Cell Structure part 2
Objectives
• Description of the major organelles of the cell and their functions
(continue)
Nucleus
• Understanding the the life cycle of proteins:
1. Translation
2. Targeting and translocation
Nucleus – post translational translocation
Mitochondria – post translational translocation
ER – co-translational translocation
3. Folding
4. Degradation
The ubiquitin-proteasome pathway
Lysosomal degradation
Cell Structure Part 2
THE NUCLEUS THE LIFE CYCLE OF PROTEINS
Structure
• The nuclear envelope (=nuclear membrane) is comprised
out of two membranes
• The nuclear envelope is a continuation of the ER
• The nucleus contains our genetic material
Function
• The nucleus is involved in all stages of the
biological central dogma of information flow:
DNA → RNA → proteins
1. Translation
• It is the site for:
Translation requires 3 types of RNA
1. DNA storage and replication
molecules:
2. RNA transcription
mRNA= messenger RNA- codes for
3. Ribosome biogenesis (* translation occurs in the
proteins
cytosol)
rRNA= ribosomal RNA- component
• DNA is packed in chromatin - a complex of
of ribosomes
DNA and proteins
tRNA= transfer RNA- delivers and
• Transcription occurs in the active form of
incorporates amino acids into the
chromatin= euchromatin.
growing polypeptide chain
• Heterochromatin = dense, inactive form of
Polysome= mRNA translated
chromatin:
simultaneously by many ribosomes
Visible with light microscopy
Comprises ~90% of total chromatin Translation- steps
Chromatin packing
• Nucleosomes= octamer of histone proteins wrapped with
two complete turns of the
DNA molecule (beads on a string)
• Helical coiling of consecutive nucleosomes allows a Initiation:
dense packing of chromatin 1. The small subunit binds to the 5’ cap of the mRNA
2. Methionine (1st amino acid) tRNA and the large subunit join
The nucleolus 3. Ribosome moves along the mRNA until a AUG codon matches the Met tRNA
The nucleolus is a dense region within the nucleus where
ribosomal RNA synthesis and ribosome biogenesis
occurs
The nucleus functions - Ribosome biogenesis
• Ribosomes are a molecular machine comprised of a
large and a small subunit Elongation:
• Both subunits contain multiple protein and rRNA 1. New tRNA is recruited to the A site (aminoacyl site)
(ribosomal RNA) components 2. The growing polypeptide is attached to the amino acid in the A site
1. Ribosomal proteins are translated and transported 3. Ribosome movement brings: a) the tRNA from P site (peptidyl site) to E site (exit
to the nucleus site) and it is released, b) the tRNA from A site to P site
2. rRNA is transcribed in the nucleolus 4. New tRNA is recruited to the A site
3. Ribosome biogenesis starts in the nucleolus-continues
in the nucleus and cytosol
Termination:
1. The A-site of reaches a stop codon
2. Release factor binds to the A site
3. The tRNA moves from P site to E site and is released
4. The release factor assists releasing the polypeptide from the ribosome
5. The ribosome leaves the mRNA and dissociates into a small and a large subunit.
2. Targeting and traslocation 3. Folding - Chaperones facilitate proper folding of proteins
• Chaperones bind to polypeptide chains that
exit the ribosomes
• They hold the protein, which allows proper
folding and prevents misfolding
Diseases related to protein folding – Prion diseases
• Fatal neurodegenerative diseases
Protein targeting - nucleus • Affect humans (e.g. Creutzfeldt-Jakob disease) and animals
• Protein complexes embedded in nuclear envelope form (e.g. mad cow disease)
9-11 nm pores that are • A pathogenic form of the prion protein is the sole causative
called nuclear pores agent of the disease: the
• The two membranes are united at the nuclear pores infectious protein native protein have identical sequence
• PrPSc seeds the misfolding of PrPC, leading to an
Nuclear pores exponential increase in PrPSc in
• Nuclear pores allow passive diffusion of small proteins the brain and spinal cord that eventually leads to neuronal
• Large proteins are transported by transport factors death
(importins)
4. Protein degradation - the ubiquitin-proteasome pathway
• Nuclear proteins contain a nuclear localization signal
1. Ubiquitin-activating enzyme (E1) activates ubiquitin
(NLS)
2. Activated ubiquitin is transferred to the active-site of an ubiquitin-
conjugating enzyme (E2)
Nuclear Import: the Ran GTPase Cycle
3. Ubiquitin ligase (E3) facilitates the transfer of ubiquitin from E2
Ran GTPase Cycle:
to the
1. Free importin binds a NLS containing protein
target substrate
2. Importin enter the nucleus with the protein
3. Ran-GTP binds importin →protein is released
Ubiquitin labels proteins for degradation and more…
4. Ran-GTP mediates exit of importin from the nucleus
The type of the ubiquitination determines the affect of the
5. GTP hydrolysis triggers release of importin
modification
6. Ran-GDP enters the nucleus. GDP is exchanged to
the substrate differently (localization, interactions, degradation, etc.)
GTP
Summary
The ubiquitin-proteasome pathway
Importin – transports proteins into the nucleus
• Poly ubiquitination chains are often a signal for degradation
Ran mediates the recycling of importin:
• Ubiquitinated proteins are degraded by the proteasome: comprised
Ran GTP (active) - transports importin from the nucleus
of a 20S catalytic
to the cytosol
core and two 19S regulatory complexes
Ran GDP (inactive) – cannot bind importin
• Ubiquitinated protein are recognized by the proteasome → ubiquitin
chain is removed → protein enters the 20S barrel → protein
undergoes unfolding and degradation
Protein targeting and traslocation - Mitochondria
• Mitochondrial proteins contain a targeting
ER associated degradation (ERAD)
“barcode”=Mitochondrial Targeting Sequence
• ERAD = proteasomal degradation of misfolded/damaged ER
(MTS)
proteins
• MTS is recognized by mitochondrial surface
• The ERAD machinery ubiquitinates and pulls out proteins from the
receptors
ER to the cytosol for degradation
• MTS containing proteins are translocated while un-
folded through TOM and TIM,
Cystic fibrosis and Gaucher’s disease- the harmful side of
• Inside the lumen - MTS is cleaved and protein gets
ERAD and protein degradation
folded
• Cystic Fibrosis - mutations in the CFTR gene, which encodes a
TOM=translocase of the outer membrane
plasma membrane ion channel
TIM=translocase of the inner membrane
• Gaucher’s disease is caused by mutations in β-glucosidase, which
encodes a lysosomal enzyme responsible for degradation of lipids.
Protein targeting and traslocation - ER
• Some of the mutations in these genes, (i.e. CFTR ΔF508) lead to
1. How do ribosomes know if they need to stay in the
protein misfolding and degradation by ERAD
cytosol or go to the ER ?
• The mutant proteins are partially functional but ERAD prevents
2. How do proteins that are translated in the cytosol know
them from reaching their cellular distination
where to go in the cells?
Lysosomal protein degradation: Endocytosis
Protein targeting and translocation - ER
• Following endocytosis, receptors can be transported to the
1. Protein synthesis begins and the signal sequence is
lysosomes for degradation
synthesized
• Degradation switches off signaling by eliminating the ligand-bound
2. Binding of Signal Recognition particle (SRP) to the
receptor
signal
Lysosomal protein degradation: Autophagy
sequence stops protein synthesis
• Cytosolic proteins can undergo lysosomal degradation via
3. SRP delivers the ribosome to the ER by interacting with
autophagy
the SRP receptor that is couples to the ER translocase.
• In autophagy a vesicle nucleates in the cytosol, entrapping cytosolic
The targeting signal is inserted into the translocase
components
4. SRP is released and protein translation resumes
• Following sealing of the vesicle, it is targeted and fuses with
5. The targeting signal is cleaved
lysosomes, where its content is degraded
6. The newly synthesized protein is released in the ER
lumen
