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MCB 2050 - Second Half - Mullen > Lecture 3 - nucleus part 2 > Flashcards

Lecture 3 - nucleus part 2 Flashcards

1
Q

What is imported into the nucleus from the cytoplasm?

A

all proteins required for DNA replication, txn, splicing, ribosome assembly, chromatin packing, nuclear matrix proteins, lamins etc

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2
Q

What are some characteristics of nucleocytoplasmic transport via the NPC?

A
  • most congested bi-directional trafficking pathways in the cell
  • includes variety of cytoplasm-to-nucleus (import) and nucleus-to-cytoplasm (export) pathways
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3
Q

What are exported out of the nucleus into the cytoplasm?

A

RNA, partially assembled ribosomes, and some proteins

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4
Q

What are the molecular mechanisms of nucleocytoplasmic transport via the NPC?

A

requires energy, specific protein receptors and unique targeting signals

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5
Q

What is a feature of cytoplasm to nuclear transport?

A

most nuclear imported protiens contain a nuclear localization signal (NLS)

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6
Q

What is an NLS?

A

specific stretch of AA recognized by nuclear receptor proteins - serves as a zipcode to mediate targeting of protein from cytoplasm to nucleus
- several different NLSs

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7
Q

What are two types of NLSs?

A

classic NLS - most common
- short stretch of positively charged basic AA residues e.g. (-KKQRKK-)

bipartite NLS
- two short stretches of basic AA and 7-10 AA long space sequence
e.g. (-KR[PAATAGQA]KKKK-)

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8
Q

Can proteins have more than one NLS?

A

yes

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9
Q

Can proteins have more than one NES?

A

yes

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10
Q

Why are NLSs necessary and sufficient for cytoplasm to nuclear targeting?

A

necessary - if sequences is mutated, then modified protein fails to target to nucleus
sufficient - if sequence linked to non-nuclear protein is capable of redirecting resulting fusion protein to nucleus

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11
Q

What is ARC1?

A

protein required for plant pollination, involved in signal transduction pathway for recognition of self-incompatible pollen at plasma membrane

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12
Q

What does ARC1 do in NPCs?

A

shuttles between the nucleus and the cytoplasm

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13
Q

How is ARC1 localized to the nucleus?

A

contains putative classic NLS and putative NESs

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14
Q

What are the two experiments done to identify an NLS in ARC1?

A

Experiment 1: mutation of residues 261-266 results in myc-epitope-tagged ARC1 mislocalized exclusively to cytoplasm
Experiment 2: fusion of residues 261-266 in ARC1 to cytoplasmic protein CAT results in fusion protein redirected to nucleus

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15
Q

What are transport receptors?

A

mobile proteins responsible for moving protein cargo across nuclear envelope

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16
Q

What are karyoferins?

A

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

17
Q

What did the characterization of different NLSs lead to?

A

identification of factors necessary for nuclear import of proteins from cytoplasm such as transport receptors and karyoferins

18
Q

What are the steps to cytoplasm-to-nucleus transport?

A
  1. nascent (newly-synthesized) NLS containing cargo protein is recognized in the cytoplasm by importin
  2. cargo protein-importin receptor complex moves through cytoplasm, towards nucleus (via importins ability to bind cytoskeleton), at the surface of the nucleus, importin beta binds to cytoplasmic filament at NPC
  3. cargo protein importin receptor complex is translocated through central channel of NPC, cargo receptor complex successively interacts with hydrophilic and FG domains of FG nups in central channel
  4. cargo receptor complex associates with nuclear basket on inner surface of NPC, cargo receptor complex binds to Ran-GTP via importin beta resulting in its release from NPC and disassembly into nucleoplasm. import of the NLS containing cargo protein into nucleus is accomplished
  5. Ran GTP bound importin beta subunit moves back to cytoplasm due to Ran GTP gradient. in the cytoplasm, GTP on Ran GTP is hydrolyzed via GAP, ran GDP is released from importin beta. importin beta used for another round of import. ran GDP released from importin beta moves back into nucleus (gradient), RanGDP nucleus < Ran GDP cytoplasm. Ran Gdp in nucleus is converted into Ran GTP by GEF.
  6. Importin alpha (NES containing cargo protein) exportin complex binds to Ran GTP. Ran GTP promotes stable assembly of importin alpha exportin complex. importin alpha exportin ran GTP complex transported into cytoplasm due to ran GTP gradient
  7. in cytoplasm, GTP on ran GTP is hydrolyzed by GAP. ran GDP released from exportin and release of importin alpha or other NES containing cargo protein
    importin alpha - used for another round of import
    ran GDP - moves back into nucleus due to gradietn and converted via GEF into ran GTP
    exportin - moves back into nucleus via NLS and importin for another round of export
19
Q

What is importin?

A

heterodimeric protein
- consists of two distinct subunits importin alpha and importin beta
- importin alpha recognizes and binds to basic residues in cargo proteins NLS

20
Q

is the NLS porteolytically cleaved from the cargo protein in cytoplasm to nucleus import?

A

no, this allows for re-import of nuclear proteins

21
Q

What is Ran?

A

small GTP binding protein, its activity is regulated by GTP binding and hydrolysis

22
Q

What are the two distinct states that Ran exists as?

A

Ran-GTP (active GTP-bound form)
Ran-GDP (inactive GDP-bound form)

23
Q

Where is Ran GTP gradient in the cytoplasm vs nucleus?

A

a steep concentration gradient of Ran GTP exists between nucleus and cytoplasm
RanGTP nucleus > RanGTP cytoplasm

24
Q

What is GEF?

A

nuclear protein that promotes conversion of Ran GDP to Ran GTP, maintains a high Ran GTP in nucleus (promotes GTPase activity of ran)

25
Q

What is Gap?

A

cytoplasmic protein that promotes hydrolysis of RanGTP to RanGDP, maintains low RanGTP concentration in the cytoplasm

26
Q

What does the Ran GTP gradient do?

A

determines directionality of nucleocytoplasmic transport - GTP hydrolysis provides energy required for nucleocytoplasmic transport

27
Q

What is the fate of importin alpha in the nucleus?

A

importin alpha binds to exportin
karyopherin mediates nuclear to cytoplasm transport
release of nuclear imported cargo protein exposes nuclear export signal NES in importin alpha

28
Q

What is the fate of cargo proteins exported out of the nucleus?

A

exportin also binds to other cargo protein exported from nucleus via NESs

29
Q

Are there multiple types of NESs?

A

yes

30
Q

What is the most common NES?

A

consists of leucine rich motif
e.g. -LxxLxxL-

31
Q
A

MCB 2050 - Second Half - Mullen (14 decks)

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