Unit 2: Nucleus Flashcards
Eukaryote nucleus
- membrane bund
- nuclear envelope
- nuclear pores
- organized internal nuclear structure (highly regulated)
Characteristics of nucleus
- irregular shape
- one per cell
- largest organelle
Two main functioins:
- compartmentalization of cellular genome and activities (DNA replication, transcription, RNA processing)
- coordination of cellular activities (metabolism, protein synthesis, reproduction)
Nuclear structure components
- nuclear envelope (membrane, lamina, pores)
- nuclear content (chromatin, nucleoplasm, nuclear matrix, nucleolus)
3 functions of nuclear envelope
- separates nuclear content from cytoplasm (genome and cytosol; transcription/translation)
- selective barrier (regulates certain molecules, composition of nucleus and regulates gene expression)
- binds nuclear lamina - structural framework
Nuclear Lamina structure
- thin meshwrok of long filament-like proteins
- bound to inner surface of nuclear envelope
Nuclear functions
- support structure for nuclear envelope
- scaffold for chromatin and nuclear matrix (?) attachment
Progeria
- premature aging
- point mutation
- deletion of 150 nucleotides
- progerin = mutation of lamin A
- structure of lamina compromised
Nucleoplasm
highly organized fluid-filled interior of nucleus
>30 specialized regions (subdomains) w/ special functions
Choromosomes
- during interphase organized into discrete subdomains within nucleus
- location related to activity
Interchromosomal channels
regions between domains that serve as barriers for unwanted interactions (DNA-DNA and DNA-protein)
Transcriptioin factories
active genes (chromatin) from different subdomains extend into interchromosomal channels to form “transcription factories” - where TF are concentrated
Nuclear Speckles
- subdomains where mRNA splicing factors are concentrated - i.e. mRNA processing
- located in interchromosomal channels next to TF
- numerus and highly dynamic depending on needs of cell
Nucleolus
- most obvious subdomain: irregular, dense; NOT membrane bound
- 1-5 nucleoli depending on metabolic activity
Function of the nucleolus
- ribosomal biogenesis
- site of ribosomal rDNA transcription and rRNA processing
- INITIAL stages of ribosomal subunit assembly (final assembly in cytosol)
Nuclear matrix
- insoluble fibrillar-like protein network (mesh) distributed throughout the nucleoplasm
Role of the nuclear matrix
- structural role (maintain overall shape)
- scaffold (organizing nuclear subdomains and anchoring protein factors)
Nuclear Pores
- inner and outer membranes of nuclear envelope fuse = pore
- gateway between cytoplasm and nucleoplasm
NPC Structure
- highly conserved in euk.
- octagonal symmetry of overal structure
- central scaffold (anchors complex to nuclear envelope)
- forms an aqueous central channel
- filament like proteins - FG nucleoporins (Nups) - inner surface of channel lined
FG domains
- FG repeats (FG nucleoporins)
- line inner sruface of channel
- extended and flexible organization (highly disordered sec. structure)
- extend into central channel
- hydrophobic mesh - sieve like gel limits macromolec
Cytoplasmic and Nuclear rings
- cytoplasmic and nuclear side of NPC
- linked o central scaffold and cytoplasmic filament s or nuclear basket
Cytoplasmic filaments
- long protein filaments that extend into cytosol on cytosolic side
- nucl. receptor cargo protein recognition and import
Nuclear basket
- basket like structure located on nucl side of NPC
- involved in nucl receptor-cargo protein import and export
Functions of NPC
size exclusion
- passive diffusion of small molecules (nucleotides etc)
- regulate mvt (active) of large molecules (RNAs and proteins)
What is nucleoplasmin?
- nuclear protein
Where is nucleoplasmin synthesized
- cytoplasm, associates with cytoplasmic fillaments and translocates into nucleus
Nucleocytoplasmic transport (cytosol to nuclear) requires
- energy
- specific protein receptors
- unique targeting signals
Nuclear Localization Signal is used in
- imported proteins
- mediates targeting of protein from cytosol to nucleus
What is a NLS?
- nuclear localization signal
- a.a. sequence necessary and sufficient for cytosol to nuclear targetting
- recognized by nuclear receptor proteins
Necessary? NLS
- sequence cannot be mutated or else the modified protein will fail to target nucleus
Sufficient? NLS
- if sequence linked to non-nuclear passenger protein is capable of redirecting fusion protein to nucleus
Transport receptors are also known as
Karyopherins
- importin - into nucleus
- exportin - out of nucleus
What is the role of karyopherins?
- mobile proteins responsible for moving (ferrying) protein cargo across nuclear envelope
G proteins
- molecular switch in transport process
- i.e. Ran
How are G proteins activated?
- weak intrinsic GTPase activity
- conformational change
- GTP binding and hydrolysis regulates activity and conformation
Ran-GTP and Ran-GDP(active/inactive)
Ran GTP = active
RanGDP = inactive
Where is there a high concentration gradient of Ran GTP?
- high in nucleus
- low in cytoplasm
- gradient maintained high Ran-GTP in nucleus maintained by accessory proteins
How is Ran-GDP yielded?
- Ran-GDP is yielded in the cytoplasm (deactivated)
- GTPase Activating Protein (GAP)
- HYDROLYSIS of GTP of Ran-GTP to GDP = Ran-GDP
- Ran-GDP then moves (translocates) to the nucleus
How is GTP produced?
- produced in the nucleus
- Guanine nucleotide Exchange factor (GEF)
- GDP is EXCHANGED by GEF
- to product Ran-GTP
- NOT a PHOSPHORYLATION event (the whole GDP is replaced)
What are the accessory proteins involved in maintaining the [Ran-GTP] gradient?
- RCC1 (promotes conversion of Ran-GDP to Ran-GTP (high [Ran-GTP] in nucleus]
- Ran-GAP-1 cytosolic protein promotes hydrolysis of Ran-GTP to Ran-GDP (maintains low [Ran-GTP] in cytosol)
Ran-GTP gradient determines what?
directionality of nucleocytoplasmic transport
What is required for nucleoplasmic transport (energy)?
GTP hydrolysis
Step one of Nuclear import
- nascent NLS-containing cargo protein
- recognized in cytosol by importin
- importin a recognizes and binds basic residues of cargo NLS
Importin is a ____ proteins
heterodimeric
- containing a and b subunits (two distinct)
Step Two Nuclear Import
- cargo protein- importer complex moves through cytosol towards nucleus
- cytoskeleton elements serves as highway for intracellular transport
- surface of nucleus (outside) importin b subunit binds cytoplasmic filament at NPC
- importin ability to bind to cytoskeleton
Step Three Nuclear Import
- cargo protein- importin complex translocaated through aqueous central channel of NPC
- cargo-receptor complex interacts with FG-domains
- interactions dissolve/untangle FG domain network and allow translocation through channel
Step Four Nuclear Import
- cargo-receptor complex associates with nuclear basket on inner NPC
- cargo-receptor complex binds toRan-GTP via importin b
- released from NPC and disassembled in nucleoplasm
- success import NLS - containing cargo into the nucleus
Step Five Nuclear Import
- Ran-GTP bound importin b subunit moves back to cytosol due to [Ran-GTP] gradient
- in cytosol, Ran-GTP hydrolyzed
What hydrolyzes Ran-GTP in cytosol?
RanGap1
What happens after RanGTP is hydrolyzed in cytosol?
- Ran-GDP released from importin b
- importin b used for another round of protein import
- Ran-GDP moves back to nucleus due to gradient
What converts Ran-GDP back to RanGTP?
- accessory protein RCC1 in the nucleus
What happens to importin a (and cargo proteins) in the nucleus (after import)?
- need to move back into the cytosol - nuclear export
- importin a binds exportin (karyopherin)
How are other cargo proteins brought back to the cytoplasm?
- Nuclear export signal (NES)
- specific sequence of a.a.
- LxxLxxL
- leucine motif
How does the imporitina- exportin complex move into the nucleus?
- binds Ran-GTP
- high RanGTP conc in nucleus
- RanGTP promotes stable assembly and transports to cytosol
What happens to Ran-GTP in the cytosol after nuclear export?
- GTP on Ran-GTP is hydrolyzed by Ran-Gap1
- Ran-GDP hydrolyzed releases exportin and releases importin a
What happens to importin a and exportin once they are brought back into the cytosol?
- importin a is used for another round of nuclear import Ran-GDP
- exportin moves back into the nucleus for another round of export
How do some proteins move into the nucleus without a NLS?
- piggyback nuclear protein import
- nascent protein lacking NLS binds NLS containing protein in cytosol
- targeting and import of protein-protein complex mediated by importin receptors
