Cell Bio Vesicle Transport and Lysosomes

importance of vesicle transport

- compartmentalization needs directed, selective traffic

- preserves membrane identity and precise cargo localization

- powers secretion, PM remodeling, organelle homeostasis

COPII- ER to golgi

- buds from ER exit sites (ERES) for anterograde flow

- exports ER passed-quality control cargo to the cis-golgi

- uses Sar1 (small GTPase) + inner/outer coat layers

COPII mechanism- Sar1 activation

- Sec12 (GEF): Sar1-GDP -> Sar1-GTP

- Sar1 amphipathic helix inserts -> membrane curvature

- Sec23/24 select cargo

- Sec13/31 form the cage

COPI- golgi to ER

- retrograde retrieval: golgi back to ER

- returns ER residents and recycles golgi enzymes

- helps maintain cis/medial/trans organization

COPI mechanism- ARF1

- ARF1-GTP recruits COPI on golgi membranes

- coats sort retrieal-motif cargoes (KDEL/KKXX via receptors)

- vesicles uncoat prior to fusion with ER

clathrin: endocytosis and TGN

- operates at plasma membrane (endocytosis) and TGN (to endosomes)

- adaptors read sorting signals and recruit clathrin

- triskelia assemble into polyhedral lattices

clathrin triskelion assembly

- 3 heavy + 3 light chains -> triskelion

- triskelia tile into hexagons/pentagons (cages)

- uncoat before fusion (ATP-dependent)

adaptor proteins

AP complexes

- AP1, AP2, AP3, AP4

- recognize YxxΦ and [DE]xxxLL motifs on cytosolic tails

-> short linear motifs

0 recruit clathrin and concentrate cargo

AP1

(TGN) for outbound sorting

AP2

(PM) for endocytosis

AP3

(TGN) for endosome sorting

AP4

(TGN) for transport from the TGN

AP1 scaffold

clathrin

AP4 scaffold

unknown

AP1 signals

- YxxΦ

- [DE]XXXL[LI]

- noncanonical

AP4 signals

- XY{FYL][FL]E

- noncanonical

AP1 localization

TGN/RE

AP4 localization

TGN

cargo selection signals

- tyrosine-based (YxxΦ) and dileucine ([DE]xxxLL) signals in AP

- KDEL/KKXX enable ER retrieval

- short motifs

KDEL/KKXX enable ER retrieval

via receptors/ligands -> COPI coats

short motifs

addresses parsed by adaptors/coats

Rab GTPases as identity markers

- Rab 1-11

- multiple Rabs to partition the secretory and endosomal routes

- prenylated membrane anchor

- Rab-GTP recruits effectors

Rab 1-11

zip-code GTPases

- cycle GDP(off)/GTP(on) to mark compartments/vesicles

Rab GTPases: prenylated membrane anchor

- Guanine Exchange Factor (GEF) load GTP at the right membrane

- GAPs promote hydrolysis to terminate signaling

Rab-GTP recruits effectors

- long tethers

- multisubunit tethering complexes (MTCs)

- motors (kynesin/dynein/myosin)

Rab effectors and tethering

- coiled-coil tethers (eg Golgins) reach out and capture vesicles at long range

- MTCs bridge vesicle by Rab to the target and organize SNARE assembly

- tethering raises local SNARE pairing probability without causing fusion

SNARE code

complementary v-SNARE (vesicle) + t-SNAREs (target)

SM proteins

chapterone and license correct SNARE pairing

- eg Sec1/Munc18

v-SNAREs and t-SNARES

- assembly yields a 4-helix bundle that encodes compartment specificity

- correct combinations are fast and favorable

- mismatches are kinetically suppressed

SNARE complex formation

- trans-SNARE nucleates at the contact site after tethering/uncoating

- N -> C "zippering" pulls bilayers into tight docking

- energy of bundle formation substitutes for external energy input

- precedes the hemifusion

hemifusion

fusion pore transitions

zippering mechanism of fusion

- hemifusion stalk -> diaphragm -> fusion pore -> pore dilation

- SNARE energy drives each transition

- lipids redistribute between leaflets

- ends with cis-SNARE complex on one membrane, ready for recycling

NSF/SNAP-mediated SNARE recycling

- α-SNAP binds cis-SNARE

- NSF (AAA+ ATPase) uses ATP to unwind the bundle

- recycling restores free v/t-SNARE pools for subsequent rounds

- coupling to traffic hubs keeps local SNARE availability high

neurotransmitter vesicle release

- dock/prime at active zones

- synaptotagmin binds membranes upon Ca2+ influx -> clamp release

- millisecond exocytosis with precise timing and probability control

- same core logic (Rab/tether/SNARE), tuned for speed

_____ clamps the SNARE

complexin

synaptotagmin

Ca2+ sensor

botulinum neurotoxins cause

SNARE cleavage

SNARE cleavage and botulinum neurotoxins

- clostridial Zn-endopeptidases cleave SNAP-25, syntaxin, or VAMP (SNAREs)

- loss of SNARE function

loss of SNARE function with botulinum neurotoxins leads to

exocytosis block -> paralysis

clinical use of botulinum neurotoxins

botulinum as target to block acetylcholine (which causes temporary muscle paralysis)

lysosome structure

acidic lumen

acidic lumen of lysosomes

- single membrane organelles with pH ~4.5-5.0

- contain acid hydrolases

- LAMP1/2 glycoproteins form a protective luminal (internal) glycocalyx

- ion channels/transporters maintain osmotic balance during degradation

acid hydrolases

- proteases

- lipases

- nucleases

- glycosidases

V-ATPase proton pump

- V1 + V0 drive H+ import into the lumen

- sets/maintains lysosomal acidity for hydrolase activity

- acidification also drives cargo-receptor dissociation

- inhibition (eg bafilomycin) alkalinizes lysosomes

V1

ATPase

V0

rotor

inhibition (eg bafilomycin) alkalinizes lysosomes, which leads to

functional failure

Mannse-6-Phosphate (M6P) tag

- Cis-Golgi

- NAGPA exposes M6P by removing the GcNAc

- M6P acts as an address label for lysosomal targeting

- specificity depends on hydrolase recognition motifs

Mannse-6-Phosphate (M6P) tag: Cis-Golgi

GlcNAc-1-phosphotransferase (GNPT) primes specific N-glycans to be tagged

NAGPA

- uncovering enzyme

- exposes M6P by removing the GcNAc

M6P receptor pathway

- MPRs bind M6P-hydrolases in the TGN

- packaged via AP1/clathrin -> early/late endosomes

- directionality set by pH gradient

- low pH triggers cargo release

directionality of M6P receptor pathway set by pH gradient

TGN -> endosome -> lysosome

M6P receptor pathway: low pH triggers cargo release

receptors recycle to TGN by retromer complex

TGN

sorting hub for PM, secretion, lysosomes

sorting from trans-golgi network

- decisions use signal + adaptor + coat logic

- constitutive vs regulated secretory routes co-exist

- lysosomal route relies on M6P tag (others use distinct signals)

autophagy overview

- double-membrane autophagosome -> lysosome

- recycles proteins, organelles, aggregates

- supports stress survival

- selective lysosomal receptors link cargo to LC3 interacting region on the phagophore

- complements proteasome by handling bulk/insoluble substrates

macroautophagy

cytosolic double membrane encloses cargo

microautophagy

lysosome invaginates small cytosolic portions

macroautophagy vs microautophagy

- both converge on lysosomal degradation

- choice reflects cargo size/selectivity and context

mitophagy

- selective removal of damaged mitochondria

- PINK1 accumulates on depolarized mitochondria

- Parkin ubiquitinates OMM proteins

- preserves bioenergetic quality

- defects link to neurodegeneration

PINK1 accumulates on depolarized mitochondria ->

recruits Parkin

Parkin ubiquitinates OMM proteins ->

receptors that bind LC3 on the autophagosome

lysosomal storage diseases

- enzyme/trafficking defects

- tissue-specific phenotypes

- ex: Tay-Sachs

lysosomal storage diseases: enzyme/trafficking defects

substrate buildup in lysosomes

lysosomal storage diseases: tissue-specific phenotypes

- CNS

- liver

- bone

- muscle

lysosomal storage diseases: strategies

- enzyme replacement

- substrate reduction

- chaperones

vesicle transport pathways

- anterograde

- post TGN branches

- retrograde

- Rab/tether/SNARE layers

anterograde vesicle transport pathways

ER -> cis -> medial -> trans-Golgi -> TGN

post-TGN branches: vesicle transport pathways

- PM/constitutive

- regulated granules

- endosome/lysosome

retrograde vesicle transport pathways

- COPI returns ER/Golgi residents

- carriers return M6P receptors to TGN

Rab/tether/SNARE layers in vesicle transport pathways

enforce route specificity

ER -> Golgi -> lysosome routes

- secreted/PM cargo

- lysosomal hydrolase

- pH gradient orchestrates receptor hand-offs

- uncoating enables fusion

- COPI retrieval prevents enzyme drift

- MPR recycling sustains lysosomal supply

ER -> Golgi -> lysosome routes: secreted/PM cargo

ER fold -> golgi process -> TGN -> constitutive/regulated pathways

ER -> Golgi -> lysosome routes: hysosomal hydrolase

ER fold -> M6P tagging in Golgi -> MPR/AP1/clathrin -> endosome -> lysosome

quality control in lysosomal targeting

- tag fidelity

- receptor cycle

- acidification

- safety rails

tag fidelity

correct M6P addition (GNPT) + unvovering (NAGPA)

receptor cycle

- M6P capture at TGN

- release in endosome

- return to TGN

acidification

V-ATPase endures hydrolase activity and receptor handoffs

safety rails

- misfolded hydrolases retained/ERAD

- mis-sorted enzymes secreted (diagnostic clue)

failure modes and phenotypes

- V-ATPase/ion channel defects

- MPR/retromer defects

- coat/SNARE defects

- GNPT deficiency (I-cell)

V-ATPase/ion channel defects

alkaline lysosomes -> storage, mis-sorting

MPR/retromer defects

receptor recycling loss -> hydrolase delivery inefficiency

coat/SNARE defects

bud/fusion bottlenecks -> global traffic delays

GNPT deficiency (I-cell)

- hydrolases secreted

- empty lysosomes

I-cell disease

- GNPT (GlcNAc-1-phosphotransferase) deficiency -> no M6P tag

- hydrolases are secreted

- lysosomes enzyme-poor

- severe skeletal/craniofacial and developmental abnormalities

lab clue of I-cell disease

- high plasma hydrolase activity

- low intracellular

build -> aim -> fuse

- clathrin coats select/shape

- Rabs/tethers target

- SNAREs merge

Golgi/TGN impose ___

order

pH gradients impose _____

direction and allow selection

lysosomal delivery hinges on _____

M6P/MPR and V-ATPase

failures map to checkpoints

- tagging

- pH

- receptors

- coats/SNARes