Part 1: Intracellular trafficking Week 1: Transport through the nuclear pore complex Week 2: RNA...
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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)
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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
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High concentration of nuclear pore complexes (NPCs) on nuclearenvelopes from Xenopus oocytes
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Signals are necessary and sufficient for nuclear import
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Npl-coated gold particles can translocate across NPCs
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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
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In vitro transport assay to nuclear import I
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In vitro transport assay to nuclear import II
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In vitro transport assay to nuclear import III
Extract + energy Fraction A
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RanGTP displaces importin from importin
importin
importin
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The NLS receptor importin
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The NLS receptor importin
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Ran-GTP
NPC
cargo
Importin
The import receptor importin
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RanGTP displaces import cargoes from importins
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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
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Conformational changes that regulate cargo binding
Cse1 with cargo Cse1 without cargo
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Structure of the exportin Cse1 with RanGTP and cargo
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Establishment of a “Ran-gradient”
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Ran cycle is coupled to transport cycle
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Consumption of one GTP per transport cycle
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Inversion of the Ran gradient leads to an inversion of the
direction of transport
export cargo
+ exportin1
cytoplasmic RanGTPnuclear RanGTP
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Import-Export Cycle: the role of adaptors
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Ran gradient throughout the cell cycle
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G1G2
S
M
RanGTPase
Replicationlicensing
Nucleocytoplasmictransport
Chromatincondensatio
n
Mitotic Spindle Assembly
KinetochoreAssembly
Nuclear Pore
Assembly Nuclear Envelope Fusion
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THE METAZOAN NPC
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Rout et al.(2000) JCB 148:635
THE YEAST NPC
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Interaction between nuclear transport receptors and the FG-repeats within the NPC
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Organization of the NPC
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Brownian Gate Selective Phase ‘Oily Spaghetti
Models for NPC function
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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