Source: PCB3023L Assigned Reading, University of Florida
Tags: endosomal sorting, early endosome, late endosome, lysosome, MVB, ESCRT, ubiquitin, ubiquitination, Rab5, Rab7, Rab GTPases, EGFR degradation, EGFR recycling, receptor trafficking
Difficulty: Intermediate Prerequisites: Part 1 of these notes (vesicular transport, coat proteins, clathrin-mediated endocytosis). Familiarity with basic protein modification concepts is helpful.
Part 1 covered how EGFR gets pulled into the cell inside a vesicle. This part covers what happens next: the sorting decision. Once EGFR reaches the early endosome, the cell has to decide whether to send the receptor back to the surface (recycling, which keeps the signal going) or send it to a lysosome for destruction (degradation, which shuts the signal off). That decision is controlled by ubiquitin tags on the receptor and by a cast of regulatory proteins including Rab GTPases and the ESCRT machinery. When this sorting system breaks down, receptors that should be degraded keep signalling, which is one of the mechanisms behind certain cancers.
After EGFR is internalised, it reaches early endosomes where the cell sorts it for recycling (back to the surface) or degradation (via multivesicular bodies and lysosomes). Ubiquitin tags mark receptors for destruction, ESCRT proteins package them into intraluminal vesicles, and Rab GTPases (Rab5, Rab7) control endosome identity and maturation.
Early endosome
The first intracellular compartment that receives cargo after endocytosis. Think of it as the sorting room: cargo arrives here and is directed onward to recycling, degradation, or other routes.
Late endosome
A more acidic compartment that forms as early endosomes mature. Late endosomes are on the path toward fusion with lysosomes. The transition from early to late endosome is marked by a switch from Rab5 to Rab7.
Multivesicular body (MVB)
A type of late endosome that contains smaller vesicles (intraluminal vesicles, or ILVs) inside its lumen. Cargo destined for degradation is sorted onto these internal vesicles by ESCRT proteins. When the MVB fuses with a lysosome, the ILVs and their cargo are digested.
ESCRT (Endosomal Sorting Complex Required for Transport)
A set of protein complexes (ESCRT-0, -I, -II, -III) that recognise ubiquitinated cargo on the endosome membrane and package it into intraluminal vesicles within MVBs. In simple terms, ESCRT is the machinery that pushes marked-for-destruction cargo into the interior of the MVB.
Ubiquitin / ubiquitination
Ubiquitin is a small regulatory protein that cells attach to other proteins as a sorting tag. When EGFR is ubiquitinated, it is flagged for sorting into the degradative pathway. Think of ubiquitin as a "destroy me" label.
Rab GTPases (Rab5, Rab7)
Small GTP-binding proteins that act as molecular switches controlling vesicle identity and trafficking. Rab5 marks early endosomes and recruits effectors for early sorting. Rab7 marks late endosomes and drives maturation toward lysosome fusion.
Lysosome
An acidic organelle packed with hydrolytic enzymes that degrades proteins, lipids, and other macromolecules delivered to it. The final destination for cargo on the degradative pathway.
Retrograde trafficking
Transport of proteins backward from endosomes to the trans-Golgi network, rather than forward to lysosomes. Some cargo is retrieved this way instead of being degraded.
Recycling pathway
The route by which internalised receptors are returned from early endosomes to the plasma membrane for reuse. Recycling sustains signalling; degradation terminates it.
The early endosome as sorting hub
After clathrin-mediated endocytosis, the uncoated vesicle carrying EGFR fuses with an early endosome.
The early endosome is the decision point. From here, cargo can take one of three routes:
Recycling pathway: receptor is returned to the plasma membrane.
Degradative pathway: receptor is sent to late endosomes, then lysosomes.
Retrograde pathway: cargo is sent back to the trans-Golgi network (less common for EGFR, but relevant for other proteins).
Recycling pathway
Receptors that are not tagged for degradation can be sorted into recycling tubules that bud from the early endosome and return to the plasma membrane.
This allows the receptor to be reused for another round of signalling.
Recycling is associated with transient, self-limiting signals: the receptor activates briefly, is internalised, and then returns.
Degradative pathway
Receptors marked with ubiquitin are recognised by the ESCRT machinery on the endosome membrane.
ESCRT complexes (0 through III) sequentially recognise the ubiquitin tag, cluster the cargo, and invaginate the endosomal membrane inward to form intraluminal vesicles (ILVs).
An endosome containing ILVs is called a multivesicular body (MVB).
The MVB then fuses with a lysosome, and lysosomal enzymes degrade everything inside, including the receptor. This permanently terminates signalling.
Retrograde and nuclear trafficking
Some proteins are retrieved from endosomes and sent back to the trans-Golgi network rather than onward to lysosomes.
In rare cases, EGFR or fragments of it may be translocated to the nucleus, where they can influence gene expression through non-canonical signalling pathways.
Ubiquitination as a sorting signal
Ubiquitin is a small protein (76 amino acids) that is covalently attached to target proteins by ubiquitin ligases.
On EGFR, ubiquitination serves as the primary signal that routes the receptor toward degradation rather than recycling.
ESCRT-0 contains ubiquitin-binding domains that recognise and capture ubiquitinated cargo on the endosome membrane.
Rab GTPases as identity markers
Rab5 is active on early endosomes. It recruits tethering factors and promotes fusion of incoming vesicles with the early endosome.
As the endosome matures, Rab5 is replaced by Rab7 (a process called Rab conversion). Rab7 drives the transition to late endosome identity and eventual fusion with lysosomes.
This Rab5-to-Rab7 switch is a key regulatory checkpoint: it is what converts a sorting station into a degradation-bound compartment.
Other regulatory players
Small GTPases of the Arf family help regulate coat assembly and membrane dynamics.
Adaptor proteins link cargo to sorting machinery at multiple steps.
Kinases phosphorylate trafficking components to modulate their activity in response to signalling cues.
Cytoskeletal components (actin, microtubules, motor proteins) physically move vesicles and endosomes to the correct location in the cell.
Putting it all together: EGFR trafficking from start to finish
EGF binds EGFR at the cell surface, triggering dimerisation and autophosphorylation.
AP2 and clathrin assemble a coated pit around the activated receptor.
Dynamin pinches off the clathrin-coated vesicle.
The coat is removed; the vesicle fuses with an early endosome (Rab5-positive).
At the early endosome, a sorting decision is made:
If EGFR is not ubiquitinated (or only lightly), it enters recycling tubules and returns to the plasma membrane.
If EGFR is ubiquitinated, ESCRT complexes package it into intraluminal vesicles, forming an MVB.
The early endosome matures (Rab5 is replaced by Rab7) into a late endosome.
The late endosome/MVB fuses with a lysosome, and EGFR is degraded, terminating the signal.
Many cancers involve mutations that prevent EGFR from being properly ubiquitinated or sorted for degradation. The receptor keeps recycling to the surface and signalling indefinitely, driving uncontrolled cell proliferation. Drugs like cetuximab (a monoclonal antibody) and gefitinib (a small-molecule inhibitor) target EGFR signalling, and understanding the trafficking pathway helps explain why some tumours develop resistance: if the degradation machinery is intact, blocking the receptor works; if the trafficking is broken at a different step, the drug may be less effective.
Students often think ubiquitin always means "destroy the protein via the proteasome." In the context of EGFR trafficking, ubiquitin is a sorting signal for the endosomal/lysosomal degradation pathway, not the proteasomal pathway.
Students sometimes assume Rab5 and Rab7 are on the endosome at the same time. They are not (in the standard model): Rab conversion means Rab5 is replaced by Rab7 as the endosome matures.
The ESCRT machinery is sometimes described as though it degrades cargo. It does not. ESCRT sorts and packages cargo into intraluminal vesicles. The actual degradation happens later, inside the lysosome.
Recycling is sometimes treated as a default or passive process. It is actively regulated, and whether a receptor recycles or is degraded depends on specific signals (principally ubiquitination).
Be able to trace the full EGFR trafficking pathway from ligand binding to lysosomal degradation.
Know what ubiquitination signals in this context (sorting to MVBs, not proteasomal degradation).
Understand the Rab5-to-Rab7 switch and what it means for endosome maturation.
Know the role of ESCRT proteins: recognise ubiquitinated cargo, form intraluminal vesicles in MVBs.
Expect questions that ask you to predict what happens if a step fails (e.g. "What happens to EGFR signalling if ubiquitination is blocked?" Answer: EGFR recycles instead of being degraded, and signalling persists).
True or false: Ubiquitination of EGFR signals for its degradation via the proteasome. (False. In this context, ubiquitination signals sorting into MVBs and degradation in the lysosome.)
Fill in the blank: The protein complexes that package ubiquitinated cargo into intraluminal vesicles within MVBs are called ______. (ESCRT complexes)
True or false: Rab5 is associated with late endosomes. (False. Rab5 marks early endosomes. Rab7 marks late endosomes.)
Fill in the blank: The switch from ______ to ______ on an endosome marks its maturation from early to late. (Rab5 to Rab7)
True or false: If EGFR ubiquitination is blocked, the receptor will be preferentially recycled to the plasma membrane instead of degraded. (True.)
Q: What are the three possible fates of EGFR once it reaches the early endosome?
A: It can be recycled back to the plasma membrane, directed to late endosomes/lysosomes for degradation, or (less commonly) trafficked retrogradely to the Golgi or translocated to the nucleus.
Q: Explain the role of the ESCRT machinery in EGFR degradation.
A: ESCRT complexes (0 through III) sequentially recognise ubiquitinated EGFR on the endosomal membrane, cluster the cargo, and invaginate the membrane inward to form intraluminal vesicles within the MVB. When the MVB fuses with a lysosome, the ILVs and their cargo (including EGFR) are degraded by lysosomal enzymes.
Q: What is the functional significance of the Rab5-to-Rab7 conversion?
A: It marks the transition from an early endosome (a sorting station that can still recycle cargo) to a late endosome (committed to lysosomal fusion and degradation). The switch changes the identity and effector proteins on the endosome, moving it irreversibly toward the degradative pathway.
Q: Predict what would happen to EGFR signalling in a cell where ubiquitin ligase activity toward EGFR is lost.
A: EGFR would not be tagged for degradation. It would be recycled back to the plasma membrane instead of being sorted into MVBs. The result is sustained, excessive EGFR signalling, which can drive uncontrolled cell proliferation and is associated with cancer.
Q: Distinguish between the recycling and degradative pathways in terms of their effect on EGFR signalling.
A: The recycling pathway returns EGFR to the cell surface, allowing it to bind ligand again and continue signalling. The degradative pathway sends EGFR to lysosomes for destruction, permanently terminating that receptor's signalling capacity. The balance between the two pathways determines the duration and intensity of the EGFR signal.
This material connects to the ubiquitin-proteasome system (a different degradation route for cytoplasmic proteins, covered elsewhere in cell biology). It also ties into cancer biology: many oncogenic mutations affect EGFR trafficking or the proteins that regulate it. The lysosome content here links to autophagy (another route by which cellular material reaches lysosomes), and the Rab GTPase family appears again in secretory pathway regulation and synaptic vesicle cycling.
Endosomal sorting, early endosome, late endosome, multivesicular body, MVB, intraluminal vesicle, ILV, ESCRT, ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, ubiquitin, ubiquitination, ubiquitin ligase, Rab GTPase, Rab5, Rab7, Rab conversion, lysosome, lysosomal degradation, receptor recycling, receptor degradation, EGFR trafficking, EGFR signalling, signal termination, retrograde trafficking, trans-Golgi network, adaptor protein, dynamin, cancer, receptor tyrosine kinase, cell biology, PCB3023L