cell bio module 4

• Welcome back to Biology 2B03: Cell Biology. Today, we will start with Module 4: Protein targeting, with Lecture 1: Post-translational transport to the peroxisome.
• Peroxisome


  • Bound by a single membrane; responsible for oxidative and synthetic functions in the cell; does NOT have its own genetic information; able to reproduce by the process of fission that is very similar to the process that we see in bacterial cells
• In this transmission electron microscopy or TEM image of the interior of the cell is a close-up of three peroxisomes. Each is seen to have a single bilipid membrane. Inside the peroxisomes are dense regions that contain protein aggregates. This protein aggregate is composed almost exclusively of one protein, the catalase enzyme.
• Functions of peroxisomes


  • In animal cells, peroxisomes are responsible for cholesterol synthesis; In nerve cells, peroxisomes synthesize plasmalogen for cell membranes; In the liver cells, the peroxisome is the site of oxidation of toxins, such as alcohol; In plant cells the peroxisomes are also required for the conversion of fatty acids into carbohydrates; In all cells, peroxisomes are responsible for the catalysis of fatty acids
• A major function of the peroxisome is the breakdown of fatty acids that are very long, through beta-oxidation. In animal cells, the long fatty acids are oxidized to medium chain fatty acids. These shorter fatty acids are then subsequently shuttled to the mitochondria for further processing so that they can be used as a source of energy.
• When compared to other macromolecule classes (such as carbohydrates and proteins), fatty acids yield the most ATP on an energy per gram basis. So, the oxidation of fatty acids in the peroxisome is required for the eventual release of metabolic energy. This is true and essential for both plants and animals.
• The unfortunate things, is that in this process of fatty acid beta-oxidation, the biproduct hydrogen peroxide is made, and this is very toxic to cells. It is the catalase enzyme, which is so abundant in peroxisomes, that is able to convert the toxic hydrogen peroxide into non-toxic oxygen and water molecules.
• Since catalase is so abundant in these organelles, we can use its presence, as a useful marker for the visualization of peroxisomes in the cell.
• Fluorescence microscopy images of catalase in the cell show that each individual dot is representing a peroxisome. An advantage to using fluorescence over TEM is that we can look at living cells.
• The movie illustrates how dynamic the cell is. In particular, we see that the peroxisomes are not static, but rather they are moving around, changing shape, and undergoing both fission and fusion.
• How peroxisomal proteins get to the peroxisome

  Peroxisomal proteins are synthesized in the cytosol and then transported to the peroxisome; Peroxisomal membrane proteins, such as PMP70, are targeted to precursor membranes first to create something called a peroxisomal ghost; Once proteins are present in the peroxisomal membrane, they are then used to transport peroxisomal matrix proteins, such as catalase, into the interior of the organelle; As more and more proteins are synthesized, the peroxisome can grow and new peroxisomes can then form by a process of fission
• Luciferase is an enzyme that allows the bioluminescence that we see in fireflies. Specifically, the luciferase enzyme is found in the peroxisomes of the cells in the abdomen of all fireflies.
• A fascinating and novel study was conducted in which luciferase was expressed in mammalian cells. Remarkably, the luciferase went specifically to the peroxisomes of the mammalian cells. This told researchers that the pathways for protein transport from the cytosol to the peroxisome are conserved from fireflies to mammals.
• Five rules for protein transport

  • a signal sequence on the transported protein, (2) a receptor for that signal sequence on the target organelle or membrane, (3) a translocation channel which helps get the protein across the membrane into the organelle, (4) required energy (like ATP) at some step in the process, and (5) there has to be a way of targeting a protein to specific and different locations within an organelle
• PTS1


  Peroxisomal-transport sequence 1, a tripeptide made up of three amino acids: serine, lysine, and leucine
• PTS1 is found at the C-terminus of the translated peroxisomal protein upon the completion of translation in the cytosol of the cell.
• If the PTS1 sequence is deleted


  The luciferase protein will no longer go to the peroxisome
• Experiments showed that deleting the PTS1 signal sequence disrupted luciferase transport to the peroxisomes, demonstrating that the signal is necessary for transport.
• If the PTS1 sequence is replaced with Ser-Ala-Leu


  This single amino acid substitution would also disrupt protein transport to the peroxisome
• DHFR is normally a cytosolic protein and can be seen distributed homogeneously throughout the cytosol of this cell.
• Adding the C-terminus from luciferase, which contains the PTS1 sequence, to DHFR would test whether the PTS1 sequence is sufficient for transport of a protein to the peroxisome.
• PTS1 sequence


  Sequence of 3 amino acids necessary for protein transport to the peroxisome
• Replacing PTS1 sequence


  1. Ser-Lys-Leu with Ser-Ala-Leu

  2. Disrupts protein transport to the peroxisome
• Sufficiency of PTS1 sequence

  Whether the PTS1 sequence is the only sequence needed to direct a protein to the peroxisome
• DHFR is normally a cytosolic protein
• Adding PTS1 sequence to DHFR

  DHFR-PTS1 is transported to the peroxisome
• Adding PTS1 sequence to GFP

  GFP-PTS1 is transported to the peroxisome
• Pex5


  Cytosolic protein that recognizes the PTS1 sequence
• Pex5 and Pex14 transport PTS1-containing proteins to the peroxisome


  1. Pex5 binds to PTS1 sequence

  2. Pex5 associates with transmembrane Pex14 protein

  3. Pex5 and Pex14 target PTS1-containing proteins to the peroxisome
• Pex5 structure


  Contains 7 TPR motifs that form a PTS1 binding pocket
• Transport of peroxisomal proteins into the peroxisome

  1. Pex5 with target protein translocates through Pex14 translocon

  2. Pex5 dissociates from target protein inside peroxisome

  3. Pex5 is recycled back to the cytosol
• Recycling of Pex5


  1. Pex5 is ubiquitinylated

  2. Pex2, Pex10, Pex12 complex exports Pex5 out of peroxisome

  3. Ubiquitinylation requires ATP hydrolysis
• Identification of peroxisomal transport pathway components

  1. Genetic screens to identify mutations that prevent normal peroxisomal transport

  2. Mutant cell lines with GFP-PTS1 remaining in cytosol were further studied to identify Pex proteins
• Pex12 mutation


  Disrupts transport of catalase into peroxisomal matrix but not PMP70 into peroxisomal membrane
• Pex3 mutation


  Disrupts transport of both PMP70 and catalase
• Pex3 is required for formation of peroxisomal membrane
• Zellweger's syndrome is caused by mutations in peroxisomal transport proteins
• Zellweger's syndrome


  Accumulation of very long chain fatty acids due to impaired peroxisome function, leading to disruption of neuronal migration, positioning, and brain development
• Restoring Pex19 function can rescue peroxisomal transport defects in Zellweger's syndrome cells
• Rules of post-translational protein targeting

  • There is a signal sequence on the transported protein
  • Receptors recognize the signal sequences
  • Translocation channels transport proteins into the organelle
  • Energy (e.g. ATP hydrolysis) is required
  • There are ways to target proteins to the matrix or membrane of an organelle