Biogenesis Flashcards
Which cellular components have temporally continuous membranes?
Rough and smooth endoplasmic reticulum & outer nuclear membrane Golgi apparatus Secretory vesicles Plasma membrane Lysosomes Endosomes
Where does the synthesis of protein and lipids for temporarly continuous membranes occur?
In the ER
Which cellular components have temporally distinct membranes (i.e. not connect by vesicle traffic) and where are their proteins and lipids synthesized?
Inner & outer mitochondrial membranes
Peroxisomes
Proteins synthesized on cytoplasmic ribosomes but lipids may be synthesized on ER.
What is the purpose of SER?
Lacks ribosomes
Site of lipid synthesis
Detoxifies hydrophobic molecules using enzymes including Cytochrome P450
How does lipid synthesis proceed?
Free fatty acids in cytosol are activated by CoA. The free fatty acids insert into the outer leaflet of SER. Enymes for lipid syn is in the leaflet. Aceyl transferase transfers fatty acids on outer leaflet to a glycerol 3 phosphate group (forms backbone for phospholipid). Then phsophate is cleaved and another tranferase enzyme in leaflet thats facing cytosol fa transfers activated choline molecule to the phospholipid precursor to form phosophotylcholine. All on leaflet facing cytosol of ER.
Vesicle delivery of lipids to the plasma membrane
Lipids on cytosolic leaflet need to be transfered to lumenal leaflet.
How do lipids get to other organelles?
Go to syllabus
What proteins are synthesized on free ribosomes?
Soluble cytoplasmic proteins Peripheral membrane proteins Intranuclear proteins Mitochondrial proteins Peroxisomal proteins ***These all require organelles to be translocation competent.
What happens when mRNA + ribosome + RER?
We get secreted proteins, lumenal organelle proteins, synthesis of membrane proteins, golgi proteins, and plasma membrane. Proteins in golgi and ER have retention signals so they stay there.
What proteins are synthesizes on RER?
All other membrane proteins
Proteins inside other organelles
Secreted proteins
Note:ER sequesters calcium which can be released upon triggering to put Ca in cytosol
What is the Signal hypothesis for synthesis of secreted proteins?
Consider first the synthesis of a protein destined to be secreted.
- This is encoded by an mRNA that begins to be translated on free cytoplasmic ribosomes.
- The first stretch of amino acids in this protein generates what is known as the “signal sequence”or signal peptide. This is bound by the Signal Recognition Particle (SRP) as it emerges from the ribosome. The SRP contains proteins and RNA.
- Binding of the SRP to the signal sequence halts translation. This is important because it would be disastrous for the cell to have a digestive enzyme, which is destined to be secreted, released free into the cytoplasm.
- Translation starts again when the SRP binds to the SRP receptor (SRPR) in the membrane of the RER. The SRP has thereby targeted the ribosomes synthesizing a membrane protein to the RER.
- The signal sequence binds to a signal sequence binding protein, which forms part of a protein translocator channel in the RER membrane.
- N-(aspargine)- linked oligosaccharide may be added via an oligosaccharide tranferase from a high energy lipid, dolichol diphosphate almost as soon as nascent peptide emerges in RER lumen. Dolichol is a long-chain, polyisoprenoid lipid. The oligosaccharide is first synthesized on the dolichol in the RER.
- The SRP is released and the newly synthesized protein is passed through the
translocator channel in the RER membrane into the lumen (cistema) of the RER. - Passage of the newly synthesized polypeptide chain continues into the RER lumen until the complete protein has passed through the membrane via the protein translocator channel. Chaperone proteins [e.g. Calnexin, calreticulin, BiP] prevent misfolding and aggregation of proteins in the lumen of the RER
- The signal sequence is cleaved off by a signal peptidase. The resulting protein is now free within the lumen of the RER and topologically outside the cell.
Cleavage of the signal peptide is followed by release of the new protein into the lumen of the RER
Describe signal peptides
They are located at N terminus of proteins being synthesized and are highly hydrophobic sequences preceded by 1 or more basic residues. Typically 13-36 residues.
Describe a translocon
Protein translocator or Sec 61 is composed of 3 subunits that form a donut which only opens when signal sequences binds and release the plug
Describe the signal hypothesis in transmembrane proteins
The process is a little more comoplicated. We have tranlation from 5 to 3 the srp binds to the emerging signal seq pauses trans srb receptor is bound and trans ensues carb is added and as you go on eventually there is a stretch in newly formed peptide that is stop protein transfer seq that cause sprtoein to be stuck in the lumen 5 in cytosol and 3’ in lumen.
Essentially, the same steps occur as for a secreted protein and transfer through the RER membrane, via the protein translocator channel, takes place until a “stop transfer sequence” is reached within the polypeptide. This is a hydrophobic stretch of amino acids destined to become the transmembrane domain of the protein.
Continued protein synthesis will generate the cytoplasmic domain of the protein. When synthesis of the protein has been completed, the ribosomal subunits will dissociate from each other and the mRNA. It is important to note that a single mRNA may have many ribosomes simultaneously bound. This is known as a polyribosome or polysome. The longer the mRNA, the more ribosomes may be bound.
The signal sequence is usually cleaved off by the signal peptidase.
The insertion of a membrane protein into the RER membrane during translation is known as cotranslational import.
Protein modification occurs in the lumen of the RER:
a) correct folding of the protein
b) formation of disulfide bonds occurs in the RER lumen
c) glycosylation begins in the lumen of the RER
Consider the synthesis of a membrane protein that spans the membrane multiple times:
The steps described above are repeated, but following the stop transfer sequence and the synthesis of some cytoplasmic sequence, there will be another signal sequence or start transfer sequence. Alternating start transfer and stop transfer sequences can generate proteins that span the membrane multiple times. Each start transfer and stop transfer sequence gives rise to a hydrophobic transmembrane domain in the mature protein
Describe signal hypothesis for multi-spanning proteins
First signal anchor sequence anchors peptide to translocon and membrane.
First stop transfer signal causes ribosome to translate on the cytosolic side where another signal anchor sequence and stop transfer sequence are produced.
On the cytosolic side and anchor sequence and then a stop transfer sequence are synthesized which then spontaneously insert….and so on.
You have signal seq binding to translocon and now whe have ather seq which is downstream which is stop anchor seq and makes complex dissociate. In mRna IS ANOTHER STOP TRANSFER SEQ THAT IS MADE and then there is transfer and anchor sque. The section on cytosolic side of ER inserts in membrane and ribsoomes can come off and syntehsis double helicies.
Describe protein modification in lumen of ER
Glycosylation
Formation of disulfide bonds-using protein disulfide isomerase (PDI) to catalyze free -SH groups to disulfide bonds [-S-S-]
Correct folding of the protein
Summarize the five steps in ER processing
1) N-(aspargine)- linked oligosaccharide is added via an oligosaccharide tranferase from a high energy lipid, dolichol diphosphate almost as soon as nascent peptide emerges in ER lumen. Dolichol is a long-chain, polyisoprenoid lipid
2) Two Calcium dependent chaparones, calnexin and calretculin, as well as BiP bind to protein to prevent aggregation and to hold unfolded proteins in the ER. They recognize terminal glucose residues and target those proteins for degradation if these residues are not removed.
3) Sugars are trimmed and chaparones release from correctly folded protein
4) Incorrectly folded proteins are recognized and degraded
5) Correctly folded proteins go to Golgi
Note: This describes Calnexin (protein embedded in membrane) /calreticulin (protein in lumen) cycle for glycoprotein folding in the endoplasmic reticulum
