Cytoplasmic Membrane Systems I
Lecture 11
The Cellular Compartmentalization
Problem of Eukaryotic Cells
Cytoplasm:
Cytosol plus
Organelles
Excluding
Nucleus
How Do Proteins Get Imported Into
Membrane Enclosed Organelles?
Import Requires Input of Energy to Occur!
Pathways for Protein Trafficking
• Default Localization
(Remain in Cytosol)
• Signal Mediated
Localization
– Gated Transport
– Transmembrane
Transport
– Vesicular Transport
Secretory
Pathway
The Eukaryotic Nucleus
Why Have One?
• Separation of mRNA synthesis/processing
from translation- Allows for greater
regulation of gene expression
• Separate Fragile Chromosomes from
Cytoplasmic Cytoskeletal Filaments
Involved in Cell Movement
The Eukaryotic Nucleus
Distinctive Features:
Nuclear Envelope
Nuclear Lamina
Chromatin Organization
Subcompartments that
lack Membranes,
including Nucleoli
Nuclear Pore Complexes
The Nucleus is Bound by the Nuclear Envelope
Double Membrane
Nuclear Pore Complexes
The Only Channels for
Transport Between
Nucleus and Cytoplasm
Lumen Contiguous
With ER Lumen
The Nuclear Lamina Underlies the Nuclear
Envelope and Provides Structural Support
Composed of Intermediate Filaments called
Lamins
Located Beneath the Inner Membrane and
Physically Connected by:
-Integral Lamina Associated Proteins
-Lipid Anchors on Lamins
Also Associated with Chromatin via proteins
- May be important in organizing Replication
and Transcription
Nuclear Lamina
Chromatin
Chromosomes Occupy Distinct Areas of The
Nucleus Called Chromosome Territories
Chromosomal
Territory
Interchromosomal
Domain
Nucleoli are Sites of RNA synthesis and Processing
rRNA
tRNA
Additional RNA
Assembly of Ribosomal Subunits Occurs
in the Nucleolus
rRNA Synthesis and Processing and Ribosomal
Subunit Assembly Occur in Nucleoli
Speckles are Sites of snRNP Storage
Nucleoli
Speckles
(and Cajal Bodies)
Bulk
Chromatin
Cajal
Bodies
Nuclear Pore
not simple “holes”
in the membrane
Selective Gateways
for two-way traffic
Nuclear Pore Complexes are
Embedded in the Nuclear Membrane
Nucleus
1 µm
Nucleolus
Nucleus
Nuclear envelope:
Inner membrane
Outer membrane
Nuclear pore
Pore
complex
Surface of nuclear
envelope.
Ribosome
1 µm
0.25 µm
Close-up of
nuclear
envelope
Pore complexes (TEM).
Nuclear lamina (TEM).
EM view of Xenopus Nuclear Pores
Nuclear
(Basket)
Cytoplasmicside
Cytoplasmic
Large Molecules are Actively Transported
Between Nucleus and Cytoplasm
Active Transport through the Nuclear Pore Complex
has the following Features:
Energy Dependent
Signal Dependent
Temperature Dependent
Can Be Saturated
These are Features of a Carrier Mediated Process
Large Molecules are Actively Transported
Between Nucleus and Cytoplasm
Active Transport: Translocation
Diffusion
Small Molecules
And Proteins up to
40 kDA
ribosomal
Proteins
snRNPs
nuclear
proteins
Small
uncharged
molecules
tRNAs
mRNPs
snRNAs
miRNAs
Nucleus
ribosomal
subunits
Nuclear Pores Are Busy!
A Single Human Cell contains 10 million Ribosomes, has
~4000 Nuclear Pores and divides every 24 hours
Needs 400, 000 ribosomal proteins imported every
minute (~100/pore)
Needs 12, 000 Ribosomal Subunits Exported every
minute (~3 per pore)
If DNA is Synthesized- needs 1 million new Histone
Proteins every 3 minutes
Nuclear Pores Contain a Open Channel to
Allow for Small Molecules to Diffuse
120 nm
9-10 nm
Nuclear Pore Complexes are Symmetrical Structures
Located Where Inner and Outer Membranes are Fused
Composed of
30-50 Different
Pore Proteins
called
Nucleoporins
Present in
Multiple Copies
Proteins Are Selectively Transported through Nuclear Pores
1) Signal Dependent
NLS- Nuclear Localization Signal –Basic Amino Acids
NES- Nuclear Export Signal - Leucine Rich Sequence
2) Receptor Mediated Process
Involves Receptor Transport Proteins that Recognize NLS or
NES of Cargo Proteins
Nuclear Transport Receptors are often part of the large family of
Karyopherin proteins
Importins- Involved in Nuclear Import
Exportins- Involved in Nuclear Export
3) Regulated by G-protein RAN
Nuclear Localization Signals are Necessary
and Sufficient for Nuclear Import
General Characteristics but No Elaborate Sequence Requirements
Classical NLS Sequences
1. Short Stretch of Basic Amino Acids
Lysine and Arginine Rich
4-8 residues
Best Characterized: NLS of SV 40 T-Antigen
PPKKKRKV
2. Bipartite NLS sequences
Two stretches of Basic Amino Acids about 10 aa Apart
Example: NLS of nucleoplasm protein
KRPAATKKAGQAKKKK
Mutation of the
SV-40 T-Antigen
NLS Abolishes
Nuclear Import
Cytoplasmic Nuclear Transport Receptors
Mediate Nuclear Protein Import
(Importin)
Karyopherins Transiently Bind the Nucleoporins
That Line the Route through the Nuclear Pore
Nup358
Nup214
Nup62
Nup98
Nup153
Movement of Proteins through
NPC is regulated by the G-protein RAN
Distribution of RAN-GTP Across
the Nuclear Envelope
GAP:
Stimulates
RAN GTP
Hydrolysis
GEF:
Stimulates
Exchange
of GDP
for GTP
by
RAN
Nuclear Import:
Dissociation of Importin-Cargo Complexes
Requires Ran-GTP binding
1.
Recognition of NLS of Cargo
Protein by Importin
(Nuclear Transport Receptor)
2.
Importin-Cargo Complex
binds specific nucleoporin
proteins of cytoplasmic
filaments
3.
Complex translocated
4.
Dissociation of Complex
at nuclear side requires
RAN-GTP binding
Nuclear Export:
The Assembly of Exportin-Cargo Complexes
Requires RAN-GTP
IMPORT
EXPORT
1.Protein with Nuclear Export Signal (NES)
Bound by Export Receptor (Exportin)
And RAN-GTP
Cytosol
2. Complex Associates with Nucleoporins
And Translocates to Cytoplasm
3. Once in Cytosol Exportin-Cargo
Complex Dissociates
Ran-GTP hydrolysis  RAN-GDP
4. Exportin recycled back to Nucleus
Nucleus
Why Aren’t Nuclear Localization Signals Removed?
Nuclear Proteins Undergo Repeated Nuclear Entry
• Some proteins shuttle between nucleus and cytoplasm Nuclear Proteins are capable of repeated entry into the
nucleus
• When mitosis occurs, the nuclear envelope breaks
down- cytosolic and nuclear proteins mix- when it is
reformed- nuclear proteins need to be reimported
Multiple Classes of RNA are
Exported from the Nucleus
• The Same NPCs used for protein transport are used for
RNA export
• Transport of most RNA is unidirectional from nucleus to
cytoplasm
• Export of RNA is receptor mediated and energy dependent.
• Different soluble transport factors are required for
transport of each class of RNA
mRNA Are Exported from the Nucleus
as RNA–Protein Complexes
Most mRNP require TAP/MEX67
Functions like Exportin
Regulated Nuclear Export of Unspliced
RNA by the HIV Rev Protein
How does HIV
Assemble Progeny
Virus Containing
A Single Stranded
Unspliced
RNA Genome?
Transport of Various RNAs and RNPs
• Ribosomes
– Need to Import Ribosomal Proteins
– Need to Export Ribosomal Subunits
– Involves Karyopherin family of Receptor proteins
– RAN dependent
• tRNA
– Dedicated transport receptor- Exportin-t- only one binds RNA directly
– Requires RAN
– Aminoacylated and fully processed tRNA are the preferential substrate for
Exportin-t
• microRNA
– Dedicated transport receptor Exportin- V
– Requires RAN
• snRNA– Exported to Cytoplasm to form snRNPS- then imported by Snurportin and
karyopherin family members