Class 10 transport

Nuclear Pore Complex (structure, function, composition)

protein complex present at nuclear pores. Regulate passage.

Cytoplasmic Side:

• Cytoplasmic fibrils points straight up

FG repeat proteins in the middle (spaghettic filling up the pore space)

• form a hydrogel

Nucelplasmic Side:

• nuclear basket. forms a basket

Passive Diffusion through nuclear pore

can only be done by small molecuels <50kda

no directionality

no energy

Active transport

How to large moleculs >50kda get through nuclear pore

but <50kda can also use

reuiqres energy

directional

require a signal (highly regulated)

Transport receptors

bind to cargo to faciliatet cargo active transport through nuclear pore

Ran

helps with some types of active transport

it is a GTPase that when active (GTP bound) binds to transport receptors

provides directinoality to transport process

molecules transported through ran dependent transport

proteins and RNAs (tRNAs, rRNAs)

molecules transported through ran independent (active still) transport

mRNAs

Nuclear import steps

Cargo protein has an NLS

NLS binds importin

complex moves through nuclear pore complex (thanks to affinity of the importin to nuclearporins)

dissociate in nucleoplasm.

association/dissociation driven by RAN

RAN GTP competitively binds importin, competing with the cargo proteins NLS. Therefore, since RANGTP conc. greater in nucleus, once the cargo protein-importin complex enters the nucleus, RANGTP is preferred to bind.

importins

import receptor protein for active transport

importins or exportins bind to transport signals on cargo molecules, they also bind RAN GTP, and also nucleoporins.

exportins

export receptor protein for active transport

importins or exportins bind to transport signals on cargo molecules, they also bind RAN GTP, and also nucleoporins.

Nuclear localization signal (NLS)

the signal that a cargo protein has when it is to be imported (binds to importins)

Nuclear export signal (NES)

The signal that a cargo protein has when it is to be exported (binds to exportins)

which side is ran gtp conc. high on

nucleoplasm

nuclear export steps

cargo protein in nucleus with NES

NES binds exportin

binding requires presence of RAN GTP

this triple complex moves through nucelar porins

in cytoplasm, RAN hydrolyzes GTP to form RAN GDP.

then this dissociates them

Mechanisms of nuclear transport regulation

1. Regulate binding of transport signal to transport receptor (importin/exportin)

2. Regulate binding of the cargo to an anchoring protein

Regulation binding of transport signal to transport receptor (importin/exportin)

mechanisms

1. Covalent modification (like phosphorylation) of nuclear transport signal on cargo protein

2. Regulatory protein binding to the cargo protein

these can either up or down regulate (increase transport or decrease)

• ex. it could even be that cargo doesn’t bind at all, unless it has covalent mod. or regulatory protein bound.

REgulating binding of cargo protein to anchoring protein mechanisms

a. cytoplasmic anchoring protein or nuclear anchoring protein

how is RAN GTP/GDP gradient created across cytoplasm/nucleoplasm

GAPs (favor inactive), GEFS (favor active)

GEFs in the nucleus - the RAN GEF is actually bound to chromatin

GAPs in the cytoplasm - the RAN GAP is bound to the cytoplasmic fibrils

what are RAN GAPs bound to

cytoplasmic fibrils of NPCwh

what are ran gefs bound to

chromatin in the nucleus.

how does the transport receptor (importin/exportin) actually help cargo through the FG repeat proteins

they have specific binding sites for the FG repeat proteins

Remember most proteins start in the cytosol. So if they are in the nucleus it must have gotten there somehow (passive diffusion or NLS)

Remember most proteins start in the cytosol. So if they are in the nucleus it must have gotten there somehow (passive diffusion or NLS)