Transcript Lecture 5&6 Date: 12/01/03

Lectures 5 & 6
Nuclear Import, Export and
Targeting
Nuclear Import and the Role of Nuclear
Pore Complex Proteins
• Nuclear Localization sequences (NLS’s) are
necessary & sufficient signals for a protein to
be imported into the nucleus from the
cytoplasm.
• Simple SV-40 type: PKKKRKV 7-mer
necessary and sufficient for nuclear import.
• Bipartite or split NLS’s: KR-[PATKKAGQA]KKK: Spacers can be mutated by SDM and still
have import but not NLS.
Discovery of the SV-40 7-mer NLS
• SV-40 is a DNA virus that infects mammalian cells and takes over the
genomic machinery of the cell nucleus.
• SV-40 (T antigen) antigen is a viral protein essential for the regulation
of viral DNA replication and transcription.
• In normal cells the antigen is made in the cytoplasm and is imported to
the nucleus where it is concentrated 10 fold more than in the cytoplasm.
• During a mutagenesis study of the SV-40 virus, a class of mutants were
isolated that had a higher concentration of T-antigen in the cytoplasm
than in the nucleus.
• It was then shown that a single amino acid substitution (K-128) was
sufficient enough to block nuclear import.
• In vitro site directed mutagenesis defined a 7-mer sequence around K128 that was critical for nuclear import.
Infected mammalian cells
Cell membrane
Immunofluorescence using
anti-T-antigen & FITC
conjugated 20 antibody
Nuclear membrane
T antigen
Normal T
Mutant T
The 7-mer NLS is Necessay and Sufficient for Nuclear Import
• A DNA construct was engineered in which the 7- mer peptide was
ligated to the 3’ end of the pyruvate kinase gene.
• Pyruvate kinase is a cytoplasmic protein not found in the nucleus.
• Transfection into cells tested whether the 7-mer is both necessary and
sufficient for nuclear import.
• Results along with the control experiment are shown below.
Pyruvate kinase control construct
Pyruvate kinase - NLS construct
In vitro Systems for Nuclear Import
•  DNA
oocytes in vivo
mininuclei
NPC
Inner nuclear membrane
Chromatin
Outer nuclear
membrane
•  DNA or sperm chromatin + Xenopus oocyte extract
or permeablized cells + cytosol
• real nuclei + Xenopus oocyte extract
Nuclear Source
Synthetic/ real nuclei +
Xenopus extract or
permeabilized cells +
cytoplasmic extract
synthetic nuclei
NUCLEAR IMPORT
NUCLEAR IMPORT
Nuclear import systems
Labeled proteins
Import detection
Radiolabeled
Biochemical quantitation
Fluorescent labeled
Fluorescence microscopy
Colloidal gold labeled
Electron microscopy
Demonstration of ATP and Cytosol Dependent Transport
in Permeabilized Mammalian Cells
• Digitonin was used to permeabilize cells
• The transport of a normally cytoplasmic protein into the nucleus
was observed when it was conjugated at the N-T to a 7-mer NLS.
- Lysate
Newmeyer and Forbes Experiment
[Newmeyer & Forbes, Cell 52 (1988) 641-653]
• Xenopus Ext + real nuclei fluor/c.g. probeimportFluor Mic or EM
• 3 major findings: (1) 7-mer NLS is sufficient & necessary; (2) Role of ATP;
(3) Effect of WGA
• Protein Probes: 7-mer(norm or mutant)-HSA-FITC (Green) [FM]
• HSA= human serum albumin“ “
“
-HSA-colloidal gold [EM]
• Import Assay: 7-mer-HSA (FITC or CG) + Xenopus ext + rat liver nuclei 
examine
• Exp. Results: normal 7-mer: (1) Binds to NPC (2) Accumulates inside nucleus
Mutant 7-mer: (1) No binding to NPC (2) No accumul. inside the nucleus
Normal 7-mer
Mutant 7-mer
ATP Requirement
• Xenopus ext + apyrase  ATP depleted
extract + 7-mer-HSA + nuclei  Binds to
NPC but no import
Identifies Two Steps for Nuclear
Import
ATP Independent Step: Binding to the NPC
ATP Dependent Step: Translocation Through the NPC
Wheat Germ Agglutinin (WGA) Effect
•
WGA Effect: WGA (Wheat germ agglutinin) – plant lectin that binds Nactylglucosamine (GlcNc): Many NPC proteins have 0-linked GlcNc
• Exp: 7-mer probe + extract + WGA + nuclei  7-mer binds to NPC but
no import. Expect that WGA sterically blocks the GlcNcs of NUPs.
NUP
+
NUP
WGA
NUP
WGA sterically blocks
GlcNc’s of NUP’s
Further Distinguishes Nuclear Import into Two Steps:
(1) Binding to NPC (ATP independent and WGA
resistant) ; (2) Translocation through the NPC (ATP
dependent and WGA sensitive)
Finley and Forbes Experiment
[Finley & Forbes, Cell 60 (1990) 17-29]
Depletion experiment to determine the role of NPC
glycoproteins (NPC-GPs) in nuclear import
Xen ext  WGA sepharose  CENTRIFUGE  beads  ext GlcNc NPC-GPs
beads
supernatant
(Xenopus extract depleted of NPC-GPs)
Depletion Exp:
sperm chromatin + depleted extract  synthetic nuclei (depleted)
Synthetic nuclei (depleted) + 7-mer HSA-probe  No NPC binding or import
The absence of binding
to the NPC indicates that
the NPC-GP’s are
required for NPC binding
but that the GlcNc
residues on the GPs are
not required.
Remarkably, the NPCs
reassemble in this
depletion experiment
and “look the same” as
“normal” NPCs
Finley and Forbes experiment contd…
Reconstitution Exp: sperm chromatin + depleted ext. + NPC-GP’s 
synthetic nuclei (reconst) + 7-mer HSA-probeNPC binding and
import
CONCLUSION: Glycosylated (GlucNc) NPC proteins are required
for both steps of nuclear import in vitro [NPC binding and
translocation] but are not essential for the overall architecture
of the NPC.
Mechanisms of Nuclear Import and Export
Gene Expression in Prokaryotes:
DNA  RNA  Protein
Gene Expression in Eukaryotes:
NE
NUCLEUS
DNA  RNAN ----- RNAC  Protein
export
--------- Nuclear Proteins
import
CYTOPLASM
---------
Import
Export
Shuttling Proteins
NUCLEAR IMPORT/EXPORT ARE ESSENTIAL PROCESSES
FOR GENE REGULATION IN EUKARYOTES AND ARE
LIKELY HIGHLY REGULATED PROCESSES
• Proteins to be imported into the
nucleus have Nuclear Localization
Signals (NLS’s) that enable
nuclear import.
Nuclear Import Mechanisms
• NLS’s bind to importin α subunit
of an importin α-β complex.
• Transport through the NPC is
mediated by interaction of
degenerative sequences in the NPC
proteins with the importin β
subunit.
• Key to function and regulation
are RAN GTP [high in nucleus by
RCC1 (Ran nucleotide exchange
factor)] & RAN GDP [high in
cytoplasm by RAN GAP (RAN
GTP activating protein)].
• The asymmetric distribution of
RCC1 in the nucleus and RAN
GAP in the cytoplasm drives the
nuclear import process.
Ran
• The exporting proteins
have special sequences
called Nuclear Export
Signals (NES’s) that
mediate export through
binding to a class of
proteins that function in
export called exportins.
• Exportins are typically
monomeric and function
in a reverse manner to
importin under the
control of RAN.
• Thus the cargo complex
requires RAN-GTP which
is found only in the
nucleus.
• Disassociation of the
‘cargo” from the exportin
requires RAN-GDP
which occurs only in the
cytoplasm.
NUCLEAR EXPORT
Machinery of nuclear import/export contd…
RNA Export:
• In the current model, RNA export occurs by the export of
multiple RNP proteins that cover the mRNA during transport.
• So a more precise term is: “RNP (ribonucleoprotein) export”.
• In the cell nucleus pre-mRNA is packaged into particles
called pre-mRNP or hnRNP particles.
protein
RNA
pre-mRNA
RNP particle
Machinery of Nuclear Import/Export contd…
• During RNA splicing changes occur in the proteins
associated with the hnRNP particles.
• Most dramatic change occurs at the moment of nuclear
export where some of the RNP proteins are “nuclear
restricted” and are therefore released from the RNP
particles while others stay associated with the RNA.
• CBC (cap binding protein complex) initiates the export of
RNA from the 5’ end.
• In the cytoplasm the remaining nuclear RNP proteins are
removed and the RNA gets associated with cytoplasmic
specific proteins which enable the mRNA to associate with
ribosomes and carry out protein synthesis.
• The disassociated RNP proteins are imported back into the
nucleus where they associate with other pre m-RNA’s.
• The RNP proteins imported back into the nucleus “shuttle”
between nucleus and the cytoplasm through the NPC.
• Shuttling proteins have both a NES and NLS sequences.
NLS
NES
Shuttling protein
HETEROKARON ANALYSIS TO DETERMINE IF A NUCLEAR
PROTEIN SHUTTLES BETWEEN NUCLEUS AND CYTOPLASM
1. Construct heterokaryons
Cell Fusion
+
Human cell
Polyethylene
glycol (PEG)
Mouse cell
Heterokaryon
2. Do IF with Ab to protein of interest – Ab must recognize only the species
of interest, e.g., human specific Ab to protein X and see if X is found only in
the human cell nucleus of heterokaryon [nuclear restricted protein] or in
both the human and mouse nucleus [a shuttling protein].
Nuclear restricted
Shuttling protein
Demonstration of a nuclear restricted (hnRNP C, green)
versus a shuttling (hnRNA A, red) protein by heterokaryon
analysis after cell fusion of human (HeLa) and Xenopus
laevis (frog) cells using antibodies specific for the human
proteins
HeLa
H
HK
Xenopus
H
HK
HK
Xen
Xen
HeLa
HeLa
Xenopus
Heterokaryon - solid arrow
HeLa cell – arrowhead
Xenopus cell – dotted arrow
Nuclear Targeting
• Aside from NLS’s and NES’s
there is growing evidence that
many nuclear proteins contain
Nuclear Targeting Sequences
(NTS’s) that target individual
proteins to the sites of genomic
function/organization.
• A classic example is the DNA
methyl transferase (MTase)
which is an enzyme associated
with replication sites in cells and
is responsible for maintaining
the methylation patterns of the
DNA from cell generation to
generation.
• This is important for regulation
of transcription ( highly
methylated genes are generally
not transcribed).
Leonhardt et al. Cell (1992) 71:865-873
Co-localization of MTase (red)
with BrdU labeled (green) RS.
Leonhardt et al. Cell (1992) 71:865-873
Question: How is MTase targeted to RS? Is there a specific region of
the MTase protein that is responsible for targeting the MTase to RS??
• Construct a series of deletion mutants of MTase
• Transfect mammalian cells with MTase constructs fused to the betagalactosidase (β-gal) gene.
• Use anti-β-gal antibodies to detect localization of the fusion protein in the
nucleus and with RS labeled with BrdU method.
Mtase
1-455**
**
1-385
207-511**
**
245-511
[207- 455 REQUIRED FOR TARGETING TO RS]
Fusion p
Nuclear Targeting contd…
Results: A region of the N-terminal MTase is necessary and sufficient
to target β-gal to RS. The targeting sequence is a 248 aa track from
aa 207-455 of the 1,502 aa sequence of the whole protein.
1-1490
1-109;
308-1490
Anti-MTase
Anti-β-gal
Merged