amino acid residues
• NES> NLS(p)- localized in cytoplasm
  
  • Functions in the ubiquitin-mediated turnover of other proteins, leading to pollen rejection (no fertilization)
  • Phosphorylation of NLS results in ARC1 being mislocalized to the cytoplasm
  Mutation of NES results in ARC1 being mislocalized to the nucleus

Question 14

Cell cycle checkpoints

Answer 14

Mid G1 (Cell commits to DNA replication and organelle duplication in S phase)

End of G2- Cell commits to enter Mitosis

End of metaphase- cell commits to chromosome segregation

Question 15

Cyclins

Answer 15

Transition through checkpoints is controlled by cyclins and CDKs

CDKs phosphorylate target nuclear proteins when bound to cyclin

Concentration varies in a cyclic fashion during cell cycle (synthesized and degraded during each cell cycle)

Question 16

Maturation Promoting Factor (MPF)

Answer 16

□ Heterodimeric protein consisting of a kinase and regulatory cyclin subunit

Critical for G2/M transition during cell cycle

Question 17

Phosphorylation of histones and condensins

Answer 17

Phosphorylation leads to chromatin packing and chromosome condensation

Question 18

Phosphorylation of lamins

Answer 18

Phosphorylation leads to disassembly of the nuclear lamina

Question 19

Phosphorylation of nups

Answer 19

Phosphorylation leads to disassembly of NPC

Question 20

Open Mitosis

Answer 20

Dynamic process in higher eukaryotes

   - Lower eukaryotes exhibit "closed mitosis"
           - Nuclear envelope remains intact

Entire nucleus disassembles by metaphase
-Nuclear outer and inner membranes fragment, lamina and NPCs disassemble, all soluble nuclear proteins are released into cytoplasm

2 daughter nuclei reassemble during telophase (after chromosomal division)

Low [cyclin] = low CDK activity = dephosphorylation of nups and lamins= reformation of nuclear lamina, envelope, and NPC re-import of soluble NLS-proteins from the cytoplasm

Question 21

Binding of Different Cyclins

Answer 21

Different cyclins can bind to the same CDK (Cyclins cause a conformational change that activates the CDK)

Question 22

Cdc2

Answer 22

CDK that controls the transition from G1 to S (START) and G2 to M phase by binding to different cyclins

End of G1
-High [G1 cyclins] = High cdc2 activity= cell enters S phase

End of G2
-High [mitotic cyclins] = high cdc2 activity = cell enters M phase

Question 23

Cyclin Oscillation

Answer 23

Oscillation in different cyclin concentration during the cell cycle leads to changes in CDK activity

Oscillation due to rates of protein synthesis and degradation at different points of the cell cycle

Decrease in [cyclin] after the start of S and M phase due to decreased synthesis of new cyclin proteins and degradation of pre-existing cyclin proteins

Question 24

CDK Inactivation

Answer 24

CDKs themselves are inactivated by phosphorylation from CDK kinases

Pre-existing cyclins also prevented from targeting to the nucleus
-Can’t activate CDKs in nucleus

Question 25

Localization of Mitotic Cyclin B1 in Mammals:

Answer 25

Cyclins normally shuttle between nucleus and cytoplasm

Mitotic Cyclin B1

Up to and during G2

 - Cyclin B1 shuttles between nucleus and cytoplasm
 - Localizes primarily in cytoplasm 
 - NES > NLS

End of G2

  - NES in cyclin B1 is phosphorylated
   - NLS > NES
   - Cyclin B1 localized to nucleus

Nuclear localized cyclin B1 activates CDK2s required for transition from G2 to M phase