protein sorting overall

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.

What is the default location of a protein without a signal?

Cytosol

What determines where a protein goes in the cell?

Targeting signals and targeting factors

What are the two mechanisms for protein movement between compartments?

Translocation and vesicular transport

What is the entry point to the secretory pathway?

The endoplasmic reticulum (ER)

What is the signal hypothesis?

Secretory proteins have an N-terminal signal peptide that directs them to the ER

What type of sequence targets proteins to the ER?

An N-terminal hydrophobic signal sequence

What feature defines ER signal sequences?

A hydrophobic core of ~8+ hydrophobic residues

What rule defines signal peptidase cleavage?

The –1 and -3 position of small non-charged residues from the cleavage site

Is ER import co-translational or post-translational?

Co-translational

What are microsomes?

Vesicles derived from fragmented ER used in in-vitro experiments

What does adding microsomes during in-vitro translation reveal?

Signal cleavage and ER import

Why can’t Coomassie staining detect low-abundance in-vitro translated proteins?

Too many background proteins; low sensitivity

Why use radiolabelling in in-vitro translation?

It is sensitive and allows detection of small amounts of protein

What does “necessary” mean when testing a signal sequence?

Removing the sequence prevents ER import

What does “sufficient” mean when testing a signal sequence?

Adding the sequence to a cytosolic protein causes ER import

What indicates signal peptide cleavage on SDS-PAGE?

A lower molecular-weight band

What does protease protection indicate?

Protein is inside microsomes and protected by the membrane

What happens when detergent is added in a protease protection assay?

Microsomes dissolve and the protein is degraded

What is the conclusion of signal sequence experiments?

ER signals are necessary and sufficient for ER targeting

What tool predicts signal peptides?

SignalP

What is SRP?

Signal Recognition Particle

What domain of SRP binds the signal sequence?

The methionine-rich M-domain of SRP54

What does SRP binding to the ribosome cause?

Translation pause

Where does SRP dock the ribosome?

The SRP receptor (SR) on the ER membrane

What is the ER translocon?

Sec61 channel

What resumes translation during ER targeting?

Handover of ribosome to Sec61

What removes the signal peptide?

Signal peptidase

What drives SRP–SR interactions?

GTP binding and hydrolysis

What does GTP hydrolysis accomplish in SRP targeting?

Release of SRP and commitment to translocation

Do ribosomes stay permanently bound to the ER?

No

What enables SRP to bind diverse signal sequences?

Flexible methionine-rich hydrophobic groove

Where are soluble ER-targeted proteins delivered?

Into the ER lumen

Where are membrane ER-targeted proteins inserted?

Into the ER membrane via Sec61

What is the classical pathway for targeting proteins to the ER?

The SRP-dependent targeting pathway.

What reporter was used to identify SRP-independent ER targeting?

A fluorescent protein fused to the Gas1 signal sequence.

What did mislocalization of the reporter in the yeast deletion screen indicate?

Loss of a gene required for ER targeting.

What pathway was discovered as a new SRP-independent route to the ER?

The SND pathway.

What does the SND pathway primarily target?

Proteins with internal signal sequences.

What is the main site of membrane protein biogenesis?

The endoplasmic reticulum (ER).

What is the core ER translocon?

The Sec61 complex.

What three subunits make up Sec61?

Sec61α, Sec61β, Sec61γ

What blocks the Sec61 translocon when it is closed?

A plug helix.

What structural feature allows hydrophobic segments to enter the membrane?

The lateral gate.

What method enabled high-resolution structures of active Sec61?

Cryo-EM with stalling sequences.

Why are stalling sequences used in structural studies of Sec61?

They freeze ribosome–nascent chain complexes at specific stages.

What powers co-translational translocation?

Energy from peptide chain elongation.