Nuclear Function and Trafficking

1

• Nuclear Function and Trafficking
• Lecture Outline
• 1. The Nucleus in Context
• A. Compartmentalizing DNA into the nucleus
• B. Origin of the nucleus
• C. ER is continuous with nuclear membrane
• 2. Organization and Function of the Nucleus
• A. Comparison of complexity of
  transcription/translation in eukaryotes
  prokaryotes
• B. DNA synthesis
• C. RNA synthesis and processing
• D. Non-membraneous compartments in the
  nucleus
• E. Splicing
• F. Ribosome assembly
• G. Processing of eukaryotic mRNA for export
• 3. The Nuclear Membrane
• A. Function
• B. Structure
• 4. The Nuclear Pore Complex
• A. Structure of the NPC

2

• Trafficking In and Out of the Nucleus
• Lecture Outline, cont.
• 5. Trafficking through the Nuclear Pore
• A. Functional relevance
• B. Mechanism of nuclear trafficking
• C. Mechanism of nuclear import
• D. Mechanism of nuclear export
• E. Shuttling proteins
• F. Specific nuclear export proteins
• G. Specific nuclear export pathways
• 6. Experimental Approaches used to Study Nuclear
  Trafficking
• 7. Examples from Pathobiology
• A. Regulation of NF-kB transport
• B. NFAT, a shuttling protein
• C. How HIV-1 exploits cellular trafficking
  machinery for genomic RNA export
• D. How other retroviruses exploit cellular
  trafficking machinery for RNA export
• E. How nuclear import machinery is exploited by
  adenovirus for viral entry

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Trafficking In and Out of The Nucleus

• 1. The Nucleus in Context
• A. Compartmentalization of the nucleus
• A key feature of eukaryotic cells
• 1. Eukaryotic but not prokaryotic cells
  contain a nucleus that sequesters DNA
• 2. Other differences between eukaryotes and
  prokaryotes
• Larger cell volumes in eukaryotes
• Presence of cytoskeleton in eukaryotes
• Elaborate set of internal membranes and
  organelles in eukaryotes
• Larger amounts of DNA in eukaryotes, and more
  regulatory non-coding DNA
• Metabolism of eukaryotes is dependent on
  oxidation occurring in mitochondria
• 3. Note single cell eukaryotes are called
  protists include yeast and protozoa

Prokaryotic cell
Eukaryotic cell
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Trafficking In and Out of The Nucleus

• 1. The Nucleus in Context, cont.
• B. Origin of the Nucleus Most likely, from
  invagination of PM of an ancient bacterium.
  Note Topology of a compartment can be determined
  from its evolutionary origins. Thus the interior
  of the nucleus is the topological equivalent of
  the cytoplasm.
• C. The ER is continuous with the nuclear
  membrane. ER lumen is continuous with the space
  between the inner and outer membrane, and
  topologically equivalent to the extracellular
  space.

Evolution of the Eukaryotic Nucleus
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Trafficking In and Out of The Nucleus

• 2. Organization and Function of the Nucleus

A. Comparison of Complexity of
Transcription/Translation in Eukaryotes and
Prokaryotes
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Trafficking In and Out of The Nucleus

• 2. Organization and Function of the Nucleus
• B. DNA replication in mammalian cells occurs
  in clustered sites throughout nucleus
• C. RNA synthesis and processing
• Types of RNA
• Ribosomal RNAs (rRNA ) made/processed/assembled
  in nucleolus made by RNA Pol I and III
  constitutes 80 of RNA in some cells methylated
  using snoRNAs
• Small nucleolar RNAs (snoRNA) guide RNAs used
  for processing rRNAs, snRNAs
• Messenger RNA (mRNA) made by Pol II are
  processed in nucleus (see below), then exported
  and undergo translation in cytoplasm
• Transfer RNA (tRNA) 80 nt synthesized by Pol
  III different ones for different aas,
  aminoacylated then export to cytoplasm provide
  aas for translation
• Small nuclear RNAs (snRNA) lt 200 nt
  components of the spliceosome U1-U6

tRNA structure
SnoRNAs are used as guides in the processing of
rRNA precursors
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Trafficking In and Out of The Nucleus

• 2. Organization and Function of the Nucleus,
  cont.
• D. Non-membraneous compartments in the
  nucleus
• Nucleolus Site where rRNAs, tRNAs and some
  snRNPs are synthesized, processed, and
  assembled into RNPs. Also contains snoRNAs that
  act as guides for RNA modifications. Site of
  assembly of ribosomes.
• Cajal bodies/ GEMS sites where snRNAs and
  snoRNAs undergo modification and final assembly
  events.
• Speckles or interchromatin granular clusters
  storage site for mature snRNPs

Bright pink Cajal bodies Dark pink
nucleoli Green speckles Blue bulk chromatin
Compartments in the Nucleus
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Trafficking In and Out of The Nucleus

• 2. Organization and Function of the Nucleus,
  cont.
• E. Splicing
• 1. UsnRNAs
• Transcribed in nucleolus, exported via Crm1
  pathway to cytoplasm where they associate with
  snRNP proteins (see below).
• Import back into nucleus for final modification
  and assembly in Cajal bodies and Gems.
• Storage in nuclear speckles.
• 2. Machinery consists of
• 5 small nuclear RNAs (snRNAs), U1, 2, 4, 5, 6
• Each snRNA is complexed with proteins to form
  an snRNP
• 3. Critical snRNP proteins include
• SMN, mutations in which cause spinal muscular
  atrophy
• Gemins
• Sm proteins
• 4. snRNPs form the spliceosome core

Biogenesis of UsnRNPs
From Dreyfus et al., Current Opinion in Cell Bio
14 305 (2002)
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Trafficking In and Out of The Nucleus

• 2. Organization and Function of the Nucleus,
  cont.
• F. Ribosome assembly
• 1. Nucleolus is not surrounded by a membrane.
• 2. Three rRNAs (5.8S, 18S, and 28S) are
  transcribed as a unit and then processed within
  nucleolus by RNA pol I.
• 3. One rRNA (5S) is transcribed outside
  nucleolus by RNA pol III is brought into
  nucleolus for assembly.
• 4. Ribosomal proteins translated in cytoplasm
  transported to nucleolus.
• 5. Assembly of ribosomal proteins with rRNA
  occurs in nucleolus.
• 6. Export of fully assembled large and small
  ribosomal subunits to the cytoplasm

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Trafficking In and Out of The Nucleus

• 2. Organization and Function of the Nucleus,
  cont.
• G. Processing of Eukaryotic mRNA for Export.
  Only a small percentage of pre-mRNAs are
  exported. Events that mature mRNAs must undergo
  in order to be exported include
• 1. Marking of exon boundaries with SR proteins
  
• 2. Proper splicing of introns
• 3. Addition of 5 methyl-guanosine CAP by 3
  enzymes
• 4. Addition of 3 poly A tail by cleavage and
  polyadenylation specificity factors
• 5. hnRNPs that help distinguish between mature
  and immature mRNAs some stay in nucleus, some
  exit with mRNA into cytoplasm
• 6. Exit via interaction with specific nuclear
  transport receptors (i.e. TAP)

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Trafficking In and Out of The Nucleus

• 3. The Nuclear Membrane
• A. Function of the Nuclear Membrane
• 1. Separation of transcription from
  translation allows for post- transcriptional
  processing.
• 2. Limiting access of genome by proteins
  allows regulation of transcription.
• 3. Acts as a barrier for the passage of
  molecules between nucleus and cytoplasm.
• B. Structure of the Nuclear Membrane
• 1. TWO lipid bilayers outer nuclear membrane
  (OM) and inner nuclear membrane (IM).
• 2. Outer nuclear membrane is connected to the
  ER.
• 3. Perinuclear space is continuous with the ER
  lumen.
• 4. Different composition of OM and IM, with OM
  similar to ER membrane.
• 5. IM and OM are joined at the nuclear pore
  complex.
• 6. Nuclear lamina underlying IM provides
  structure is site of chromatin attachment.

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Trafficking In and Out of The Nucleus

• 4. The Nuclear Pore Complex - Except during
  mitosis, essentially all traffic between nucleus
  and cytoplasm in higher eukaryotes occurs via
  nuclear pore complexes.
• A. Structure of the Nuclear Pore Complex
  (NPC)
• 1. Definition channels for trafficking of
  small polar molecules, ions, macromolecules.
• 2. Size diameter 120 nm, 8-fold
  rotational symmetry molecular mass 125 million
  daltons composed of 50 -100 different proteins
  called nucleoporins.

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Trafficking In and Out of The Nucleus

• 4. The Nuclear Pore Complex, cont.
• A. Structure of the Nuclear Pore Complex (NPC),
  cont.
• 3. Number Average cell has over 4000 NPCs.
• 4. Channel Pathway for free diffusion is 9
  nm x 15 nm. Can open to a diameter of more than
  25 nm allowing passage of large complexes.
• 5. Passage across the NPC Proteins lt 40 kD
  pass through the NPC by diffusion, but this is
  often inefficient. Larger proteins and RNA pass
  thru NPC only selectively
• (Gated Transport).

6. Microscopic structure Eight spokes around a
central channel connected to a ring anchored into
nuclear membrane (on cytoplasmic and nuclear
sides). Filaments extend from both sides
forming basket on nuclear side. NPC
extensions may be initial cargo-docking sites
during nucleo-cytoplasmic transport. A subset of
NPC components contain phenylalanine glycine (FG)
repeats that act as docking sites for transport
factors.
From Alberts,Molecular Biology of the Cell, Fig.
12-10
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Trafficking In and Out of The Nucleus

• 4. The Nuclear Pore Complex, cont.
• B. Molecular components of the NPC
• 1. Nucleoporins (NPs)
• Make up the cytoplasmic filaments, nuclear
  basket, and line the pore
• Contain different types of FG (Phe/Gly)
  repeats
• Nuclear transport factors make multiple
  sequential contacts with distinct NPs,
  resulting in docking and translocation of
  nuclear transport complexes
• Exact mechanism by which NPs mediate
  translocation of complexes unclearbut may
  involve sequential association with of
  receptor plus cargo with FG repeats lining pore

From Bayliss, R. et al. Traffic 1448 (2000)
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Trafficking In and Out of The Nucleus

• 4. The Nuclear Pore Complex, cont.
• B. Molecular components of the NPC, cont.
• 2. Soluble nuclear transport receptors in
  cytosol and nucleus
• Nuclear transport receptors include
  karyopherins
• Bind to nucleoporins (NPs)
• Different factors for export and import,
  i.e. Exportin Importin, see below
• Nuclear transport factors bind to cargo
  (or adaptors that bind cargo) that have
  localization or export signals (NLS, NES)
• Cargo can be protein (with NLS, NES) or
  RNA
• Nuclear transport factors bind to and are
  regulated by RanGTP

J. Lingappa, 2003
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Trafficking In and Out of The Nucleus 4. The
Nuclear Pore Complex, cont. B. Molecular
components of the NPC, cont.

• 3. Ran-GTP
• A small GTPase that gives directionality to
  nuclear transport, see below
• Members of the Ras oncogene family (like Rab,
  which regulates vesicle fusion)
• Binds to nuclear transport receptors
• 4. Lamins make up the nuclear lamina
• Lamins are 80 kD proteins related to
  intermediate filaments of cytoskeleton
• Form dimers that polymerize to form filaments
  and a lattice
• Associate with the inner nuclear membrane via
  prenylation
• Give structural support to the nucleus and may
  be involved in recruitment of nuclear envelope
  components

Structure of the nuclear lamina
From Hutchison, Nature Rev. Mol. Cell Bio. 3 849
(2002)
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Trafficking In and Out of The Nucleus

• 5. Trafficking through the Nuclear Pore
• A. Functional relevance of trafficking
• 1. Bring into nucleus transcription
  factors, proteins for ribosome and spliceosome
  assembly, and other proteins needed for nuclear
  functions.
• 2. Export RNAs and ribosomes out of nucleus in
  a regulated manner. Each is exported via a
  specific pathway.
• 3. Shuttling of cellular proteins that go back
  and forth between nucleus and cytoplasm (nuclear
  transport receptors, HnRNPs, etc.).
• 4. Pathogens (mainly viruses) usurp nuclear
  trafficking machinery
• Viral genome import into and export out of the
  nucleus
• Virus entry into nucleus
• Virus exit from nucleus
• Shuttling proteins encoded by viruses
• 5. Pathogens can also destroy cellular nuclear
  trafficking machinery.

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Trafficking In and Out of The Nucleus

• 5. Trafficking through the Nuclear Pore, cont.
• B. Mechanism of Nuclear Trafficking
• 1. Ran GTPase
• a. Little hydrolysis or exchange activity
  on its own.
• b. Regulated by GTPase-activating protein
  (RanGAP) that promotes hydrolysis that is
  predominantly in cytoplasm and guanine exchange
  factor (RanGEF) that promotes GTP exchange that
  is predominantly in the nucleus
• c. So, in the cytoplasm Ran exists in
  inactive GDP-bound form
• d. In contrast, in the nucleus, Ran is
  largely bound to GTP
• e. Ran gradient is critical for most
  nuclear transport, except bulk mRNA transport
  which is mediated by factors that are not
  karyopherins
• f. Ran gradient is created by having
  RanGap (which promotes GTP hydrolysis) only in
  cytoplasm and RanGEF (which promotes loading of
  Ran with GTP) only in nucleus

from Nalkieny and Dreyfus, Cell 99677 (1999)
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Trafficking In and Out of The Nucleus

• 5. Trafficking through the Nuclear Pore, cont.
• C. Mechanism of Nuclear Import
• a. Cargo and/or adaptors encode nuclear
  localization signals (NLS)
• b. NLS can be composed of short stretches of
  basic amino acids, not always contiguous (can be
  bipartite). Other types of NLS exist.
• c. NLS containing proteins (NLS proteins)
  bind directly or via an adaptor to nuclear import
  receptors (i.e. Importin)
• d. Cargo/adaptors bind to import receptors,
  some of which are karyopherins (importins), in
  the absence of RanGTP.
• Note that protein cargo is completely folded
  (unlike in mitochondrial import).
• e. Importin composed of an a subunit that
  binds NLS and a b subunit that binds nucleoporins
  in cytoplasmic filaments.
• f. After translocation thru NPC, import
  receptors release cargo upon binding to Ran-GTP
  in nucleus.

Nuclear Import
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Trafficking In and Out of The Nucleus

• 5. Trafficking through the Nuclear Pore, cont.
• D. Mechanism of Nuclear Export
• a. Cargo and/or adaptors encode nuclear
  export signals (NES)
• b. NLS often composed of leucine rich
  regions.
• c. NES-containing proteins bind nuclear
  export receptors, some but not all of which are
  karyopherins (i.e. exportin).
• d. Nuclear export factors require RanGTP to
  bind cargo in nucleus.
• e. Nuclear export factors release cargo and
  Ran-GTP upon translocation to cytoplasm where
  hydrolysis of RanGTP is induced by Ran-GAP.

Nuclear Export
Nuclear Import
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Trafficking In and Out of The Nucleus

• 5. Trafficking through the Nuclear Pore, cont.
• E. Shuttling Proteins (includes HIV-1 Rev, Vpr,
  and many other viral proteins)
• 1. Proteins contain both nuclear localization
  and export signals (NLS NES).
• 2. These proteins shuttle back and forth
  between nucleus and cytosol.
• 3. Rate of export and import determines in
  which compartment it resides.
• 4. Export/ import of shuttling proteins can be
  regulated, i.e. by phosphorylation-dephosphoryl
  ation of residues adjacent to NLS or NES
  signals resulting in blockade/exposure of signals.

from Nalkieny and Dreyfus, Cell 99677 (1999)
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Trafficking In and Out of The Nucleus

• 5. Trafficking through the Nuclear Pore, cont.
• F. Specific Nuclear Export Proteins
• 1. Crm1 (Exportin), a karyopherin, mediates
  export of most cellular proteins that shuttle,
  several U snRNAs, all rRNAs, and a subset of
  mRNAs.
• Crm1 discovered because it is required for
  the nuclear export of late HIV-1 mRNAs (see
  below), other lentiviruses, and HTLV
• Retroviral Rev and HTLV Rex proteins are
  adaptor proteins that recruit Crm1 to retroviral
  mRNA
• Crm1 pathway specifically inhibited by
  leptomycin B (LMB) which modifies a cysteine
  residue present on the Crm1 protein
• 2. NXF proteins (include Tap) are not
  karyopherins these mediate export of most
  mRNAs. MPMV and other simple retroviruses use
  Tap/Nxt as export factors. However, unlike
  HIV-1 and lentiviruses that encode adaptor
  proteins like Rev that recruit Crm 1, simple
  retroviruses encode a cis-acting RNA sequence
  (consititutive transport element CTE) that
  recruits NXF protein transport factors
• 3. Exp-t (Exportin t), a karyopherin, mediates
  export of tRNA by binding directly to tRNA
  unlike other karyopherins that bind RNA via
  adaptors only binds mature / aminoacylated tRNAs

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Trafficking In and Out of The Nucleus

• 5. Trafficking through the Nuclear Pore, cont.
• G. Specific Nuclear Export Pathways

from Cullen
24
Trafficking In and Out of The Nucleus

• 6. Experimental approaches used to study nuclear
  trafficking
• A. Permeabilized cells/ Cell-free assay
• Digitonin perforates PM (because of high
  cholestrol conc.) releasing cytosol
• Nuclear membrane, nucleus, and other organelles
  remain intact
• Allows add back of cytosolic fractions,
  biochemical manipulations, antibody blockade
• B. Immunofluorescent Tags
• Transfect cells with proteins tagged with GFP,
  RFP, YFP, etc. Assess nuclear vs. cytoplasmic
  location by IF.
• C. Use of chimeric proteins
• Import/Export signals can be engineered into
  heterologous proteins (usually encoding
  fluorescent tags) to demonstrate effect of the
  signal
• Or, fuse an adaptor onto a cargo that doesnt
  bind transport receptor
• D. Heterokaryon assay
• For proteins that are predominantly nuclear and
  shuttle in and out of nucleus.
• Nuclei transfected/injected to express tagged
  protein ( marker protein that stays in "donor"
  nucleus)
• Cells are then fused to another set of cells
  using PEG.
• Examine whether tagged protein shows up in
  cytoplasm alone (export), or in recipient
  nucleus as well (export followed by import,
  implying shuttling).
• E. Biochemical Fractionation
• Ultracentrifugation to separate nuclei from
  cytoplasm
• F. Inhibitors Leptomycin B

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Trafficking In and Out of The Nucleus

• 7. Examples from Pathobiology
• A. Regulation of NF-kB Nuclear Transport
• 1. Masking NLS by binding with an inhibitory
  protein e.g. transcription factor NF-kB.
• I-kB is an inhibitory protein that forms an
  inactive complex with NF-kB in cytosol.
• I-kB binding masks the NLS of NF-kB,
  preventing its translocation into the nucleus.
• Upon stimulation of lymphocytes, I-kB is
  phosphorylated and ubiquitinated resulting in
  proteasome-mediated degradation of I-kB.
• Once released from I-kB, NF-kB can translocate
  into nucleus activate transcription.
• 2. Note that for other proteins, NLS can be
  masked by direct phosphorylation of residues
  adjacent to the NLS.

26
Trafficking In and Out of The Nucleus7.
Examples from Pathobiology, cont. B.
Regulation of NF-AT, a shuttling protein

• Phosphorylated NF-AT is found in the cytosol of
  resting T cells. T cell activation leads to
  increased intracellular Ca2 and
  dephosphorylation of NF-AT (via calcineurin, a
  phosphatase). This exposes an NLS and possibly
  masks an NES, resulting in nuclear import and
  activation of transcription by NF-AT.
• Decreased Ca2 leads to re-phosphorylation of
  NF-AT which inactivates the NLS and re-exposes
  the NES, causing NF-AT to relocate to the
  cytosol.
• Some immunosuppresive drugs act by blocking the
  ability of calcineurin to dephosphorylate NF-AT
  and thereby block nuclear transport of NF-AT.

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Trafficking In and Out of The Nucleus

• 7. Examples from Pathobiology, cont.
• C. How HIV-1 exploits cellular machinery
  for genomic RNA trafficking
• 1. Cellular RNAs almost always exported from
  the nucleus in a fully spliced form (albeit
  sometimes spliced in alternate ways), with
  retention of incompletely spliced RNA in nucleus.
  Question why is this important for the cell?
• 2. But HIV-1 exports incompletely spliced RNA
  out of nucleus. How?
• a. HIV-1 produces multiply-spliced mRNAs
  encoding Tat, Rev, Nef (early proteins)
  singly-spliced mRNAs encoding Vif, Vpr, Vpu, and
  Env (regulatory proteins) and an unspliced mRNA
  encoding Gag GagPol (late proteins).
• b. Initially, only multiply-spliced mRNAs
  are exported out of the nucleus, resulting in
  translation of Tat, Rev, and Nef.
• c. Rev is transported back into nucleus,
  and binds to RRE stem loop present in HIV-1
  singly and multiply-spliced genomic RNA retained
  in nucleus. Rev has an NES that recruits the
  nuclear export factor Crm1 to the Rev/RRE
  complex. Thus, Rev acts as a virally-encoded
  adaptor to mediate nuclear export of genomic RNA
  via Crm1 pathway, resulting in later expression
  of regulatory and late proteins.

28
Trafficking In and Out of The Nucleus 7.
Examples from Pathobiology, cont. C. How
HIV-1 exploits cellular machinery for genomic RNA
trafficking, cont.
29
Trafficking In and Out of The Nucleus 7.
Examples from Pathobiology, cont. D. Other
retroviruses use a different mechanism to export
unspliced genomic RNA Lentiviruses as well
as HTLV-1 use a Rev-like mechanism for nuclear
export of genomic RNA. Type D retroviruses
and avian type C retroviruses have structured
sequences in their genomic RNAs (the
constitutive transport element, or CTE) that
binds directly to transport receptors TAP and
Nxt, which mediate most mRNA export.
Thus, these simple retroviruses bypass the need
for a virally-encoded adaptor protein like Rev.
Direct binding of CTE in MPMV genomic RNA to
export receptors
Rev interacts with a specific loop in the RRE in
the HIV-1 genome via an arginine-rich
RNA-binding motif
J. Lingappa, 2003
Cullen, J. Cell Sci. 116 587 (2003)
30
Trafficking In and Out of The Nucleus 7.
Examples from Pathobiology, cont. E. How
nuclear import machinery is exploited by
Adenovirus (Ad) for viral entry into the nucleus
J. Lingappa, 2003
31
Trafficking In and Out of The Nucleus 7.
Examples from Pathobiology F. Herpesvirus
Egress
Herpesvirus Egress

• Herpesvirus capsids assemble in nucleus.
• Envelopment occurs at inner nuclear membrane.
• One model gaining favor proposes that capsids
  then get de-enveloped at the outer nuclear
  membrane resulting in release of unenveloped
  capsids in the cytoplasm.
• These capsids acquire a new envelope at the
  Golgi.
• Study of mechanism involved in de-envelopment may
  reveal nature and function of proteins that
  reside in space between the two nuclear
  membranes.
• Currently, mechanism of de-envelopment is not
  understood.

From Mettenleiter, J. Virol. 76 1537 (2002)
32
Trafficking In and Out of The Nucleus 8.
Additional Reading

• Cullen, B. R. Nuclear RNA export. J. Cell
  Science 116 587 (2002).
• Bayliss, R. The molecular mechanism of transport
  of macromolecules through nuclear pore
  complexes. Traffic 2000 1448 (2000).
• Nakielny, S. and G. Dreyfuss. Transport of
  proteins and RNAs in and out of the nucleus.
  Cell 99 677 (1999).
• Paushkin, et al. The SMN ocmplex, an
  assemblyosome of ribonucleoproteins. Current
  Opinion in Cell Bio. 14 305 (2002).
• Cullen, B. R. Retroviruses as model systems for
  the study of nuclear RNA export pathways.
  Virology 249 203 (1998).
• Whittaker, G. and A. Helenius. Nuclear import
  and export of viruses and virus genomes.
  Virology 246 1-23 (1998).
• Trotman, L. Import of adenovirus DNA involves
  the nuclear pore complex receptor CAN/Nup214 and
  histone H1. Nat Cell Biol. 31092-100 (2001).
• Mettenleiter, T. C. Herpesvirus assembly and
  egress. J. Virol. 76 1537 (2002).

33

• Digitonin cell membranes that are rich in
  cholesterol, like PM, are permeabilized, but
  internal membranes that are low in cholesterol,
  like nuclear envelope, remain in tact.
• Point out that substrates that traffic in and out
  of nucleus are FOLDED
• Nuclear transport is gated selective transport
  across the NPC as well as diffusion
• Actinomycin D binds to DNA and blocks movement of
  Pol along DNA