Perixosomes
Introduction
Instructor Information:
Dr. Triana Amen
Email: t.amen@soton.ac.uk
Office: Building 85, Room 3035
Course Details:
Course Code: BIOL2056
Topic: Cell Biology Protein Targeting: Peroxisome
Recommended Literature:
Alberts, Molecular Biology of the Cell, 6th Edition, Chapter 12
Learning Objectives
Understanding the role of peroxisomes in:
Redox homeostasis
Lipid metabolism
Knowledge of peroxisomal protein import machinery and targeting signals
Mechanisms of protein translocation into peroxisomes
Characteristics of protein translocation specific to peroxisomal import
Case studies on peroxisome biogenesis disorders
Importance of Peroxisomal Protein Targeting
Cellular Universality:
All eukaryotic cells contain peroxisomes
Functional Roles of Peroxisomes:
Synthesize essential lipids (e.g., ether lipids)
Catalyze reactions transforming electrons to oxygen, producing hydrogen peroxide (H2O2)
Bioluminescence: Light produced by luciferase in peroxisomes
Peroxisome Targeting Signals (PTS)
Types of Peroxisome Targeting Signals:
PTS1 (Peroxisomal Targeting Signal-1):
A C-terminal tripeptide sufficient for directing proteins to peroxisomes
Sequence: [SAGCN]1-[RKH]2-[LIVMAF]3
Localized at the C-terminus
PTS2 (Peroxisomal Targeting Signal-2):
An N-terminal sequence providing a second import pathway
Sequence: [RK]-[LVIQ]-X-X-[LVIHQ]-[LSGAK]-X-[QH]-[LAF]
Localized close to the N-terminus
Note: Only one PTS (either PTS1 or PTS2) is sufficient for import into peroxisomes.
Peroxisomal Import Machinery
Reception and Recognition:
PEX5: The receptor for PTS1 and PTS2
Interacts with PTS signals using tetratricopeptide repeats (TPRs) within its C-terminal half
Interaction with PEX7 for PTS2
Structure: C-terminal tetra-helical 34 aa motifs
Translocation Complex:
PEX13 and PEX14: Subunits of the peroxisomal protein translocation complex
Identified complexities not previously observed
PEX5-cargo complex binds the PEX13/14 translocation complex to facilitate internalization into peroxisomes
Mechanism of Peroxisomal Import
Import Process:
Peroxisomes are capable of importing large folded proteins, protein complexes, and even particles coated with PTS
Cargo size: Particles around 0.2µm
Example: Import of 9 nm gold particles coated with PTS (Walton et al., 1995)
Pore Complexes:
Speculation on large pore complexes enabling import has not been visually confirmed
PEX13 features YG repeats akin to FG repeats in nuclear pore complexes facilitating nuclear import
Import Models
Three Models:
NPC-like Model:
Proteins synthesized in the cytoplasm
Limited diffusion between cellular compartments
Condensation Model:
PEX5-cargo complexes cluster through interactions with PEX13
YG-rich repeats create structure aiding in cargo import
Energy Considerations
Energy Requirement:
Peroxisomal import is energy-independent
PEX5 Recycling:
PEX5 recycling requires ATP energy through PEX1/PEX6 ATPase
Peroxisome Dysfunction & Development
Brain Development Impact:
Loss of PEX5 results in impaired import and complete loss of peroxisomal functions
Related to Peroxisome Biogenesis Disorders (PBD) characterized by neurological defects such as demyelination
Reference: Zellweger spectrum of PBDs (Powers and Moser, 1998)
Dual Localization Potential
Proteins with multiple targeting signals can localize to different compartments
Example: Yeast phosphatase Ptc5 with both N-terminal mitochondrial localization sequence and C-terminal PTS1
Piggyback Mechanism
External Interaction for Import:
Proteins can utilize interaction with other peroxisomal cargoes to gain entry
Example: Malate dehydrogenase Mdh2 enters via interaction with Mdh3 that contains PTS1 (Gabay-Maskit et al., 2020)
Summary of Peroxisomal Import
Recognition and Translocation:
PEX5 recognizes PTS-containing proteins and transports them to peroxisomes
PEX13 and PEX14 facilitate the transport of PEX5-cargo complexes
Proteins can localize to multiple places within cells
Recommended Literature & Further Questions
Literature for Further Reading:
Alberts, Molecular Biology of the Cell, 6th Edition, Chapter 12
Identified studies on nuclear pore-like phases in protein import, and mechanisms via protein phase separation
Research on the piggybacking mechanism for peroxisomal localization
Questions for Further Exploration:
Can a protein change its localization to a different cellular compartment?
What is the mechanism of the piggybacking approach for protein translocation?
Assessment & Feedback
Engagement Encouraged:
Students are invited to express their views via an anonymous Assessment Environment Questionnaire
Opportunity for additional involvement with a focus group leading to gain rewards for feedback participation.
Introduction
Instructor Information:
Dr. Triana Amen
Email: t.amen@soton.ac.uk
Office: Building 85, Room 3035
Course Details:
Course Code: BIOL2056
Topic: Cell Biology Protein Targeting: Peroxisome
Recommended Literature:
Alberts, Molecular Biology of the Cell, 6th Edition, Chapter 12: "Intracellular Compartments and Protein Sorting"
Learning Objectives
Metabolic Roles:
Mastery of peroxisomal roles in redox homeostasis, specifically the management of reactive oxygen species (ROS).
Understanding lipid metabolism, focusing on $\beta$-oxidation of very-long-chain fatty acids (VLCFA) and ether lipid synthesis.
Molecular Mechanisms:
Detailed knowledge of peroxisomal protein import machinery (PEX proteins).
Identification of Peroxisome Targeting Signals ($PTS1$ and $PTS2$).
Analysis of the unique translocation characteristics (e.g., importing fully folded proteins).
Pathology:
Evaluation of Peroxisome Biogenesis Disorders (PBDs) such as Zellweger Syndrome through case studies.
Importance of Peroxisomal Protein Targeting
Cellular Universality:
Present in nearly all eukaryotic cells. Unlike mitochondria or chloroplasts, they lack their own genome and must import all proteins from the cytoplasm.
Functional Diversity:
Lipid Synthesis: Synthesis of plasmalogens (the most abundant class of phospholipids in myelin), essential for insulating nerve cells.
Oxidative Reactions: Peroxisomes contain enzymes that remove hydrogen atoms from specific organic substrates ($R$) in an oxidative reaction that produces hydrogen peroxide ($H{2}O{2}$):
$RH{2} + O{2} \rightarrow R + H{2}O{2}$
Catalase Action: Peroxisomes contain high concentrations of the enzyme catalase, which utilizes the $H{2}O{2}$ to oxidize other substrates (phenols, formic acid, alcohol) or converts it to water: $2H{2}O{2} \rightarrow 2H{2}O + O{2}$.
Bioluminescence: In fireflies, the enzyme luciferase is targeted to peroxisomes to produce light.
Peroxisome Targeting Signals (PTS)
PTS1 (Post-translational):
The most common signal found in the majority of peroxisomal matrix proteins.
Sequence: A C-terminal tripeptide ($SKL$ motif: Serine-Lysine-Leucine) or variants described by $[SAGCN]{1}-[RKH]{2}-[LIVMAF]_{3}$.
Position: Must be at the extreme C-terminus; addition of extra amino acids after the signal usually abolishes targeting.
PTS2:
Used by a smaller subset of proteins.
Sequence: A nonapeptide sequence near the N-terminus: $[RK]-[LVIQ]-X-X-[LVIHQ]-[LSGAK]-X-[QH]-[LAF]$.
Processing: Often involves the cleavage of a pro-peptide after import, unlike $PTS1$.
Efficiency: Only one signal (either $PTS1$ or $PTS2$) is required for successful translocation.
Peroxisomal Import Machinery
Receptor Recognition:
PEX5: The primary soluble receptor for $PTS1$. It utilizes tetratricopeptide repeats (TPRs) to bind the C-terminal cargo.
PEX7: The soluble receptor for $PTS2$. In many organisms, PEX5 acts as a co-receptor or "adapter" for PEX7.
Structure: PEX5 is a "shuttling receptor," meaning it moves between the cytosol and the peroxisome interior.
Docking and Translocation Complex:
PEX13 and PEX14: These are integral membrane proteins that form the docking site for the receptor-cargo complex.
Convergent Pathways: Both $PTS1$ and $PTS2$ pathways converge at the PEX13/PEX14 translocation complex.
Mechanism of Peroxisomal Import
Folded Import: Unlike the Sec64 or TOM/TIM complexes (ER and mitochondria), peroxisomes import proteins in their fully folded and even oligomeric states.
Evidence: Experiments showed that $9 \text{ nm}$ gold particles conjugated to $PTS1$ signals were successfully imported into the matrix ($Walton \text{ et al., } 1995$).
Cargo Size: Peroxisomes can accommodate particles up to approximately $0.2 \mu m$ in diameter.
Pore Dynamics:
There is no permanent large pore. Instead, a "transient pore" or "cargo-triggered pore" is hypothesized to form upon the binding of PEX5-cargo complexes to PEX14.
Y-G Repeats: PEX13 contains tyrosine-glycine ($YG$) repeats, similar to the $FG$ (phenylalanine-glycine) repeats in nuclear pore complexes, suggesting a shared evolutionary strategy for moving large complexes across membranes.
Energy and Receptor Recycling
Energy-Independent Import: The actual translocation of the cargo into the matrix does not directly require $ATP$ hydrolysis.
The Recycling Phase: $ATP$ is required to remove the receptor (PEX5) from the peroxisomal membrane back into the cytosol.
Mechanism: PEX5 is mono-ubiquitinated at a conserved cysteine residue to signal its release.
The AAA-ATPase Complex: $PEX1$ and $PEX6$ are $AAA$ (ATPases Associated with diverse cellular Activities) proteins that use the energy of $ATP$ to pull PEX5 out of the membrane, resetting the system for the next round of import.
Peroxisome dysfunction and Pathology
Peroxisome Biogenesis Disorders (PBD):
Zellweger Spectrum Disorders (ZSD): Caused by mutations in PEX genes ($PEX1, 2, 3, 5, 6, 10, 12, 13, 14, 16, 19, 26$).
Clinical Manifestations: Characterized by "empty" peroxisomes (ghosts) where the membrane exists but matrix enzymes remain in the cytosol and are degraded.
Symptoms: Severe neurological deficits, hypotonia, demyelination, and liver dysfunction. Death often occurs within the first year of life ($Powers \text{ and } Moser, 1998$).
Metabolic Impact: Failure to oxidize $VLCFAs$ leads to their accumulation in the brain and adrenal glands, disrupting myelin formation.
Advanced Targeting: Piggybacking and Dual Localization
Dual Localization: Some proteins contain multiple signals. Example: Yeast $Ptc5$ has a mitochondrial signal at the N-terminus and a $PTS1$ at the C-terminus, allowing it to function in both organelles depending on cellular metabolic state.
Piggybacking: Proteins without a $PTS$ can enter the peroxisome by binding to a protein that does have one.
Example: In yeast, Malate dehydrogenase $2$ ($Mdh2$) lacks a targeting signal but enters the peroxisome by forming a complex with $Mdh3$, which possesses a $PTS1$ ($Gabay-Maskit \text{ et al., } 2020$).
Summary of Peroxisomal Import
Recognition: Soluble receptors (PEX5/PEX7) bind cargo in the cytosol.
Docking: The receptor-cargo complex binds PEX14/PEX13 at the membrane.
Translocation: The complex enters a dynamic pore; folded proteins pass through.
Release: Cargo is released into the matrix.
Recycling: PEX5 is ubiquitinated and extracted by $PEX1/PEX6$ in an $ATP$-dependent manner.
Recommended Literature & Research Questions
Reading: Alberts $MBOC$, Ch $12$.
Future Research Topics:
Exploring the mechanism of "protein phase separation" in forming the peroxisomal translocation pore.
How do cells regulate the ratio of dual-localized proteins between mitochondria and peroxisomes?