3 - nucleocytoplasmic transport

Question 1

what is nucleocytoplasmic transport?

Answer 1

• nucleocytoplasmic transport via the NPC is one of the most congested bidirectional trafficking pathways in the cell
• includes cytoplasm to nucleus import and nucleus to cytoplasm export pathways
• various cargo
• all proteins involved with the genome must be imported into the nucleus from the cytoplasm
• all RNA, partially assembled ribosomes, and some proteins must be exported out of the nucleus into the cytoplasm
• the molecular mechanism is well understood
• • requires energy, specific protein receptors, and unique targeting signals

Question 2

what is cytoplasm to nuclear transport?

Answer 2

• most nuclear imported proteins have a nuclear localization signal
• • it is a specific sequence of amino acids that are recognized by nuclear receptor proteins
• • • mediates the targeting of the protein from the cytoplasm to the nucleus
• • • there are different types of NLSs
• characterization of different NLSs led to the identification of factors necessary for the nuclear import of proteins from the cytoplasm
• • transport receptors: mobile proteins that move protein cargo across the nuclear envelope
• • karyopherins: large family of receptor proteins responsible for moving macromolecules either into the nucleus or out of the nucleus
• there are five main steps in protein import into the nucleus

Question 3

what is the 1st step of cytoplasm to nucleus transport?

Answer 3

1. in the cytoplasm, nascent/newly synthesized cargo protein with NLS is recognized by importin
   - - importin is heterodimeric
   - - - has two distinct subunits: importin a and b
   - - importin a recognizes and binds to the basic residues in the cargo protein’s NLS

Question 4

what is the 2nd step of cytoplasm to nucleus transport?

Answer 4

2. cargo protein importin complex moves towards the nucleus via importin’s ability to bind to the cytoskeleton
   - - cytoskeleton is a highway for intracellular transport
   - - at the surface of the nucleus, importin b of the cargo protein/importin complex binds to a cytoplasmic filament at the NPC

Question 5

what is the 3rd step of cytoplasm to nucleus transport?

Answer 5

3. protein/receptor complex is translocated through the NPC
   - - current understanding of how this happens: cargo/receptor complex successively interacts with hydrophilic and FG domains of FG nups in central channel
   - - - interactions untangle the FG domain network and allow the complex to move through the channel

Question 6

what is the 4th step of cytoplasm to nucleus transport?

Answer 6

4. cargo/importin complex associates with the nuclear basket on the inner surface of the NPC
   - - complex binds to ran-GTP via importin b which makes it release from the NPC and disassemble in the nucleoplasm
   - - - import is accomplished

Question 7

what is the 5th step of cytoplasm to nucleus transport?

Answer 7

5. importin b with ran-GTP goes back to the cytoplasm due to the ran-GTP gradient
   - - since ran-GTP is high in the nucleus, the ran/importin complex want to go to an aera of low concentration so it goes to the cytoplasm where there is less ran-GTP
   - - once in the cytoplasm, GAP hydrolyzes the GTP and the converted ran-GDP releases from importin b
   - - - can be used for another round of nuclear protein import
   - - ran-GDP that was released goes back to the nucleus due to the gradient
   - - - ran-GDP is high in the cytoplasm and low in the nucleus
   - - once in the nucleus, ran-GDP is converted into ran-GTP by GEF

Question 8

what is the 1st step of nucleus to cytoplasm transport?

Answer 8

1. importin a binds to exportin
   - - exportin is a karyopherin that mediates nucleus to cytoplasm transport
   - - release of cargo protein in the nucleus exposes a nuclear export signal (NES) in importin a
   - - exportin also binds to other cargo proteins that need to be exported to the cytoplasm via their NESs

Question 9

what is an NES?

Answer 9

• NES: specific sequence of amino acids that are recognized by exportin and help target the protein to go to the cytoplasm
• different types of NESs, all necessary and sufficient
• most common is a leucine rich motif
• • LxxLxxL

Question 10

what is the 2nd step of nucleus to cytoplasm transport?

Answer 10

2. importin a or cargo with NES bound to exportin also binds to ran-GTP as there is lots of it in the nucleus
   - - ran-GTP helps with the stable assembly of the importin a/exportin complex

Question 11

what is the 3rd step of nucleus to cytoplasm transport?

Answer 11

3. importin a/exportin/ran-GTP complex follows the ran-GTP gradient through the NPC and into the cytoplasm

Question 12

what is the 4th step of nucleus to cytoplasm transport?

Answer 12

4. in the cytoplasm, GTP is hydrolyzed to GDP by GAP and the converted ran-GDP is released from exportin
   - - importin a or the cargo protein with an NES is also released
   - - - importin a can be used for another round of import

Question 13

what is the 5th step of nucleus to cytoplasm transport?

Answer 13

5. ran-GDP goes back to the nucleus due to the gradient and is converted by GEF into ran-GTP
   - - exportin also goes back into the nucleus via importin for another round of export

Question 14

what is ran?

Answer 14

• ran is a small GTP binding protein
• • its conformation and activity is regulated by the binding of GTP and its hydrolysis
• • GTP hydrolysis provides the energy required for the transport
• • exists in two states
• • • ran-GTP: active GTP bound form
• • • ran-GDP: inactive GDP bound form

Question 15

how does the ran-GTP concentration gradient work?

Answer 15

• there is a concentration gradient of ran-GTP between the nucleus and cytoplasm
• • ran-GTP is high in the nucleus and low in the cytoplasm
• • the gradient is mediated by two accessory proteins
• • • GEF: nuclear protein that promotes the conversion of ran-GDP to ran-GTP and maintains a high ran-GTP in the nucleus
• • • GAP: cytoplasmic protein that promotes the hydrolysis of ran-GTP to ran-GDP and maintains low ran-GTP in the cytoplasm
• • the gradient determines the directionality of nucleocytoplasmic transport

Question 16

what is an NLS?

Answer 16

• NLS: an amino acid sequence that is both necessary and sufficient for cytoplasm to nuclear targeting
• • necessary: if the sequence is mutated then the modified protein fails to target to the nucleus
• • sufficient: the sequence present on a non nuclear protein can still target the protein to the nucleus
• most common NLS has a short stretch of positively charged/basic amino acid residues
• • KKQRKK
• bipartite NLS: has two short stretches of basic amino acids and a 7 to 10 amino acid long spacer sequence
• • KR(PAATKAGQA)KKKK
• proteins can have more than one NLS and a nuclear export signal (NES)
• NLSs are identified in proteins based on mutational analyses

Question 17

what is ARC1?

Answer 17

• ARC1 is a protein required for plant pollination
• • shuttles between the nucleus and cytoplasm
• • has an assumed classic NLS and classic NES
• experiment 1: mutation of the NLS residues in myc epitope (immunodetection) tagged ARC1 being mislocalized exclusively to the cytoplasm
• experiment 2: fusion of NLS residues in ARC1 to the cytoplasmic protein CAT results in the fusion protein being redirected to the nucleus