Lecture 13 - ER and Proteins

Describe the endoplasmic reticulum. What are its two parts?What are cisternae?

• Networks of membrane-enclosed tubes and flattened sacks called cisternae

  • Largest organelle in most eukaryotic cells

• The ER has two contiguous domains that perform different functions:

  • Rough ER - studded with ribosomes, more flattened sheet-like

  • Smooth ER - no ribosomes, more tube like

What is made in the smooth endoplasmic reticulum (SER)

• Major site of phospholipid and cholesterol synthesis

  • Abudant in cells with active lipid metabolism

  • Steroid hormones are synthesized from cholesterol in the ER

  • Some are converted to glycolipids or sphingomyelin in the Golgi apparatus

Where are lipids made and why? What are flipases?

• Most lipids are synthesized in the membrane of the ER

  • Cannot be made in an aqueous environment because they are highly hydrophobic

• More specifically they are made in the cytosolic portion of the ER bilayer

  • This would lead to only the outer layer growing

• Flipases are enzymes that help phospholipids “flip” from the cytosolic layer to the lumenal layer

Describe the rough endoplasmic reticulum functions

• Protein synthesis for membrane-bound, lumenal proteins or proteins that need to leave the cell

• Folding and ensuring inocrrectly folded proteins are removed

• Packaging of exported proteins in vesicles

• Addition of glycosylation and glycolipid anchors

  • Glycosylation carries information (like a label on a package)

What is in the cytosol?

• Contiguous with the inside of the cell

  • Nucleus

  • Cytosol

What is the lumen?

Contiguous with the outside of the cell

• ER

• Golgi

• Inside of vesicles

• Outside of cell membrane

Describe protein synthesis in the ER

• Proteins are made in one location depending on where they will end up

  • Ribosomes can be free or membrane bound

• Domains of membrane proteins that are exposed on the cell surface correspond to regions of polypeptide chains that are exposed to the ER and golgi lumen

What are the two different methods of protein translation?

• Translation on Free Ribosomes

  • Proteins are free to move nucleus and cytosol. Sometimes also mitochondria, chloroplasts, and peroxisomes

• Translation on ER-bound ribosomes

  • Proteins will end up inside the ER, so can only go where it is continuous with the ER

How do proteins get in the ER?

1. Contranslation Translocation - ribosomes bind to ER while they are translating the protein, so it actively dumps protein into the ER lumen

2. Post translational relocation - Free ribosomes translate proteins, which are then transported to ER lumen afterwards

How do free ribosomes know when to associate with the ER membrane to complete cotranslation relocation?

• It’s in the mRNA they are translating

  • As the amino acid polypeptide chain is being translated, it will contain a segment called the “signaling sequence” that signals this protein needs to be translated with the ER, and so the ribosome must relocate

What are the different steps of cotranslation?

• Step 1 - A signal recognition particle (SRP) binds to the signal sequence on the polypeptide chain

• Step 2 - The SRP protein binds to an SRP receptor that is attached to a translocon

• Step 3 - The ribosome binds to the translocon, which causes the SRP protein to unbind to the SRP receptor. Ribosomes binding to the translocon also causes the translocon to open

• Step 4 - Translation resumes. Signal peptidase cleaves the signal sequence off the growing polypeptide chain

• Step 5 - Continued translating causes the growing polypeptide chain to grow into the lumen of the ER

• Step 6 - The finished product is “spit out” into the lumen of the ER

Now that the protein is in the ER lumen, what happens next?

Folding and quality control

• Inside the lumen there are chaperone proteins, like “BiP” that bind to the newly translated polypeptide chain and aid in the folding process. These chaperones do not unbind until the protein is properly folded. BiP stays in the ER lumen

What is protein glycosylation - a type of protein modification. What is it function?

• Inside the ER we can add sugars to proteins - protein glycosylation

  • This will happen on asparagine residues while translation is happening

Glycosylation:

• helps prevent protein aggregation

• Promotes protein folding

• Helps with protein sorting

What are lipid anchors - a type of protein modification

• Inside the ER, we can add lipids anchors to newly formed proteins

• Protein is held in the membrane by a C-terminal hydrophobic sequence

• Anchors are assembled in the ER

• C-terminal sequence is cleaved and exchanged for the lipid anchor

What prevents/help proteins from misfolding?

Chaperones - they dont just help proteins fold correctly, but they also detect when a protein is misfolded

• A chaperone sends a misfolded protein to the ER membrane, where it:

  • Binds to a ubiquitin ligase complex

  • Gets tagged with ubiquitin

  • And then sent off into cytosol to be broken down by a Proteasome

This process is known as ERAD - ER-Associated Degradation

What is the unfolded protein response

• If too many unfolded proteins accumulate in the ER, they will bind to receptors on the ER membrane

  • These receptors iniate pathways that lead to increased production of:

    • Increased production of chaperones

    • Increased production of proteasomes

    • Limit the number of new unfolded proteins entering the ER

  • If cells can’t do this apoptosis will occur