endosome

What is the main purpose of the endocytic pathway?

To monitor and adjust the cell surface by internalizing proteins and deciding whether to recycle or degrade them.

What compartments form the route of the endocytic pathway?

Plasma membrane → endocytic vesicle → early endosome → recycling pathway or lysosomal degradation.

What proportion of the cell surface can immune cells internalize per hour?

Approximately the entire surface area every hour.

What happens to transferrin and LDL receptors after delivering cargo?

They recycle back to the plasma membrane.

What determines whether a receptor is recycled or degraded?

Sorting decisions made inside the early endosome.

Why is EM required to visualize endocytic vesicles?

Light microscopy cannot resolve vesicles (50 nm)

What is a pulse–chase experiment?

A method where cargo is briefly labeled (pulse) and then tracked over time (chase) to observe transport.

Why was ferritin-LDL used in early experiments?

Ferritin contains iron

What phenotype was seen in familial hypercholesterolemia patient JD?

LDL bound to receptors but was not internalized.

What mutation caused the JD patient’s defect?

A single tyrosine mutation in the NPXY internalization motif of LDLR.

What is the function of sorting motifs such as YxxΦ

Binds to AP2 which will selectively recruit cargo proteins into curvature.

What is AP2?

A tetrameric adaptor protein that binds cargo motifs

Why is AP2 normally inactive in the cytosol?

Its cargo and clathrin binding sites are hidden (‘closed’ conformation).

What activates AP2?

Binding to PIP2 (PI4,5P2)

How many roles does PIP2 have

5 roles across CME

What does clathrin do?

Forms a stabilizing scaffold around budding vesicles but does not initiate curvature.

What generates initial membrane curvature?

Insertion of amphipathic helices from proteins like epsin.

What are amphipathic helices?

Helices with hydrophobic and polar faces that insert into one leaflet to induce curvature.

What is epsin’s function?

Binds PIP2 to induce membrane curvature

What are BAR-domain proteins?

Banana-shaped dimers that bind and stabilize highly curved membranes

What does amphiphysin do?

Recognizes high curvature via its BAR domain and recruits dynamin.

What is dynamin?

A GTPase that forms a collar around the vesicle neck and performs membrane scission.

What phenotype is seen in shibire (dynamin) mutants?

At non-permissive temperatures, these mutants appear causing loss of synaptic vesicles at nerve terminals

How does dynamin mediate scission?

GTP hydrolysis triggers a conformational twist that constricts and severs the vesicle neck.

What triggers coat disassembly after vesicle formation?

Conversion of PIP2 → PI4P by dephosphorylation. Further helped by Hsc70 and auxilin.

Why must coat proteins fall off after budding?

To allow vesicle fusion with the early endosome.

What experimental evidence supports timing of coat assembly?

Live-cell imaging of fluorescently tagged cargo

When is dynamin recruited during vesicle formation?

Very late

What does coincidence detection mean in CME?

Proteins require multiple simultaneous signals (e.g.

Besides clathrin-mediated endocytosis what other pathways exist?

caveolin-mediated endocytosis, clathrin and caveolin independent routes

Why is CME considered the best understood uptake pathway?

It is highly selective

What is the early endosome’s main role?

A decision point for cargo to be recycled or sent for degradation.

What structures define endosome morphology?

A vacuolar region with internal vesicles and a network of recycling tubules.

What does gold-labelled transferrin highlight in EM?

Recycling tubules within the endocytic pathway.

Why must the EGF receptor be sorted into ILVs?

To terminate signalling by targeting it for lysosomal degradation.

What protects the lysosomal limiting membrane from proteases?

Mucin-like heavily glycosylated proteins.

Which phosphoinositide defines early endosome identity?

PtdIns3P.

Which protein domains bind PtdIns3P?

FYVE and PX domains.

How were MVB mutants first identified in yeast?

Through a CPY-Invertase genetic screen selecting for missorting.

What characterises Class E vps mutants?

A collapsed endosomal compartment and defective MVB formation.

What happens to CPS cargo in Class E mutants?

It remains on the limiting membrane and fails to enter ILVs.

What is the sorting signal for CPS to enter ILVs?

Ubiquitination on cytosolic lysines.

Which ubiquitin linkage is used for MVB sorting?

K63-linked polyubiquitin.

What is the role of Vps23?

It binds ubiquitin to sort cargo into the MVB pathway.

Which complex does Vps23 belong to?

ESCRT-I.

What is the function of ESCRT-0

Initial ubiquitin recognition and clustering.

What does ESCRT-III do?

Forms polymers that drive membrane invagination and ILV scission.

What happens to LDL receptors in endosomes?

They dissociate from LDL and recycle back to the membrane.

What complex drives endosomal tubule-based recycling?

Retromer (Vps26/29/35).

What domains in sorting nexins help generate tubules?

PX domains (bind PtdIns3P) and BAR domains (induce curvature).

What overrides retromer peptide-motif recycling?

Cargo ubiquitination.

How are recycling tubules thought to elongate?

By pulling forces from actin/microtubule cytoskeleton and motor proteins.

Which ATPases may mediate scission of recycling tubules?

EHD proteins.

How is endosomal sorting linked to Alzheimer’s?

APP processing depends on endosomal trafficking; defects cause pathological imbalance.