Cell biology/Membrane Assembly: Topography

Here is the link to the ITunes U Lecture from Berkeley. Membrane Assembly: Topography

ER Translocation Membrane Channel

• Schekman's lab figured out that there is also a channel necessary using genetic research. He used genetic methods to identify the part of genetic sequence that creates this channel.
• Background:Cells must be able to secrete in order to grow, or to rephrase they must be able to perform protein translocation.
• His lab found a temperature sensitive lethal secretion mutant at sec61 (the normal gene is SEC61). This mutation allows the cell to function at room temperature but the cell dies at higher temperatures. Additionally the mutation causes the cell to produce secretory precursor protein in the cytoplasm (rather than fully functional secretory protein within the ER lumen).
• His laboratory introduced bits of genes to this mutant until it was restored to wild-type function. In other words they took the broken (mutant) yeast cell and added genes until it was fixed.
• Experimental Steps
  1. Use a plasmid vector with a selectable marker (complements a mutation in the host enzyme (i.e. Leu-creates leucine)-restore function that the host does not have without integrating this plasmid).
  2. Create a library of DNA from the yeast cell (by cutting up the genome with restriction enzymes).
  3. Insert pieces of the wild-type yeast DNA (from the library) into the plasmid.
  4. Use a double mutant yeast cell (sec61ts and leu mutation-cell cannot create leucine and dies at a high temperature) and infect with the plasmids created.
  5. Grow infected yeast cells on leucine deficient petri plate at room temperature. If the yeast cell did not take up a plasmid, they will die. If they took up any plasmid they will grow colonies on this plate.
  6. Take the plate and make a copy (by using filter paper or velvet to touch the plate transferring colonies onto the paper/velvet and then placing the paper/velvet onto another petri dish in the same orientation).
  7. Incubate the second plate at body temperature, 37 degrees.
  8. Only a few colonies will grow on the second plate but they are likely to have the wild-type SEC61 gene and the LEU gene that the yeast cell was missing.
  9. Now to get the DNA sequence. Clone the surving colony, extract plasmid, use original restriction enzyme, and perform DNA sequence analysis.
• Result: this gene produces a 53KD multi-spanning hydrophobic membrane protein, which is completely separate from SRP and SRP receptor and similar to SecY (Sec Y is required for secretion of proteins in bacteria).
• An antibody can be created to this protein and this antibody binds to and precipitates with 3 proteins called Sec61α,β,γ in a trimeric complex.

• This channel has been crystallized (from a thermophile) and the structure reveals an 8 angstrom pore aligned with secretory proteins.

Can charged ions (glutamate) penetrate a polar channel responsible for protein translocation?

• Use a two part chamber that measures conductance through a membrane channel. This chamber has 2 halves filled with buffer and the divider has a small aperture connecting the two sides.
  • This aperture is covered with an artificial lipid bilayer.
• Experiment 1: Add ER membrane vesicles (with ribosomes and nascent chains) and a chemical that will fuse vesicles to the artificial bilayer. Add glutamate and if there are ionic channels in the ER vesicles that are fused to the lipid bilayer, they will flow into the second chamber and create a change in ionic concentration and conductivity.
  • When nascent chains are present the ions will not transfer to the next chamber.
• Experiment 2: Same procedure except the ER vesicles are treated with puromycin (so ribosomes are attached but nascent chains are removed).
  • Under these conditions, the glutamate ions will transfer to the next chamber.
• Experiment 3: Same as above except this time remove the ribosomes and nascent chains with puromycin and high salt solution.
  • There will be no transfer of glutamate to the second chamber.

Comprehension Question

• What if you did this reaction with the vesicles of a sec61 temperature mutant (ts) and fused them with the bilayer at room temperature? In the puromycin treated trial, what would you expect to happen?
  • At room temperature, the experiment should progress but at higher temperatures it should not work.

Mechanism of signal hypothesis

• Secretory protein is being produced in the cytoplasm by a ribosome and forms a hairpin loop through the membrane the N-terminus remains on the cytoplasmic side and the apolar amino acids slip through a hole in the channel and enter the lipid bilayer (escaping the polar environment of the channel).
• The rest of the chain continues to grow into the ER lumen.
• When a certain sequence is presented to an enzyme, signal peptidase, resting on the lumen side of the channel signal peptidase clips the polypeptide chain at the signal polypeptide, leaving the secretory protein growing into the ER lumen. The signal peptide then floats into the lipid bilayer and is degraded into amino acids.

How do sugars get attached

• Some proteins acquire N-glycosylatedly linked oligosaccharides.
• The oligosaccharide is ready to be linked to the polypeptide waiting for the consensus sequence for N-glycosylation (Asn-X-Ser or Asn-X-Thr).
• The N-terminus of asparagine is acted upon by the dolichol polysaccharide creating an N-linkage.
• Many cytoplasmic proteins have this sequence but they are not glycosylated because they are translocated into the endoplasmic reticulum.

Types of membrane proteins

• Type I proteins have their N-terminus facing the outside of the cell.
• Type II proteins have their C-terminus facing the outside of the cell.
• Polytopic proteins loop through the cytoplasm multiple times.

Please feel free to add details or make changes where necessary. Contact me via email if you need help. Thanks, April

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