Chapter 12

Intracellular Protein Sorting

• most proteins are synthesized in cytoplam, some in ER, few in mitochondria

1. Major intracellular components

   1. Rough ER

      1. protein synthesis, storage of Ca2+ ions

   2. Smooth ER

      1. stores Ca2+, lipid synthesis

   3. Golgi Apparatus

      1. package nad process proteins

   4. Mitochondria

      1. makes ATP, powerhouse; home of cellular respiration

   5. Lysosomes

      1. take out waste and recycle materials

   6. Peroxisomes

      1. Redox center

   7. Chloroplast

      1. photosynthesis

2. Major intracellular compartments

   1. many vital processes take place in or on membrane

   2. Create enclosed compartments

      1. Labyrinthine ER comprises 50% of membranes

      2. organelles vary in abundance in cell types

3. Theory for evolution in eukaryotic phones

   1. all other organelles come from plasma memberane

      1. other than the mitochondria and chloroplasts

   2. 4 general conditions

      1. general conditions in nucleus and cytosol are similar

      2. secretory and endocytic

      3. mitochondria

      4. plastids in plants

4. Topological Equivalency

   1. similar environments

      1. inside of organs and organelles are called lumen

5. Protein trafficking

   1. gated and selective 

      1. between nucleus and cytoplasm

   2. transmembrane

      1. use translocator proteins, are topologically distinct

         1. includes plastids, mitchondria, ER, peroxisomes

6. Signal Sequence

   1. Cells have a signal for where they are to go

   2. import to nucleus - NLS

      1. a short and positive stretch of amino acids

   3. Export nucleus

      1. NES

   4. import to mitochondria

      1. alternating positive and hydrophobic

   5. import to peroxisomes

      1. present on C terminus

      2. ser-lys-leu (SKL)

   6. import to ER

      1. present on N terminus

      2. consists of 5-10 hydrophobic amino acids

   7. Return to ER

      1. present on C terminus

      2. lys-asp-glu-leu (KDEL)

7. Nuclear Transport

   1. transport is bidirectional

      1. import    

         1. histones, polymerases, transcription regulators

      2. export

         1. mature mRNA destined for translation

      3. Nucleoporins

         1. Unstructured nucleoporin domains form a tangle (like kelp), preventing passive diffusion of large molecules

         2. scaffold nucleoporins aid in bending the membrane

      4. importins bind to NLS and exportins bind NES

8. Structure of Nucleus

   1. double membraned

      1. the nuclear lamina is the skeletal structure

      2. inner membrane bends around nuclear pore

         1. baskets are located on the nuclear side (inside)

   2. importins bind to NLS and the NPC

9. Ran GTPase confers directionally on NPC transport

   1. Ran is a G protein that can go through the nuclear pore complex

   2. Ran-GAP is located in the cytosol

      1. takes off phosphate (GTP→GDP)

   3. Ran-GEF is in nucleus

      1. importin grabs cargo and goes into nucleus

      2. drops cargo and picks up Ran-GTP

      3. Takes Ran-GTP into cytosol and Ran-GAP changes it to Ran-GDP and importin drops it

      4. Then importin returns to the nucleus to start the process again, allowing for continuous and efficient transport of proteins that need to be imported into the nuclear compartment. the binding of Ran-GTP to importin decreases its affinity for cargo

10. Export

    1. exportin does not want anything in the cytoplasm

    2. exportin goes into the nucleus and grabs Ran-GTP which increases its affinity for cargo so it grabs that too

    3. Goes into cytoplasm where Ran-GAP changes it to Ran-GDP and exportin will drop both

11. Structure of mitochondria and chloroplasts

    1. mitochondria

       1. double membranes

       2. lumen is called matrix

       3. has intermembrane space

    2. chloroplast

       1. three membranes

       2. lumen is called stroma

       3. has intermembrane space

12. Mitochondrial protein translocators

    1. translocases

       1. basically are proteins with holes

          1. if want to get in membrane have to go through them

          2. vastly smaller than nuclear pore

             1. folded proteins do no fit through

    2. In outer membrane

       1. TOM complex - what most proteins go through

       2. SAM complex - optimized for beta barrel proteins

    3. in inner membrane

       1. TIM22 and TIM23 complexes and Oxa complex

          1. Oxa is the only one that can move proteins that were synthesized in matrix

    4. Proteins are synthesized in cytoplasm, but cannot fold. Chaperones shuttle unfolded proteins to organelles. The proteins have signal sequences cleaved off then they can fold

       1. Mitochondrial signal sequence is recognized by TOM and TIM

       2. cleavage of signal sequence is done by signal peptidase

    5. Transport to inner and inter membranes

       1. protein has a signal sequence and a hydrophobic stop-transfer sequence

          1. protein goes through TOM then part of the way through TIM until it reaches the hydrophobic stop-transfer sequence then it slides out into the membrane

          2. This process ensures that the protein is properly inserted into the mitochondrial inner membrane, where it can function effectively.

             1. protease cleavage can be done on stop-transferase sequence for protein to live in intermembrane

    6. Alternative pathway for inner membrane proteins

       1. Protein will go through TOM and TIM then a second sequence is releaved in the process that can be used on oxa to make it an inner membrane protein

13. Chloroplast import is very similar but requires two signal sequences

    1. TOC on outer membrane

    2. TIC on inner membrane

       1. cleavage of chloroplast signal sequence

       2. exposes the thylakoid signal sequence

          1. there are four routes to translocate the protein into the thylakoid

14. Peroxisomes

    1. contain oxadative enzymes: catalase and urate oxidase

    2. important particularly in liver and kidney cells to detoxify harmful molecules

    3. Breakdown of fatty acids into Acetyl CoA

    4. Dangerous forms of oxygen are a large client of peroxisomes

    5. single membrane

       1. import things through oligomeric pores

       2. SKL: peroxisomal-targeting sequence 1

       3. PTS1 binds to Pex5 and whole complex through Pex14

          1. PTS1 releases and goes back out to the cytoplasm through other Pex complexes (2, 10, and 12)

             1. Pex= peroxin

15. ER

    1. Rough ER:

       1. synthesis of all membranes and secretory proteins

    2. Smooth ER:

       1. synthesis of lipids for molecules

    3. Both:

       1. Ca2+ storage

    4. Co-translational translocation

       1. ER bound ribosome synthesizes directly into the ER

    5. Post translational

       1. signal sequence is made on protein in cytosol and is recognized by translocon

    6. SRP directs ER-signal bearing proteins to Er membrane

       1. binding of SRP to signal on n terminus pauses translation

       2. SRP binds to SRP receptor in rough ER and signal sequence binds to protein translocator and translation continues

16. Free and membrane bound ribosomes

    1. dependent on the mRNA that it grabs

       1. if has one with ER sequence SRP will find it and bring it to the membrane

17. insertion of single pass transmembrane protein

    1. SRP brings protein to translocase, but protein has stop-transfer sequence (hydrophobic)

    2. this sequence will get stuck in the middle of the translocon and slide out into the membrane

    3. Charge on n and c terminus have nothing to do with where sides of the membrane proteins will be

    4. positive side of signal sequence will be toward cytosol

18. Integration of multi-pass proteins into ER

    1. all nonpolar domains = transmembrane domains (hydrophobic regions)

    2. only first domain have charges so it determines orientation (positive side in cytosol)

19. Insertion of tail-anchored proteins (post-translational)

    1. vast majority will be in cytoplasm

       1. just the tail is in the membrane

    2. synthesized completely in ribosome

       1. when hydrophobic tail synthesized, grabbed by Get3

       2. Get3 will take the protein to the Er and stick the tail into the membrane

       3. Get1-Get2 complex will recycle Get3

20. ER residents (proteins that live and work in the ER) vs. ER transient proteins (made to go somewhere else)

    1. Er resident proteins contain KDEL

       1. PDI is a resident protein

          1. it helps proteins with disulfide bonds to fold or refold corrently

          2. PDI has its own cysteine amino acid to make this possible

21. Unfolded protein response

    1. Accumulation of misfolded proteins in the ER leads to unfolded protein response

       1. if not corrected, will get exported to cytoplasm and degraded by proteosome

    2. 3 major pathways

       1. IRE1, PERK, ATF6

    3. IRE1 mechanism

       1. the signal is a misfolded protein

          1. has a kinase domain

          2. has a ribonuclease domain

       2. When a misfolded protein binds, it will bring 2 IRE1s together to make the complex active

          1. will splice intron out of mRNA to make a transcription regulator

          2. the regulator will bind to a chaperone gene and make more chaperone mRNA

          3. will get translated and bind to the misfolded protein