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Part 1: Intracellular trafficking. Week 2: RNA transport, maturation & localization. Week 3: ER translocation and vesicular transport. Week 1: Transport through the nuclear pore complex. Week 4: Secretion & cell polarity.

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part 1 intracellular trafficking

Part 1: Intracellular trafficking

Week 2: RNA transport, maturation & localization

Week 3: ER translocation and vesicular transport

Week 1: Transport through the nuclear pore complex

Week 4: Secretion & cell polarity

Week 5: Transport into other organelles (mitochondria, peroxisomes)

slide2

Part 1: Intracellular trafficking

Week 1: Transport through the nuclear pore complex

Experimental approaches

Signals and pathways

The nuclear pore complex

Soluble transport factors: import + export

The functions of the small GTPase Ran

Mechanism of translocation

slide3

High concentration of nuclear pore complexes (NPCs) on nuclear

envelopes from Xenopus oocytes

slide6

SV40 T Ag contains small nuclear localization signal (NLS)

SV40 T Ag contains small nuclear localization signal (NLS

SV40 T Ag K128N

SV40 T Ag WT

slide13

The import receptor importin 

Importin 

Ran-GTP

cargo

NPC

slide15

SUMMARY

I. Signals target proteins into and out of the nucleus

a. active transport

b. signals are necessary and sufficient

II.Transport occurs through the nuclear pore complex (NPC)

a. NPC has aqueous channel

b. no unfolding of cargoes is required

c. transport is bi-directional

III. Signals are recognized by soluble receptors: importins

a. bind specific classes of cargo

b. shuttle between cytoplasm and nucleus

c. interact with nucleoporins

slide22

Inversion of the Ran gradient leads to an inversion of the direction of transport

nuclear RanGTP

cytoplasmic RanGTP

export cargo

+ exportin1

slide25

S

Nucleocytoplasmic

transport

G2

G1

Replication

licensing

RanGTPase

Chromatin

condensation

Mitotic Spindle Assembly

Nuclear Pore

Assembly

Kinetochore

Assembly

Nuclear Envelope Fusion

M

slide27

THE YEAST NPC

Rout et al.(2000) JCB 148:635

slide28

Interaction between nuclear transport receptors

and the FG-repeats within the NPC

slide31

Models for NPC function

Brownian Gate

Selective Phase

‘Oily Spaghetti

slide32

SUMMARY

I. Short signals target proteins into and out of the nucleus

Many proteins shuttle: regulation

II. Many transport events are mediated by the importin b

superfamily of importins and exportins

III. Directionality is conferred by nuclear RanGTP

Ran-GTP mediates substrate release (importins) or substrate

binding (exportins)

per import/export cycle ~ only one GTP is hydrolyzed

RanGTP gradient provides energy for transport

IV. NPC functions through facilitated diffusion

FG rich repeats provide docking sites for transport factors