Lec 16 - Endomembranes and Secretory

What organelles comprise the endomembrane system

The ER, Golgi, trans-Golgi network (TGN), endosomes, lysosomes, secretory vesicles, and plasma membrane.

Which organelles are not typically included in the endomembrane system

Mitochondria and peroxisomes are generally excluded (though some ER-derived components relate to peroxisome biogenesis).

How are membranes in the endomembrane system connected

By vesicle budding from donor membranes and fusion with acceptor membranes.

What is the biosynthetic-secretory pathway

Directional trafficking of newly synthesized membrane and secreted proteins from ER to Golgi to TGN to final destinations.

What maintains pathway directionality

Distinct coats, Rab GTPases, tethers, SNAREs, and organelle-specific enzymes and pH conditions.

What is the ERGIC

The ER-Golgi intermediate compartment where ER-derived vesicles first fuse before Golgi entry.

What is the TGN’s main role

Final sorting and packaging of proteins into distinct vesicles for specific destinations.

What is meant by vectorial movement

Time-ordered progression of cargo through ER → Golgi → TGN → destination, demonstrated by pulse-chase in acinar cells.

Which motors mediate long-range vesicle transport

Kinesin (plus-end, outward) and dynein (minus-end, inward) on microtubules.

Which motor mediates short-range transport

Myosins on actin for local movement.

Which motor primarily carries TGN-derived vesicles outward

Kinesin on microtubules.

What happens to vesicle movement when kinesin is inhibited

Vesicles bud but exhibit limited directed movement, largely Brownian motion.

What is topology conservation in the secretory pathway

Cytosolic-facing domains remain cytosolic; lumenal domains remain lumenal/extracellular after fusion.

What does ER lumen correspond to at the plasma membrane

The extracellular/exofacial face after vesicle fusion.

Where are secreted proteins during trafficking

In the lumen of ER, Golgi, TGN vesicles, and eventually released extracellularly.

What did Palade’s pulse-chase experiments show

Newly synthesized proteins move from rough ER (~3 min) to Golgi (~7 min) and then to secretory vesicles/extracellular space (~120 min).

Where do ER-derived vesicles first fuse

At the ERGIC near the cis-Golgi.

How do proteins traverse the Golgi

From cis to medial to trans cisternae, then to the TGN for sorting.

What two models explain intra-Golgi transport

Stable cisternae (vesicular transport) and cisternal maturation models.

Where does translation begin for all proteins

On free ribosomes in the cytosol.

What zip code targets nascent proteins to the ER

An ~20 amino acid hydrophobic signal sequence, often N-terminal with a peptidase cleavage site.

What recognizes the signal sequence

The Signal Recognition Particle (SRP), which stalls translation upon binding.

How is the ribosome-mRNA-nascent chain delivered to ER

SRP binds the SRP receptor on ER membrane near the translocon.

What activates release of the nascent chain into the translocon

GTP binding/hydrolysis on SRP and SRP receptor triggers conformational changes and plug opening.

What enzyme cleaves N-terminal signal peptides in the ER lumen

Signal peptidase.

Where does a fully secreted soluble protein end after translation

In the ER lumen after signal sequence cleavage.

What is the translocon

A protein-conducting channel with a ribosome-binding site, lateral gate, and a plug.

What is the lateral gate used for

Exit of hydrophobic segments into the lipid bilayer.

Which ER chaperone assists initial folding

BiP binds nascent chains to prevent premature folding.

What is a stop-transfer sequence (STS)

A hydrophobic segment that halts translocation and inserts laterally into the membrane.

Are N-terminal signal sequences always cleaved

Those near the N-terminus of single-pass proteins are cleaved; internal/C-terminal signals are not.

What determines orientation of membrane proteins

Position of signal sequence and distribution of charged residues within transmembrane segments.

How are internal signal sequences handled

They function as both targeting signals and stop-transfer sequences, inserting into the bilayer without cleavage.

How are multi-pass membrane proteins inserted

Through alternating internal signals and multiple stop-transfer sequences, each exiting laterally into the membrane.

Where are GPI anchors added

In the ER by GPI transamidase, replacing a transmembrane segment with a GPI anchor.

What leaflet is a GPI-anchored protein attached to in the ER

The luminal leaflet (becoming extracellular at the plasma membrane).

What is the functional result of GPI-anchoring

The protein is tethered to the membrane exofacially without spanning the bilayer.

What is N-linked glycosylation

Addition of an oligosaccharide to asparagine residues during ER translocation.

What lipid donates the oligosaccharide in N-linked glycosylation

Dolichol-linked oligosaccharide transfers to the nascent chain.

What initial trimming occurs in ER glycosylation quality control

Removal of three glucose residues by ER glycosidases.

Where does O-linked glycosylation occur

In the Golgi, on serine/threonine hydroxyls.

Which lectin-chaperones monitor glycoprotein folding

Calreticulin (soluble/secreted proteins) and calnexin (membrane proteins).

What does calreticulin recognize during ER QC

Glycoproteins with one terminal glucose remaining on the core oligosaccharide.

What happens if folding fails persistently in the ER

Retrotranslocation to cytosol, ubiquitination, and proteasomal degradation (ERAD).

What is the retrotranslocon

A channel that exports misfolded ER proteins to the cytosol for degradation.

What disease illustrates ER retention of misfolded protein

Alpha-1 antitrypsin deficiency with ER accumulation and loss of elastase inhibition in lung.

What is the KDEL signal

A C-terminal sequence (Lys-Asp-Glu-Leu) for retrieval of soluble ER-resident proteins.

How are KDEL proteins retrieved to the ER

KDEL receptor in ERGIC/Golgi binds KDEL proteins and returns them via COPI vesicles.

What is the KKXX signal

A C-terminal retrieval signal for ER-resident membrane proteins.

What does COPII do

Carries cargo from ER to ERGIC/Golgi (anterograde).

What does COPI do

Returns cargo from ERGIC/Golgi back to ER (retrograde).

What does clathrin do

Mediates trafficking between TGN, endosomes, lysosomes, and plasma membrane.

Which small GTPases initiate coat assembly

Sar1 for COPII and ARF1 for COPI/clathrin at Golgi/TGN.

What proteins facilitate membrane bending during budding

BAR domain proteins promote membrane curvature.

What protein mediates scission of clathrin-coated buds

Dynamin pinches off vesicles; GTPase-defective mutants block scission.

What do Rab GTPases regulate

Coat recruitment, cargo selection, vesicle formation, motor interaction, tethering, and docking.

What initiates docking at the target membrane

Rab-GTP activation and tethering factor engagement.

What determines fusion specificity

Cognate pairing of v-SNAREs (vesicle) and t-SNAREs (target) forms a 4-helix bundle.

What disassembles cis-SNARE complexes post-fusion

NSF uses ATP hydrolysis to dissociate SNAREs for recycling.

What are the three principal Golgi subcompartments

The cis, medial, and trans cisternae.

What key modification occurs in the cis-Golgi for lysosomal enzymes

Phosphorylation of mannose residues (precursor to mannose-6-phosphate).

What general carbohydrate processing occurs across cis → medial → trans

Trimming of mannose and addition of GlcNAc, galactose, fucose, and finally sialic acid.

What additional TGN modifications can occur

O-linked glycosylation and sulfation of certain glycans and proteoglycans.

What is the default trafficking route from the TGN

Constitutive secretion to the plasma membrane.

What are the three major TGN pathways

Constitutive secretion, signal-mediated diversion to lysosomes, and regulated secretion to storage granules.

What defines regulated secretion

Storage of cargo (e.g., insulin, histamine, neurotransmitters) with stimulus-triggered exocytosis.

How are soluble lysosomal enzymes tagged

GlcNAc-1-phosphotransferase adds phosphate to mannose in the cis-Golgi, forming M6P.

What receptor recognizes M6P-tagged cargo

The mannose-6-phosphate receptor (M6PR) in TGN membranes.

How are M6PR-cargo complexes delivered to lysosomes

Packaged into clathrin-coated vesicles, traffic to endosomes/lysosomes, then cargo dissociates in acidic pH.

What happens to M6PR after cargo delivery

It is recycled back to the TGN for further rounds of sorting.

What maintains lysosomal acidity

The V-type H+ ATPase pumping protons to achieve ~pH 5.

Why are lysosomal hydrolases acid-optimized

To protect the cytosol; enzymes are active at pH 5 but not ~7.2 if leakage occurs.

What do lysosomes digest

Materials delivered via endocytosis, phagocytosis, and autophagy pathways.

What cytosolic motifs target basolateral delivery

Tyrosine-based (NPxY or YxxØ) and dileucine motifs.

How is apical sorting often achieved

Partitioning into lipid rafts enriched in cholesterol/glycolipids and proteins with longer transmembrane domains or GPI anchors.

Why do apical rafts matter

They cluster apical cargos in specialized TGN microdomains for correct routing.

What is an example of polarized delivery importance

Distinct delivery to axon vs dendrite or apical vs basolateral membranes.

What is clathrin-mediated endocytosis

Receptor/adaptor-mediated budding of clathrin-coated pits into vesicles with dynamin-dependent scission.

What is constitutive endocytosis

Continuous membrane recycling; roughly half of the plasma membrane is recycled per hour.

What is regulated endocytosis

Stimulus-dependent receptor-mediated internalization of specific ligands.

How is LDL internalized

LDL binds LDL receptor, is internalized into clathrin-coated vesicles, dissociates in endosomes, and cholesterol is released.

How is transferrin cycled

Transferrin-iron binds its receptor, is internalized; iron is released in endosomes; receptor-transferrin recycles to the membrane and transferrin is released.

What happens to the EGF receptor upon ligand binding

The EGF:EGFR complex is delivered to lysosomes for degradation, downregulating signaling.

What is phagocytosis

Uptake of large particles or cells by professional phagocytes (e.g., macrophages, neutrophils).

What is pinocytosis

Non-specific uptake of extracellular fluid in small vesicles.

What specialized non-immune cells perform phagocytosis

Retinal pigment epithelium disposes of shed photoreceptor outer segments.

Where is insulin initially synthesized

As preproinsulin with an N-terminal signal sequence targeting to the ER.

Where is proinsulin processed to insulin and C-peptide

In the Golgi and secretory granules before regulated exocytosis.

What triggers insulin secretion

High glucose raises ATP/ADP, closes KATP channels, depolarizes the membrane, opens Ca2+ channels, triggering granule fusion.

What is C-peptide a marker for

Endogenous insulin synthesis and secretion.

How are synaptic vesicle components delivered

From TGN to plasma membrane/endosomes, with cycles of endocytosis and re-budding to form synaptic vesicles.

What triggers neurotransmitter release

Ca2+ influx drives synaptic vesicle fusion at nerve terminals.

What enzyme is defective in Gaucher disease

Glucocerebrosidase; macrophages accumulate glucosylceramide.

What defect causes I-cell (mucolipidosis II) disease

UDP-GlcNAc-1-phosphotransferase deficiency preventing M6P tagging of lysosomal enzymes.

What accumulates in Tay-Sachs disease

GM2 ganglioside due to Hexosaminidase A deficiency.

What accumulates in Hurler syndrome (MPS I)

Dermatan sulfate and heparan sulfate due to alpha-L-iduronidase deficiency.

What is defective in Pompe disease (GSD II)

Lysosomal acid alpha-glucosidase deficiency causing glycogen accumulation.

What enzyme deficiency underlies Niemann-Pick disease

Sphingomyelinase deficiency causing sphingomyelin accumulation.

How does alpha-1 antitrypsin deficiency relate to ER QC

Misfolded A1AT is retained in the ER, reducing elastase inhibition in lung and causing ER accumulation in hepatocytes.

Why does ERAD protect cells

It removes persistently misfolded proteins to prevent ER clogging and proteotoxic stress.