Peroxisomes, biogenesis, function, post-translational import of proteins into organelles Flashcards
RNA transport across NPC
(1) RNA Pol & Their products
- RNA Pol I (47S rRNA)
- RNA Pol II (mRNA & snRNA)
- RNA Pol III (5s rRNA & tRNA)
All move from N -> C, except snRNA.
SnRNA
N-> C for protein binding and modification.
C -> N to function in RNA Splicing
(2) Transport factors for RNA export.
- exportin-t (needs RanGTPase)
- Crm1 (needs Ran)
- Tap (NXF1) - used for mRNA export, Ran-independent.
What are the requirements for mRNA nuclear export?
(1) Signals
- 5’ cap
- PolyAtail
- Absence of Introns
(2) Energy (RNA helicase)
(3) Transport factors
- TAPs (NXF1)
- RNA helicases
- nucleoporins
How does HIV-1 mRNA get exported from the nucleus?
- HIV-1 mRNA can be unspliced or partially spliced. To be exported from the nucleus, it needs the Rev protein.
- The Rev protein binds to the Rev Response Element (RRE) on viral mRNA. It has a Nuclear Export Signal (NES) that helps it transport the mRNA out of the nucleus.
- Splicing factors are attached to unspliced or partially spliced mRNA, causing nuclear retention.
- The Rev protein helps to export unspliced viral mRNA out of the nucleus, allowing it to be translated in the cytoplasm.
Nuclear transport factors have many functions unrelated to nuclear trafficking. What are some examples?
(1) Importin-a.
- nuclear envelope membrane fusion.
- regulation of transcription
- stress response/ stress-granules
- regulation of cell differentiation
- cell scaling, chaperone function.
(2) Importan-b
- mitosis
-stress response/ stress-granules
- protein disaggregation, chaperone function
(3) Ran - mitosis.
(4) CAS - regulation transcription.
(5) CrmI - transport through nucleolus.
(6) Nupo89 - control of transcript stability.
(7) DDX3 (RNA Helicase)
-stress response/ stress-granules
Stress Granules (SGs)
- dynamic
- form due to oxidative stress, temp changes, pH imbalances, altered salt concentrations.
- temporary structures formed in cytoplasm when a cell is stressed.
- they survive under adverse conditions by
(1) Recruiting pro-apoptoptic factors to regulate cell death.
(2) Sequestering mRNAs to protect them from degradation. - once stress is removed, they disassemble and return to their original state.
if stress is too intense or chronic, SGs formed (stable/gel-like)
- form fibrils -> neurodegenerative disease, cancer, viral infections.
- phase seperation & droplet formation play a role in SG formation.
- SGs are made up of RNA binding proteins and cellular trafficking proteins.
- Importin-alpha-1 is critical for forming large SGs, without it, would be smaller-pore likely to die under stress.
- droplet compartments form when proteins and RNA mix together, adding more RNA can make these droplets hollow.
- many chemotherapy agents stress the cancer cells, produce SGs, less prone to apoptosis and cell death which is a problem.
What is the role of nuclear transport receptors in neurodegeneration?
Nuclear transport receptors, like Transportin-I, help limit pathological Liquid-Liquid Phase Separation (LLPS) during neurodegeneration.
What happens with mutant TDP-43 & FUS proteins? How does Transportin-I help?
- Mutant TDP-43 and FUS proteins (linked to ALS and FTD) tend to aggregate in cells, contributing to neurodegenerative diseases.
- Transportin-I, a nuclear import receptor, prevents aggregation of mutant proteins and helps remove them from the cell, limiting damage caused by protein accumulation.
Describe post translational import of proteins into different organelles.
(1) proteins synthesized in cytoplasm (nascent polypeptide, chaperones help).
(2) proteins then bind to chaperones.
(3) protein-chaperone complex move to membrane-receptor in organelle.
(4) receptor recognizes protein target sequence, helps transport protein into organelle.
(5) once inside, another chaperone helps proteins final folding.
Peroxisomes and Their Functions
- Peroxisomes: Organelles surrounded by a single membrane, enclosing the peroxisomal matrix.
- Functions:
(1) Detoxification: Catalase enzyme oxidizes compounds and removes H₂O₂ (regulates cellular redox status).
(2) B-oxidation: Breaks down long fatty acid chains.
(3) Synthesis of Plasmalogens: Important for myelin production
Peroxisomes originate from the cytosol and are involved in processes that take place in the cytoplasm, such as detoxification and fatty acid breakdown.
What mutants were instrumental in the study of peroxisomal protein sorting?
Yeast Mutants
What are the two ways that peroxisomes can form?
(1) By division of existing peroxisomes.
(2) De novo formation (from scratch)
a) Starts with membrane vesicles from ER.
b) fuse with mictochondria-derived vesicles, forming precurosor, pre-peroxisome.
c) Matures by acquiring peroxisomal membrane proteins (PMPs)
Once mature, can replicate by growing and dividing.
For protein import into peroxisomal or mitochondrial matrix, need signals, energy, cellular import apparatus.
How does protein transport work into the peroxisomes?
- all membrane and matrix proteins encoded by nuclear genes.
- protein import (mediated by transient translocon, involves phase seperation) into peroxisomal matrix is post-translational.
- FOLDED proteins can be transported into peroxisomal matrix; requires ATP.
- Peroxins (encoded by PEX genes) mediate peroxisomal biogenesis & protein import.
What are targeting signals for peroxisomal matrix?
(1) PTS-I (C-terminal Signal)
- SKL sequence (Ser, Lys, Leu)
- Sequence never cleaved of.
- Must be placed at C-terminus.
(2) PTS-2 (N-terminal Signal)
- Some cases signal can be cleaved off.
- mutations in this signal cause mislocalization of proteins to mitochondria.
Targeting of PTS-I Containing proteins to Peroxisomal Matrix
(1) Protein starts unfolded in the cytoplasm.
(2) Protein folds and binds to PTS-1 receptor (also called PEX-5)
(3) Protein-receptor complex forms and docks at peroxisomal membrane (no energy, at 4C)
(4) Receptor and cargo (protein) move into peroxisomal matrix (need energy -> translocation)
After protein is delivered, complex dissociates, PEX-5 receptor sent back to cytoplasm. (needs energy)
What is a special characteristic about the peroxisomal membrane translocon?
- it is transient
- this is a protein complex that helps with protein transport across membranes
- temporarily forms to import proteins into peroxisome and disassembles once process is complete.
How are proteins imported into Peroxisomes?
(1) PEX13 membrane protein has conserved YG region that forms a meshwork in the lipid bilayer.
- YG domains can form hydrogels in vitro.
- PEX5 binds to YG domain meshwork.
-this moves cargo across meshwork into peroxisomal lumen.
How is PEX5 recycled?
- PEX-5 recognizes and binds to proteins that contain a type 1 peroxisomal targeting signal (PTS1), which is typically a C-terminal tripeptide sequence (usually Ser-Lys-Leu).
(1) PEX5 is attached to cargo proteins, and it enters peroxisomal matrix.
(2) PEX14 binds to PEX5 receptor and associates with another protein complex in matrix.
(3) PEX5 ubiquitinated and transferred to another protein complex.
- on the cytoplasmic side, PEX1 and PEX6 form AA-ATPase complex. this complex pulls PEX5 out of peroxisome by Ub tags (retrotranslocon).
Then in cytoplasm, Ub removes from PEX5 and cycle repeats.
- Movement is driven by reversible Ub of PEX5.
- Transient pores in peroxisomes have narrow opening (around 2nm), allowing only small molecules to pas.
What does PEX5 recognize? What does PEX7 recognize?
(1) PEX5 -> SKL/C-terminal
(2) PEX7 -> Internal targeting sequence for perixosomes.
What is a Peroxisome Biogenesis Disorder?
(1) Zellweger Syndrome.
- mutation in PEX5 (imports proteins into peroxisomes)
- lack of functional peroxisomes, impairing cellular metabolism.
Targeting to Mitochondrial Matrix
- most mitochondrial proteins are synthesized in the cytoplasm from nuclear-encoded mRNAs.
- proteins must be imported into mitochondira using signal sequences.
- then, organelles have protein translocator complexes that guide proteins to their proper location.
MTS (mitochondrial targeting sequence)
- 25-35 amino acids, form amphipathic helix.
- chaperones bind to proteins in cytoplasm to keep them unfolded and transport competent.
- proteins bind to receptors on the OMM.
What are the steps of protein import into the mitochondrial matrix?
(1) Protein unfolded binds to chaperone (Hsp70) in cytoplasm, prevents premature folding.
(2) Binds to TOM complex.
(3) Moves to TIM23 complex.
(4) Mitochondrial Membrane Potential pulls protein into matrix.
(5) Signal peptidase cleaves signal sequence in matrix.
(6) Mitochondrial chaperones (Hsp70,60,10) help fold protein into functional state.
What are energy requirements for protein import into mitochondrion?
(1) ATP in cytoplasm for Hsp70 chaperones to maintain unfolded state.
(2) Membrane potential across IMM (drives protein translocation through TIM23)
(3) ATP in mitochondrial matrix -> for mitochondrial chaperones.
What are the two pathways for protein sorting to the Inner Mitochondrial Membranes?
(1) Pathway 1
- sorting to IMM via Oxa1.
a) Protein enters via TOM and TIM23, signal is cleaved.
b) Secondary signal interacts with Oxa1 complex, embedding protein in IMM. (for proteins imported from cytoplasm or proteins made in mitochondria)
(2) Pathway 2
- direct sorting to IMM via Tim22.
a) Protein enters via TOM, does not enter matrix.
b) Protein released into inter-membrane space.
c) Tiny-tims (chaperones) keeps soluble.
d) Tim22 complex inserts proteins into IMM.
e) need ATP in cytoplasm
membrane potential across IMM
no atp in matrix
How do proteins get sorted to outer mitochondrial membranes?
- protein enters TOM complex via OMM.
- tiny tims keep protein unfolded in inter-membrane space.
- SAM complex helps insert and fold protein in OMM.
- ATP in cytoplasm.
- no membrane potential/ATP in matrix.
