Where do nearly all proteins begin their synthesis?
On ribosomes in the cytosol. The only exceptions are a few proteins inside mitochondria and plastids.
How are proteins directed to their correct cellular location?
New proteins must go from a ribosome in the cytosol to the organelle where it
functions. It does so by using sorting signals in its amino acid sequence.
What are the main steps of the secretory pathway?
Protein is synthesized on a ribosome in the cytoplasm.
It enters the ER lumen (while being made).
It travels from the ER to the Golgi apparatus in a vesicle.
It transits through the Golgi.
It leaves the Golgi in a vesicle (that the ER made).
The vesicle fuses with the cell membrane.
The protein is released outside the cell.
How was the secretory pathway experimentally traced?
Labeled ratioactive amino acids and used electron microscopy to visually track the movement of newly synthesized proteins from the Rough ER → Golgi → Secretory Vesicles → Cell Exterior.
How were the key proteins in secretion discovered?
Used genetic screens in yeast to find secretion-defective (sec) mutants. These mutants identified proteins required for each step of the pathway. They mutated the yeast until it couldn’t reproduce. The mutated yeast has white empty vesicules, but they never arrived so you have a yeast cell with a bunch of them in their.
How does yeast reproduce
by budding. The proteins of the budding cell were made in the nucleus of the mother cell.
What steps can be blocked in secretion mutants?
Mutants fall into distinct categories depending on where the mutated protein was required.
Fail ER import.
Fail to produce ER vesicles. (so the ER gets super big)
Vesicles don’t fuse with the Golgi.
Fail to leave the Golgi (so the golgi gets super big).
Vesicles don’t fuse with the cell membrane.
What is the structure of the ER?
It is a netlike labyrinth of branching tubules and flattened sheets that extends throughout the cytosol. It has a single, continuous internal space called the ER lumen (once its here it can go anywhere). It has big sacs that move and changes shape.
What is the difference between the Rough ER and Smooth ER?
Rough ER: Has ribosomes bound to its surface. Site of protein synthesis for the secretory pathway.
Smooth ER: Lacks ribosomes. Dedicated to lipid biosynthesis and metabolism.
It’s just one organelle
What is an ER signal peptide?
A short sequence of 16-30 amino acids at the N-terminus of a newly synthesized protein that directs it to the secretory pathway (either the ER or cytoplasm).
What is the first key step in protein secretion?
Co-translational import into the ER. The protein is translocated into the ER lumen while it is still being synthesized on the ribosome. If a cell waits too long, then the protein will be made in the cytoplasm because its too big to come into the ER. it folds in the ER
The ER signal sequence is guided to the ER membrane by at least two
components:
Signal Recognition Particle (SRP) and SRP Receptor
How is the signal sequence recognized?
The Signal Recognition Particle (SRP) binds to the signal peptide as it emerges from the ribosome. It goes from linear to bent to hold onto the ribsosome.
What does SRP do after binding the signal sequence?
When a signal sequence binds, SRP exposes a binding site for an SRP
receptor, which is a transmembrane protein complex in the rough
ER membrane. its on the membrane of the ER
Membrane-bound ER ribosomes make what?
Proteins that are co-translocated across the ER membrane. Free ribosomes, unattached to any membrane, synthesize all other proteins.
The Polypeptide Chain Passes Through what?
a Signal Sequence–gated
Aqueous Channel in the Translocator (or Translocon)
The core of the
translocator is called
the Sec61 complex.
The lumen of the Er is what
hydrophilic
How are proteins that span the membrane multiple times inserted?
For multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly through the lipid bilayer. Specific hydrophobic transmembrane segments anchor it in the lipid bilayer. These proteins are locked forever. A protein that passes through three times will have 3 stretches of hydrophobic amino acids.
What three things in the ER help proteins fold correctly?
Chaperone Proteins (e.g., Hsp70/BiP)
Formation of Disulfide Bonds (by PDI)
N-Linked Glycosylation
Translocated Polypeptide Chains do what
Fold and Assemble in the Lumen of the Rough ER.
Where are disulfide bonds formed and by what enzyme?
Disulfide bonds are post-translational modifications that occur in the ER lumen only. It’s between cysteine residues. The enzyme Protein Disulfide Isomerase (PDI) catalyzes their formation and rearrangement. These bonds allow it to fold in the right direction.
What is N-linked glycosylation and when does it happen?
Adding sugars. It happens as soon as the proteins starts to made in lumen of ER. It is the addition of a pre-formed oligosaccharide (three glucoses) to the Asparagine (Asn) side chain of a protein spefically at the N.
Oligosaccharides Are Used as what?
Tags to Mark the State of Protein Folding. if folded, a glucosidase removes the last
glucose, then the protein exits from the ER. If not folded properly, a glucosyl transferase adds a glucose, and the cycle starts all over thing. It keeps going until the protein is folded incorrectly, if not the cell will destory it.
What is the role of Calnexin?
Binds to monoglucosylated on incompletely folded proteins and retain them in the ER.
● Calnexin is a chaperone.
● Recruits an oxidoreductase (ERp57) to add more disulfide bonds.
● If folding is good, GlsII removes the final glucose residue.
