Nuclear Structure

∑ nuclear compartment contains:

∑ chromatin: decondensed chromosomes & associated proteins

∑ nuclear matrix: ?

∑ nucleoli: site or ribosomal RNA synthesis & processing

∑ lamins (IFS) organized into cytoskeleton

∑ inner nuclear membrane specialized for lamina binding
which in turn binds chromatin

∑ outer nuclear membrane =RER
continuous with ER, often studded with ribosomes

∑ perinuclear space= lumen of ER

Nuclear Envelope: lamina, double membrane

∑ nuclear lamina: meshwork of IFs called lamins

∑ binds chromatin, pores, inner nuclear membrane=> strutural integrity

∑ mitotic phosph. breaks mesh-> vesiculation of membrane

∑ reassembles on chromatin->nuclear mem assembles and fuses-> pore assembly

∑ reimport of nuclear proteins

Selective transport in both directions:

∑ proteins syn, in cyto; RNA syn in nucleus=> transport required

∑ needed in nuclear compartment

∑ DNA and RNA polymerases, histones, lamins, transcript. factors, splicing factors

∑ also proteins that shuttle between nucleus and cytoplasm: eg snRNAs

∑ needed from nuc in cyto

∑ tRNAs, mRNPs (mature mRNA complexed w/ hnRNP), ribosom subunits

∑ a good example is ribosome syn:

∑ ribo proteins cyto->nuc, bind rRNA, complex exported to cyto

Nuclear Pores

∑ nuclear pore complex 125MD, 100 proteins, octagonal symmetry, 3-4000/nucleus

∑ aqueous channel: passive diffusion limit=60KD; size= 9nm dia x 15 nm long

∑ active transport:

∑ signal mediated

∑ NLS, short stretch rich in positive charge and a proline

∑ NES, eg in 5πcap in mRNA

∑ pore opens to 26nm

∑ ATP-dependent, Ran cycle not in book

∑ bidirectional, but selective

Introduce two state GTPase Ran

∑ GAP

∑ GEF

∑ key is localization of GAP (cyto) and GEG (nucleus)

Mechanism of export

∑ NES

∑ binds NE receptor/RanGTP complex

∑ export complex binds nuclear pore filaments (successive interactions for translocation?)

∑ ranGAP interactions-> GTP hydrolysis-> receptor conformational change-> release NES

∑ import(recycling) of NE receptor &RanGDP

Mechanism of import

∑ NLS interacts with adaptor

∑ NLS/adaptor complex binds NLSreceptor/RanGDP complex

∑ NLSreceptor binds nuclear pore (successive interactions for translocation?)

∑ ranGEF inteaction->RanGTP-> conf. change-> receptor releases NLS

∑ receptor/adaptor/RanGTP exported

Regulation of traffic

∑ binding proteins mask NLS or NES

∑ modification of binding protein unmasks signal

∑ protein tethered to cyto membrane

∑ modification of tether releases protein for import

∑ RNA anchored to polymerase or splicing machinery, released when finished

∑ splicing machinery blocks export

∑ 5π cap may act as NES

∑ assembly of ribo/protein complex exposes NES

∑

Questions:

∑ number of molecules passing through/min

∑ why is NLS not cleaved after import?

∑ continuity of ER and nucleus

Additions:

∑ clinical example for regulated import; see Adv. Cell

∑ Ran cycle; see Adv. Cell

Diagrams:

∑ topology

∑ pore

∑ lamina

∑ import/export signalling

Experimental:

∑ microinjection of beads