Part 1: Intracellular trafficking

Week 1: Transport through the nuclear pore complexWeek 2: RNA transport, maturation & localizationWeek 3: ER translocation and vesicular transportWeek 4: Secretion & cell polarity

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

Part 1: Intracellular trafficking

Week 1: Transport through the nuclear pore complex

Experimental approachesSignals and pathwaysThe nuclear pore complexSoluble transport factors: import + exportThe functions of the small GTPase RanMechanism of translocation

High concentration of nuclear pore complexes (NPCs) on nuclearenvelopes from Xenopus oocytes

Signals are necessary and sufficient for nuclear import

Npl-coated gold particles can translocate across NPCs

SV40 T Ag contains small nuclear localization signal (NLS)

SV40 T Ag K128N

SV40 T Ag contains small nuclear localization signal (NLS

SV40 T Ag WT

In vitro transport assay to nuclear import I

In vitro transport assay to nuclear import II

In vitro transport assay to nuclear import III

Extract + energy Fraction A

RanGTP displaces importin from importin

importin

importin

The NLS receptor importin

The NLS receptor importin

Ran-GTP

NPC

cargo

Importin

The import receptor importin

RanGTP displaces import cargoes from importins

SUMMARY

I. Signals target proteins into and out of the nucleusa. active transportb. signals are necessary and sufficient

II.Transport occurs through the nuclear pore complex (NPC)a. NPC has aqueous channelb. no unfolding of cargoes is requiredc. transport is bi-directional

III. Signals are recognized by soluble receptors: importinsa. bind specific classes of cargob. shuttle between cytoplasm and nucleusc. interact with nucleoporins

Conformational changes that regulate cargo binding

Cse1 with cargo Cse1 without cargo

Structure of the exportin Cse1 with RanGTP and cargo

Establishment of a “Ran-gradient”

Ran cycle is coupled to transport cycle

Consumption of one GTP per transport cycle

Inversion of the Ran gradient leads to an inversion of the

direction of transport

export cargo

+ exportin1

cytoplasmic RanGTPnuclear RanGTP

Import-Export Cycle: the role of adaptors

Ran gradient throughout the cell cycle

G1G2

S

M

RanGTPase

Replicationlicensing

Nucleocytoplasmictransport

Chromatincondensatio

n

Mitotic Spindle Assembly

KinetochoreAssembly

Nuclear Pore

Assembly Nuclear Envelope Fusion

THE METAZOAN NPC

Rout et al.(2000) JCB 148:635

THE YEAST NPC

Interaction between nuclear transport receptors and the FG-repeats within the NPC

Organization of the NPC

Brownian Gate Selective Phase ‘Oily Spaghetti

Models for NPC function

SUMMARY

I. Short signals target proteins into and out of the nucleusMany proteins shuttle: regulation

II. Many transport events are mediated by the importin b superfamily of importins and exportins

III. Directionality is conferred by nuclear RanGTPRan-GTP mediates substrate release (importins) or substrate binding (exportins)per import/export cycle ~ only one GTP is hydrolyzedRanGTP gradient provides energy for transport

IV. NPC functions through facilitated diffusionFG rich repeats provide docking sites for transport factors