chapter 5 nuclear structure and transport by charles n. cole & pamela a. silver
TRANSCRIPT
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Chapter 5
Nuclear structure and transportBy
Charles N. Cole & Pamela A. Silver
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5.1 Introduction
• The nucleus contains most of the cell’s DNA, allowing for sophisticated regulation of gene expression.
• The nuclear envelope is a double membrane that surrounds the nucleus.
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• The nucleus contains subcompartments that are not membrane-bounded.
• The nuclear envelope contains pores used for:– importing proteins into the nucleus – exporting RNAs and proteins from the nucleus
5.1 Introduction
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5.2 Nuclei vary in appearance according to cell type and organism
• Nuclei range in size from about one micron (1 μm) to more than 10 μm in diameter.
• Most cells have a single nucleus, but some cells contain multiple nuclei, and a few cell types lack nuclei.
• The percentage of the genome that is heterochromatin varies among cells and increases as cells become more differentiated.
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5.3 Chromosomes occupy distinct territories
• Although the nucleus lacks internal membranes, nuclei are highly organized and contain many subcompartments.
• Each chromosome occupies a distinct region or territory.– This prevents chromosomes from becoming
entangled with one another.
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• The nucleus contains both chromosome domains and interchromosomal regions.
5.3 Chromosomes occupy distinct territories
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5.4 The nucleus contains subcompartments that are not membrane-bounded
• Nuclear subcompartments are not membrane-bounded.
• rRNA is synthesized and ribosomal subunits are assembled in the nucleolus.
• The nucleolus contains DNA that encodes rRNAs and that is present on multiple chromosomes.
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• mRNA splicing factors:– are stored in nuclear speckles – move to sites of transcription where they function
• Other nuclear bodies can be identified with antibodies, but the functions of most of these are unknown.
5.4 The nucleus contains subcompartments that are not membrane-bounded
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5.5 Some processes occur at distinct nuclear sites and may reflect an underlying
structure• The nucleus contains replication sites where
DNA is synthesized.
• The nucleus may contain a nucleoskeleton that could help to organize nuclear functions.
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5.6 The nucleus is bounded by the nuclear envelope
• The nucleus is surrounded by a nuclear envelope consisting of two complete membranes.
• The outer nuclear membrane is continuous with the membranes of the endoplasmic reticulum (ER).
• The lumen of the nuclear envelope is continuous with the lumen of the ER.
• The nuclear envelope contains numerous NPCs.– They are the only channels for transport of molecules and
macromolecules between the nucleus and the cytoplasm.
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5.7 The nuclear lamina underlies the nuclear envelope
• The nuclear lamina is constructed of intermediate filament proteins called lamins.
• The nuclear lamina is located beneath the inner nuclear membrane.– They are physically connected by lamina-associated integral
membrane proteins.
• The nuclear lamina plays a role in nuclear envelope assembly and may provide physical support for the nuclear envelope.
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• Proteins connect the nuclear lamina to chromatin; – this may allow the nuclear lamina to organize DNA replication and
transcription.
• Yeast and some other unicellular eukaryotes lack a nuclear lamina.
5.7 The nuclear lamina underlies the nuclear envelope
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5.8 Large molecules are actively transported between the nucleus and
cytoplasm
• Uncharged molecules smaller than 100 daltons can pass through the membranes of the nuclear envelope.
• Molecules and macromolecules larger than 100 daltons cross the nuclear envelope by moving through NPCs.
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• Particles up to 9 nm in diameter (corresponding to globular proteins up to 40 kDa) can pass through NPCs by passive diffusion.
• Larger macromolecules are actively transported through NPCs and must contain specific information in order to be transported.
5.8 Large molecules are actively transported between the nucleus and cytoplasm
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5.9 Nuclear pore complexes are symmetrical channels
• NPCs are symmetrical structures that are found at sites where the inner and outer nuclear membrane are fused.
• Each NPC in human cells has a mass of ~120 106 daltons (40 times that of a ribosome).
• It is constructed from multiple copies of ~30 proteins.
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• NPCs contain:– fibrils that extend into the cytoplasm– a basket-like structure that extends into the
nucleus
5.9 Nuclear pore complexes are symmetrical channels
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5.10 Nuclear pore complexes are constructed from nucleoporins
• The proteins of NPCs are called nucleoporins.
• Many nucleoporins contain repeats of short sequences, which are thought to interact with transport factors during transport.– Such as:
• Gly-Leu-Phe-Gly• X-Phe-X-Phe-Gly• X-X-Phe-Gly
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• Some nucleoporins are transmembrane proteins that are thought to anchor NPCs in the nuclear envelope.
• All of the nucleoporins of yeast NPCs have been identified.
• NPCs are disassembled and reassembled during mitosis.
• Some nucleoporins are dynamic: they rapidly associate with and dissociate from NPCs.
5.10 Nuclear pore complexes are constructed from nucleoporins
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5.11 Proteins are selectively transported into the nucleus through nuclear pores• Mature nuclear proteins contain sequence information required
for their nuclear localization.
• Proteins selectively enter and exit the nucleus through nuclear pores.
• Information for nuclear import lies in a small portion of the transported protein.
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5.12 Nuclear localization sequences target proteins to the nucleus
• A nuclear localization sequence (NLS) is often a short stretch of basic amino acids.
• NLSs are defined as both necessary and sufficient for nuclear import.
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5.13 Cytoplasmic NLS receptors mediate nuclear protein import
• Receptors for nuclear import are cytoplasmic proteins that bind to the NLS of cargo proteins.
• Nuclear import receptors are part of a large family of proteins often called karyopherins.
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5.14 Export of proteins from the nucleus is also receptor-mediated
• Short stretches of amino acids rich in leucine act as the most common nuclear export sequences.
• A nuclear export receptor:– binds proteins that contain nuclear export
sequences (NESs) in the nucleus – transports them to the cytoplasm
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5.15 The Ran GTPase controls the direction of nuclear transport
• Ran is a small GTPase that is common to all eukaryotes and is found in both the nucleus and the cytoplasm.
• The Ran-GAP promotes hydrolysis of GTP by Ran.
• The Ran-GEF promotes exchange of GDP for GTP on Ran.
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• The Ran-GAP is cytoplasmic, whereas the Ran-GEF is located in the nucleus.
• Ran controls nuclear transport by binding karyopherins and affecting their ability to bind their cargoes.
5.15 The Ran GTPase controls the direction of nuclear transport
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5.16 Multiple models have been proposed for the mechanism of nuclear transport
• Interactions between karyopherins and nucleoporins are critical for translocation across the nuclear pore.
• Directionality may be conferred in part by distinct interactions of karyopherins with certain nucleoporins.
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5.17 Nuclear transport can be regulated
• Both protein import and export are regulated.
• Cells use nuclear transport to regulate many functions, including:– transit through the cell cycle– response to external stimuli
• The movement of the transcription factor NF-κB illustrates how nuclear transport is regulated.
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5.18 Multiple classes of RNA are exported from the nucleus
• mRNAs, tRNAs, and ribosomal subunits produced in the nucleus are exported through NPCs to function during translation in the cytoplasm.
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• The same NPCs used for protein transport are also used for RNA export.
• Export of RNA is receptor-mediated and energy-dependent.
• Different soluble transport factors are required for transport of each class of RNA.
5.18 Multiple classes of RNA are exported from the nucleus
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5.19 Ribosomal subunits are assembled in the nucleolus and exported by exportin 1
• Ribosomal subunits are assembled in the nucleolus where rRNA is made.
• Ribosomal proteins are imported from the cytoplasm for assembly into the ribosomal subunits.
• Export of the ribosomal subunits is carrier-mediated and requires Ran.
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5.20 tRNAs are exported by a dedicated exportin
• Exportin-t is the transport receptor for tRNAs.
• tRNA export requires Ran.
• tRNA export may be affected by modifications of the tRNAs.
• tRNAs may be re-imported into the nucleus.
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5.21 Messenger RNAs are exported from the nucleus as RNA-protein complexes
• Proteins that associate with mRNAs during transcription help to define sites of pre-mRNA processing.
• They are also thought to package mRNAs for export.
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• Most proteins that associate with mRNA in the nucleus are removed after export and returned to the nucleus. – A few are removed immediately prior to export.
• Signals for mRNA export may be present in proteins bound to the mRNA.
• The export of mRNA can be regulated, but the mechanism for this is unknown.
5.21 Messenger RNAs are exported from the nucleus as RNA-protein complexes
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5.22 hnRNPs move from sites of processing to NPCs
• mRNAs are released from chromosome territories into interchromosomal domains following completion of pre-mRNA processing.
• mRNAs move to the nuclear periphery by diffusion through interchromosomal spaces.
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5.23 mRNA export requires several novel factors
• Many factors required uniquely for mRNA export have been identified.
• Factors able to bind to both the mRNP and nuclear pore complex help to mediate mRNA export.
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• One factor, Dbp5, is an ATPase and may use energy from ATP hydrolysis to remove mRNP proteins during transport.
5.23 mRNA export requires several novel factors
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5.24 U snRNAs are exported, modified, assembled into complexes, and imported
• U snRNAs produced in the nucleus are– exported– modified– packaged into U snRNP RNA-protein complexes– imported into the nucleus to function in RNA
processing
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5.25 Precursors to microRNAs are exported from the nucleus and processed in the
cytoplasm
• MicroRNAs are produced by:– transcription in the nucleus– partial processing to generate a hairpin precursor– export of the precursor by exportin-V– final processing in the cytoplasm