7.1 The sorting problem #
7.1.1 One cytosol, many destinations #
A typical mammalian cell makes on the order of 10,000 different proteins. Ribosomes all work in the cytosol (or in the matrix of mitochondria and chloroplasts, for the few proteins encoded there). Without sorting information, every protein would remain in the cytosol. The information is in the protein itself: a short signal sequence, or a patch of the folded surface, recognised by a receptor that is located on the correct compartment. Gรผnter Blobel’s signal hypothesis, confirmed first for secretion, is the general principle. He received the Nobel Prize in 1999 for it.
There are two timings.
- Co-translational import. The protein is threaded into the ER while the ribosome is still making it. This is the route of the secretory pathway.
- Post-translational import. The protein is released in the cytosol and then delivered, folded or unfolded depending on the organelle, to the nucleus, mitochondrion, chloroplast, or peroxisome (Chapter 9). Some small ER proteins also use a post-translational route.
A protein with no signal stays in the cytosol. Default is not secretion. Secretion is a decision.
7.1.2 Road map of the secretory pathway #
ER โ vesicles โ cis-Golgi โ through the stack โ trans-Golgi network โ secretory vesicles or lysosomes or the plasma membrane.
Molecular Pathway Of Remsd Induced Apoptosis In Neurons
Retrieval runs in reverse at every step, so resident proteins that escape are brought home. The pathway is a set of steady-state locations, not a one-way conveyor that empties.
7.2 Signal hypothesis and translocation #
The study of Signal hypothesis and translocation provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.
Ethylene Signal Transduction
7.2.1 The signal sequence and SRP #
A typical ER signal sequence is at the N-terminus: a short positively charged region, a hydrophobic stretch of about 8โ15 residues, and a cleavage site. As soon as it emerges from the ribosome, the signal recognition particle (SRP) binds it. SRP is a ribonucleoprotein. Binding pauses elongation, which gives the ribosome time to dock before the rest of the protein folds in the cytosol.
Scheme Of Apoptosis Signal Transduction By Receptors Cd95, Tnfr1 And Dr3
The dock is the SRP receptor on the ER membrane. Both SRP and its receptor are GTPases. GTP hydrolysis hands the ribosome to the Sec61 translocon and releases SRP for another round. Translation resumes, and the growing chain goes through the aqueous channel of Sec61 into the ER lumen. The signal sequence is usually cut off by signal peptidase on the luminal side, so the mature secreted protein does not carry its address.
This is why rough ER looks rough. Ribosomes are there only while they are engaged. Free cytosolic ribosomes and membrane-bound ribosomes are the same particles at different moments.
7.2.2 Topology of membrane proteins #
A protein that will live in the membrane uses the same machinery with extra signals.
- A stop-transfer sequence is a hydrophobic segment that leaves the Sec61 channel sideways into the bilayer and becomes a transmembrane helix. The rest of the protein is then made in the cytosol. An N-terminal signal plus one stop-transfer produces a single-pass protein with its N-terminus in the lumen (and therefore outside the cell, later) and its C-terminus in the cytosol.
- An internal start-transfer sequence that is not cleaved can insert a protein whose N-terminus stays in the cytosol.
- Multipass proteins alternate start-transfer and stop-transfer sequences, one per transmembrane helix.
The positive-inside rule helps decide orientation: flanking sequences rich in positive charges tend to remain on the cytosolic side of the membrane. Once inserted, the protein does not flip.
Exceptions exist and are worth one sentence each. Tail-anchored proteins, with a single C-terminal transmembrane helix, are released in the cytosol and inserted post-translationally by the GET/TRC pathway, because their signal never emerges until translation is over and SRP has nothing left to bind. Mitochondrial and chloroplast membrane proteins do not use Sec61 at all.
7.2.3 Co-translational modifications in the ER #
As the chain enters, the ER begins to finish it.
- N-linked glycosylation. A preformed oligosaccharide, GlcโManโGlcNAcโ, is built on the lipid dolichol and transferred en bloc by oligosaccharyltransferase to the nitrogen of asparagine in the sequence Asn-X-Ser/Thr, where X is not proline. This happens during translocation, so only luminal (and therefore future extracellular) domains are glycosylated. Cytosolic proteins are not N-glycosylated by this pathway.
- Disulfide bonds. The ER lumen is an oxidising environment. Protein disulfide isomerase (PDI) forms and rearranges disulfide bonds. The cytosol is reducing, so disulfides there are rare.
- GPI anchors. Some proteins destined for the outer face of the plasma membrane have their C-terminal transmembrane segment cleaved off and replaced by a glycosylphosphatidylinositol lipid anchor.
- Folding chaperones. BiP, an Hsp70-family chaperone in the lumen, binds hydrophobic stretches of unfolded chains and uses ATP to give them further chances to fold.
7.3 Quality control #
The study of Quality control provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.
Rnase Mrp Cell Cycle Control
7.3.1 The calnexin cycle #
N-linked sugars are used as a folding report, not only as a finished decoration. Glucosidases trim the three glucoses. A glycoprotein with one glucose left binds the chaperones calnexin and calreticulin, which hold it and give it access to folding enzymes. When the last glucose is removed, the protein is released. If it has folded, it can leave the ER. If it has not, the enzyme UGGT puts one glucose back, and the protein re-enters the cycle. UGGT is a folding sensor: it recognises hydrophobic patches on incompletely folded proteins.
Regulation Of The Cell Cycle
7.3.2 ERAD and the unfolded-protein response #
A protein that fails repeatedly is marked for ER-associated degradation (ERAD). It is retro-translocated to the cytosol, ubiquitinated, and destroyed by the proteasome. The cell prefers to degrade a bad copy rather than send it onward.
If unfolded proteins accumulate faster than the ER can cope, the unfolded-protein response turns on. Three sensors in the ER membrane, IRE1, PERK, and ATF6, detect the overload, largely through competition with BiP. Their outputs slow general translation (PERK phosphorylates a translation-initiation factor), increase chaperone production, and expand the ER’s degradative capacity. If the stress continues, the same circuitry can switch the cell toward apoptosis. The ER would rather kill the cell than release a flood of misfolded protein.
Cystic fibrosis illustrates the system. The common ฮF508 mutation in the CFTR chloride channel makes the protein fold slowly. Quality control retains it in the ER and sends it to ERAD, so little channel reaches the apical membrane of airway and duct cells. The disease is, at the cellular level, a trafficking failure of a protein that would have had partial function if it had been allowed to travel. Drugs that help this mutant fold and escape the ER are a direct application of the quality-control chapter.
7.4 Key terms, summary, and self-check #
Key terms. Signal sequence; SRP; Sec61; stop-transfer; positive-inside rule; N-linked glycosylation; dolichol; calnexin; UGGT; ERAD; unfolded-protein response; BiP.
Summary. An N-terminal signal sequence, recognised by SRP, delivers a ribosome to Sec61, and the protein enters the ER as it is made. Hydrophobic segments decide which parts stay in the bilayer. The ER glycosylates, forms disulfides, and uses a glucose-dependent chaperone cycle to test folding. Failures are degraded; a general failure triggers the unfolded-protein response.
Self-check.
1. Why does SRP pause translation?
2. A single-pass plasma-membrane protein has its N-terminus outside the cell and its C-terminus in the cytosol. Which two signals produced that topology?
3. Why are cytosolic proteins not N-glycosylated on asparagine?
4. What does UGGT recognise, and what does it do to the sugar?
5. Why is the ฮF508 form of CFTR largely absent from the plasma membrane even though the gene is transcribed?
