Lecture 3 Flashcards
Nuclear cytoplasmic transport via the NPC consist of?
Cytoplasm to nucleus in import and nucleus to cytoplasm export trafficking pathways
Most nuclear imported proteins contain a __
Nuclear localization signal (NLS)
Nuclear localization, signal types:
- Classic NLS - most common NLS
consists of short stretch of positively-charged (basic) amino acid residues e.g., -KKQRKK- in large T antigen protein of simian virus 40 - Bipartite NLS – composed of two short stretches of basic amino acids and 7-10 amino-acid-long ‘spacer’ sequence e.g., -KR[PAATKAGQA]KKKK- in nucleoplasmin
What is a nuclear localization signal?
NLS is an amino acid sequence that is both necessary and sufficient for cytoplasm-to-nuclear targeting
Necessary: if sequence (or a portion thereof) is mutated, then modified protein fails to target to nucleus (i.e., mutant protein is mislocalized to cytoplasm)
Sufficient: if sequence linked to non-nuclear (‘passenger’) protein is capable of redirecting resulting fusion protein to nucleus
What are karyoferins?
large family of receptor proteins responsible for moving macromolecules (i.e., proteins or RNA) either into nucleus (importins) or out of nucleus (exportins)
What are the five mean steps in cytoplasm to nucleus transport?
Cytoplasmic-to-nuclear transport
Step 1
* nascent (newly-synthesized) NLS-containing ‘cargo’ protein is recognized in cytoplasm by importin
* heterodimeric protein consists of two distinct subunits - importin α and importin β
* importin α subunit recognizes and binds to basic residues in ‘cargo’ protein’s NLS a/b
Step 2
* cargo’ protein-importin receptor complex moves through cytoplasm, towards nucleus (via importin’s ability to bind cytoskeleton)
* cytoskeleton elements serves a ‘highways’ for almost all types of intracellular transport (e.g., movement of proteins, RNA, organelles, etc.)
* at the surface of nucleus… importin β subunit of cargo protein-importin receptor complex binds to cytoplasmic filament at NPC
Step 3
* ‘cargo’ protein-importin receptor complex is translocated through central channel of NPC
Step 4
* ‘cargo’-receptor complex associates with nuclear basket on inner surface of NPC
* cargo’-receptor complex binds to Ran-GTP (via importin β) resulting in its release from NPC and disassembly into nucleoplasm
* import of the NLS-containing ‘cargo’ protein into nucleus is accomplished
Step 5
* Ran-GTP-bound importin β subunit moves back to cytoplasm due to [Ran-GTP] gradient
[Ran-GTP] nucleus > [Ran-GTP] cytoplasm
* in cytoplasm, Ran-GTP is hydrolyzed via accessory protein GAP Ran-GDP released from importin β
* importin β used for another round of nuclear protein import
* Ran-GDP released from importin β moves back into nucleus (due to [Ran-GDP] gradient)
[Ran-GDP] nucleus < [Ran-GDP] cytoplasm
* Ran-GDP in nucleus converted into Ran-GTP by accessory protein GEF
Step 5 +
* importin α binds to exportin karyopherin mediates nuclear-to- cytoplasm transport release of nuclear imported ‘cargo’ protein exposes nuclear export signal (NES) in importin α
* exportin also binds to other ‘cargo’ proteins exported from nucleus via their NESs specific stretch/sequence of amino acids recognized by exportin and serve as ‘zipcode’ to mediate targeting of protein from nucleus to cytoplasm
* several different types of NESs all both necessary and sufficient for nucleus-to-cytoplasm targeting
* most common NES consists of leucine-rich motif
What is Ran and where does it exist?
Ran is a small GTP binding protein that is activity regulated by GTP binding and hydrolysis
- exists in two distinct states:
Ran-GTP (active GTP-bound form) Ran-GDP (inactive GDP-bound form) - a steep concentration gradient of Ran-GTP exists between nucleus & cytoplasm
Define GEF and GAP
GEF – nuclear protein that promotes conversion of Ran-GDP to Ran-GTP
maintains a high [Ran-GTP] in nucleus i.e., promotes the GTPase activity of Ran
GAP – cytoplasmic protein that promotes hydrolysis of Ran-GTP to Ran-GDP
maintains low [Ran-GTP] in cytoplasm
