Protein Processing and Transport

Calmodulin Calmodulin Hemoglobin Lysozyme

Cellular Compartments

  1. Cellular Compartmentalization
    1. Any membranes associated with these compartments or the plasma membrane
      1. Cytosol
      2. Endoplasmic Reticulum
      3. Golgi
      4. Mitochondria
      5. Chloroplasts
      6. Endosomes
      7. Lysosomes
      8. Peroxisomes
      9. Nucleus

  2. Protein Movement Between Compartments
    1. All protein synthesis begins in the cytosol and the ultimate destination depends on the primary sequence

    Mechanisms of Movement
    1. Cytosol/Nucleous: nuclear pores
    2. Transmembrane translocator proteins: across a membrane
    3. Vesicular Transport: within a vesicle system

  3. Types of Sorting Signals
    1. Signal peptides: often cleaved, not part of final protein product
    2. Signal Patches: usually remain in final product

Pathways

  1. The nuclear pore pathway
    1. Nuclear Transport
      1. The signal is 4 - 8 amino acids on the amino terminus
      2. It has a high + charge: arganine , proline, and lysine
      3. Requires recognition by transport proteins located on the margin of the nuclear pore
      4. Active Transport: Bi-directional transport in and out of the nucleus is rapid and with a large volume
      5. The signal sequence is usually not cleaved due to need for re-import when nucleus reassembles after cell divisoimn

  2. The Translocator Protein Pathway
    1. Mitochondrial Transport
      1. Import into matrix depends on "matrix targeting signal"
      2. An amino acid sequence of 20 - 80 amino acids at the amine terminus
      3. Transported through translocator protein at contact point of mitochondrial membranes
      4. Uses the electrocemical gradient for initial penetration, but subsequent transfer is fueld by ATP hydrolysis
      5. Recall: enters in unfolded state
      6. This is aided by chaperone proteins that bind to the protein on the outside of the mitochondria, as well as chaperones that bind once it starts being transfered to the inside of the mitochondria
      7. Transport to the other targets, such as the inner mitochondrial membrane or the inner membrane space requires extra sorting signals
      8. After the initial micochondrial import signal is cleaved by a signal peptidase, the secondary soting signal can take over
      9. After transfer is complete and associated sorting signals are cleaved, then the 3 - D structure can be formed

    2. Chloroplast Transport
      1. It is similar to mitochondrial transport except there is no electrochemical gradient used as an energy soure for the transport

    3. Peroxisomes
      1. Single membrane and no genome
      2. All proteins must be imported
      3. Signal is a 3 amino acid sequence at corboxy terminus
      4. Generally, peroxisomes are the sites of oxidative reactions that utilize CO2 to oxidize organic substrates to produce HO
      5. The hydrogen peroxide is then used in oxidative reactions to detoxify (by oxidation) hazardous organic compounds, producing water as a by-product

  3. The Vesicular Pathway
  1. Used for Endoplasmic Reticulum
  2. Golgi
  3. Secreted Proteins
  4. Lysosomes
  5. The membranes of these organelles
  6. Proteins destined to be secreted by the cell
  1. Initial entry point for this pathway is the ER
    1. The first step of getting the protein into the vesicular pathway in the first place (into the lumen) is actually dependent on a protein translocator: depending on the Signal Hypothesis
      1. Rough Endoplasmic Reticulum: Characterized by ribosomes
      2. Smooth Endoplasmic Reticulum: Has independent functions
        1. Carbohydrate metabolism
        2. Drug detoxification by hydroxylation: more water soluable
        3. Synthesis of neutral fats and lipids
        4. Synthesis of steroid hormones

  2. The Signal Hypothesis (Blobel and Sabatini, 1971)
    1. Protein synthesis begins with mRNA/ribosomes in cytosol
    2. Signal Recognition Particle (SRP) binds to signal sequence: first 15 - 30 amino acids at the amino terminus
    3. Ribosome complex is directed to ER membrane: a pause in translation occurs
    4. SRP/Ribosome binds to SRP receptor in ER membrane
    5. Translation continues, and protein is inserted into ER lumen co-translationally through a pore (channel protein) associated with the SRP receptor

  3. SRP detaches and is recycled
    1. If protein is supposed to be retained in ER, there is an "ER retension signal": 4 amino acids at the carboxy terminus
    2. Example: Protein disulfide isomerase catalyzes oxidation of S - S bonds, and BiP (a chaperone protein) aids in proper folding/formation of 3-D conformation
      1. Note that both of these ER-Retained proteins have a role in maintenance of membrane asymmetry

  4. Proteins can also be modified in the ER
    1. One of the most prominant is core glycosylation
      1. An oligosaccharide core of 14 sugars is added at Asn residues when the sequence Asn - X - Thr or Asn - X - Ser emerges into the ER lumen
      2. Thsi core can subsequently be trimmed and modified
      3. Note that this is also part of membrane asymmetry, since the ER lumen, Golgi lumen, and the interior of the vesicles of the pathway are topologically equivalent to the exterior of the plasma membrane

  5. For secreted proteins the origional sequence is cleaved
    1. For single and multipass membrane proteins insertion and embedment in the membrane depends on a series of start/stop signals
    2. Note that the initial signal sequence can also be internal: not on the very amine tip of the emerging polypeptice