M4L1 - Transport to Peroxisome

Peroxisome Characteristics 

• Small-oval shaped organelle 

• Bound by a single membrane 

• Responsible for oxidative/synthetic functions

• doesn’t have its own genetic info 

• Reproduces by binary fission

Peroxisome TEM

• Contains dense regions with protein aggregates

• Protein: catalase enzyme (anti-oxidant protein)

Function in Other Cells of Peroxisome

Animal Cells:

• Peroxisome responsible for cholesterol synthesis

• In nerve cells, it synthesizes plasmalogen

• In liver cells, it is the site of toxin oxidation (alcohol) 

Plant Cells: 

• Requires for conversino of fatty acid to carbs (important in seeds germination)

All Cells:

• Responsible for catalysis of fatty acids

Major Function of Peroxisome 

• Breakdown of very long fatty acids through beta-oxidation

• Animals: Long fatty acids are oxidized to medium ones

  • Goes to mitochondria for further processing

  • They can then be used as energy source

Fatty Acid Oxidation in Peroxisome

• Done through beta-oxidation 

• Fatty acids yield the most ATP on E/gram basis compared to other macromolecules 

• Oxidation of fatty acids in peroxisome helps release metabolic energy 

• This process creates hydrogen peroxide byproduct (harmful) 

• Catalase Enzyme: 

  • Abundant in peroxisomes 

  • Converts H2O2 into O2 and H2O molecules (not harmful) 

  • Catalase can be used as a marker for peroxisome visualization in cells bc it’s abundant 

Catalase Enzyme (Peroxisome and Beta Oxidation)

• Abundant in peroxisomes 

• Converts harmful H2O2 byproduct into O2 and H2O molecules (not harmful) 

• Catalase can be used as a marker for peroxisome visualization in cells bc it’s abundant

Fluorescence microscopy images of catalase in cell

• Uses catalase primary antibody (immunofluorescence)

• This is recognized by a red-flourescent secondary antibody 

• Each dot represents a peroxisome

• Not high resolution, but you can identify there functional peroxisomes exist in the cell 

fluorescence image of a single cell (DNA/actin microfilament/catalase)

• DNA stain (viswualize nucleas in red)

• Actin microfilament network (shown in blue)

• Catalase enzyme labelled with antibody (green)

• Each bright feen stain represents individual peroxisomes

• Fluorescence can look at living cells while TEM cannot

  • Peroxisomes aren’t static

  • They move around, change shape, undergo fission/fusion 

Peroxisome Biogenesis

• Peroxisomal proteins are synthesized in the cytosol then transported to the peroxisome 

• Peroxisomal membrane proteins (PMP70) attached to precursor membranes creates ‘peroxisomal ghost’ 

• They’re used to transport peroxisomal matrix proteins such as catalase inside 

• More proteins synethesized = more growth of peroxisome 

• New peroxisomes can form by fission 

• This lets cells make more peroxisomes and give them to new cells

Luciferase: Peroxisomal Protein Transport

• Enzyme that allows the bioluminescence in fireflies 

• This enzyme is found in the peroxisome of cells in firefly abdomens 

• Experiment: 

  • Inserted luciferase genes into mammals 

  • Found that after expression, it went straight to peroxisomes 

  • Showed mechanism for sending it to the peroxisome is same for both fireflies and mammals

  • Signals and transport proteins involved are the same 

5 Rules for Protein Transport

1. A signal sequence on the transported protein 

2. A receptor for that signal sequence on the target organelle/membrane 

3. A translocation channel to help get the protein across 

4. Required energy (ATP) at some step in the process

5. A way of targeting a protein to specific locations within the organelle 

Rule 1 for Protein Transport: Peroxisome Protein Ex

• PTS1: Peroxisomal-Transport Sequence 1 (aka SKL)

  • Found in many species’ peroxisomal proteins 

• Tri-peptide made of 3 amino acids 

  • serine

  • lysine

  • leucine 

• Found at C-terminus of translated peroxisomal protein 

  • N-terminus translated first 

  • C-terminus translated last 

  • Because the signal sequence isn’t available until the entire protein is finished, the protein is sent to the peroxisome after translation is complete.

  • This is called post-translational transport

• It folds in the cytosol, leaving the C-terminal sequence visible 

  • Facilitates transport 

PTS1: Necessary for Protein Import?

Basis: 

• Wild-type Luciferase

  • Signal Sequence is present so protein is visible within bioluminescent peroxisomes in this single cell 

• C-terminus Deletion 

• Hypothesis: If PTS1 is necessary for protein transport into peroxisome, then luciferse with the deletion will no longer go 

Results:

• Image B: Luciferase in spots in the cell

• Image C: Same cell stained with antibody to catalase

  • Colocalization of the spots confirms luciferase is in the same location as catalase

  • Suggests successful transport to peroxisome

• Image D: Modified luciferase protein (no signal) 

  • Bioluminescence is shown throughout the cytosol 

• Image E: Same cell as D but stained with antibody to catalase

  • Not in the same areas only 

• Conclusion: PTS1 is necessary for transport

Parallel Further Experiment

• Is each of the amino acids in the sequence necessary for transport? 

• Tested by single amino acid sub in PTS1 sequence 

PTS1: Sufficient for Protein Import? (DHFR)

Basis:

• Check is PTS1 sequence can for any protein into the peroxisome 

• Ex. DHFR

  • Cytosolic protein 

  • Wildtype: Normal without sequence

  • DHFR plus C-terminal luciferase 

• Hypothesis: If PTS1 sequence is sufficient and added to DHFR, then it will be transported to the peroxisome

Result:

• Image B: Wildtype DHFR localizes in cytosol

• Image C: DHFR-PTSL is found in punctate pattern in cell

• Image D + E: Co-localization of DHFR-PTS1 and catalase

  • Indicates the' DHFR-PTS1 has moved to the peroxisomes

• Conclusion: Yes it is sufficient

PTS1: Sufficient for Protein Import? (GFP) 

• GFP: Green fluorescent protein found in jellyfish 

• It’s a cytosolic protein 

• When modified with PTS1 sequence, it moves to the peroxisome as well 

• This is also shown in the tip of Arabidopsis plant 

  • GFP-PTS1 found in peroxisomes as they move within the root tip cells 

• Conclusion: Yes, sufficient once again!

Rule 2: Signal Receptor for PTS1?

• Pex5: Cytosolic protein 

  • Recognizes PTS1

  • Binds to the PTS1 sequence at C-terminus of target protein 

• Pex14: Peroxisomal Membrane protein

  • Pex5 associates with Pex14

Pex5 binding mechanism to PTS1 Signal

• Contains 7 TPR motifs

• Each motif has 2 a-helices connected by a loop 

• The 7 TRP motifs form a PTS1 binding pocket 

• Inside pocket: PTS1 tripeptide 

Rule 3: Translocation channel on peroxisome?

• Pex14 

  • Important for Pex5 recognition 

  • Forms translocon that transports Pex5 with target protein to the inside (peroxisomal matrix)

• When inside, Pex5 dissociates from the protein 

  • It is then recycled back out to the cytosol 

Rule 4: Energy Requirement for Peroxisomal Protein Transport?

• Yes during ubiquitinylation 

• Pex5 is ubiquitinylated and deubiquitinylated to release the protein in the matrix and be brought back to cytosol 

• Pex2 and Pex10 are ubiquitin ligases

  • Form complex with Pex12 

  • Makes another transllocon to export Pex5

• ubiquitinylation requires ATP hydrolysis 

Identification of Peroxisomal Transport Pathway Components

• Done by genetic screens to identify mutations preventing normal transport 

• Figure a: GFP-PTS1 is localized to peroxisomes

• Random mutations to genome were made, one per cell line 

• Each cell line was tested for its ability to transport GFP-PTS1 to peroxisome

• If unsuccessful, the cell line carried a mutation in a gene coding for a protein in the pathway

• The genes were named in order of identification

  • Pex1/Pex2/Pex3/etc

• Figures b-d: Examples of cell lines where GFP-PTS1 remained in cytosol 

  • Carry mutations in the pex genes

Rule 5: Protein Transport to Different Interior Compartments (Pex12 Mutation)

• 2 Places in peroxisome 

  • Membrane (Ex. components of translocon) 

  • Matrix (Ex. catalase) 

• They use different pathways to do so

Experiment: 

• Images follow antibody to membrane protein PMP70 (blue) OR antibody to catalase (green) 

• Figure a: Wild-type PMP70 and catalase shown in punctate pattern

• Figure b: Mutation in Pex12 gene

  • Catalase not transported into peroxisome

  • PMP70 successfully transported

Conclusion: There are distinct pathways of transport where Pex12 is only required for transport of proteins into matrix 

Rule 5: Protein Transport to Different Interior Compartments (Pex3 Mutation)

• Mutation in Pex3 disrupts PMP70 transport 

• Figure c: Has mutation on Pex3

  • PMP70 is found within cytosol (blue) 

• Pex3

  • Needed to help form peroxisome membrane as well 

  • Affects transport of both PMP70 and catalase

  • Proteins needed for catalase transport are peroxisomal membrane proteins as well (ex. Pex14) 

• If system to transport proteins into the membrane is faulty, the cell can’t transport proteins into the matrix as well

Zellweger’s syndrome

• Caused by failure to transport proteins which results in nonfunctional peroxisomes 

• Autosomal Recessive disorder caused by mutations in genes encoding Pex2/3/5/10/12

• Mutations in any of the genes lead to the same phenotype 

Result

• Cells accumulate long fatty acid chains 

• Impairs the normal function of many organ systems by disrupting

  • neuronal migration

  • nueronal positioning

  • brain development

Image:

• Figure a: Human cells carrying mutation in Pex19

  • Impaired pathway as catalase is found in cytosol

• Figure b: Rescued cells by addition of functional Pex19 gene 

  • Catalase is now localized 

• There are no treatments to the condition though :(