Intracellular endosomal trafficking and cargo routing — clathrin/AP-2 uptake, V-ATPase acidification, EHD1 recycling vs. ESCRT/RAB7A late-endosomal maturation, CTSD lysosomal proteolysis, and cytosolic escape

Receptor-bound macromolecular cargo and therapeutic nanoparticles (including diferric transferrin bound to TfR1/TFRC and apolipoprotein-coated lipid nanoparticles) enter cells via clathrin- and AP-2-mediated endocytosis into nascent endocytic vesicles that mature into early sorting endosomes. V-ATPase (catalytic subunit ATP6V1A) acidifies the early endosomal lumen (pH ~6.0-5.5), triggering ligand uncoupling (such as iron release from transferrin) and protonation of ionizable lipids. From the acidified sorting endosome, cargo partitions across three competing fates: (1) tubular endocytic recycling back to the plasma membrane driven by the membrane-remodeling ATPase EHD1; (2) multivesicular body (MVB) sorting by ESCRT-I (TSG101) and early-to-late endosomal maturation governed by the small GTPase RAB7A, which delivers cargo to lysosomes for luminal proteolysis by aspartic cathepsins (CTSD); and (3) endosomal membrane destabilization and productive escape into the cytosol. Because active RAB7A-driven late endosomal maturation sequesters cargo into the degradative lysosomal pathway, loss of RAB7A diverts endosomal flux away from lysosomal degradation and markedly increases cytosolic escape of lipid nanoparticle cargo (Jozić et al. 2026, PMID:41814093).

MODULE:endosomal_trafficking_routingDRAFTTransport Stepmodules/endosomal_trafficking_routing.yaml

receptor-mediated endocytosisGO:0006898 endosomal lumen acidificationGO:0048388 endocytic recyclingGO:0032456 endosome to lysosome transportGO:0008333

GO:0006898

receptor-mediated endocytosis

Clathrin/AP-2-dependent uptake of receptor-bound cargo at the plasma membrane.

GO:0048388

endosomal lumen acidification

V-ATPase-driven proton translocation acidifies early and late endosomal compartments.

GO:0032456

endocytic recycling

EHD1-dependent tubular recycling returns receptors and recycling cargo to the plasma membrane.

GO:0008333

endosome to lysosome transport

ESCRT-I (TSG101) and RAB7A route sorting-endosome cargo through MVBs/late endosomes to lysosomes.

PMID:41814093

In vivo endosomal escape assay identifies mechanisms for efficient hepatic LNP delivery.

LysoTag in vivo lysosomal barcoding shows ~8% baseline cytosolic LNP escape within 30 min and demonstrates that loss of Rab7-mediated late endosomal maturation increases cytosolic LNP escape.

Loss of Rab7, a mediator of late endosomal maturation, increased LNP escape.

file:human/TFRC/TFRC-ai-review.yaml

TFRC gene review (human)

Cell-surface receptor binding and internalization step (UniProtKB:P02786, GO:0004998, GO:0006898) matches the completed human TFRC review.

file:human/AP2M1/AP2M1-ai-review.yaml

AP2M1 gene review (human)

Cargo-motif recognition at clathrin-coated pits (UniProtKB:Q96CW1, GO:0005048, GO:0072583) matches the completed human AP2M1 review.

file:human/CLTC/CLTC-ai-review.yaml

CLTC gene review (human)

Clathrin lattice assembly and coated-vesicle internalization (UniProtKB:Q00610, GO:0005198, GO:0006898) matches the completed human CLTC review.

file:human/ATP6V1A/ATP6V1A-ai-review.yaml

ATP6V1A gene review (human)

Endosomal and lysosomal lumen acidification (UniProtKB:P38606, GO:0046961, GO:0048388) matches the completed human ATP6V1A review.

file:human/EHD1/EHD1-ai-review.yaml

EHD1 gene review (human)

Tubular recycling endosome fission and return to the plasma membrane (UniProtKB:Q9H4M9, GO:0016887, GO:0032456) matches the human EHD1 review.

file:human/TSG101/TSG101-ai-review.yaml

TSG101 gene review (human)

ESCRT-I ubiquitinated cargo recognition and MVB sorting (UniProtKB:Q99816, GO:0043130, GO:0036258) matches the completed human TSG101 review.

file:human/RAB7A/RAB7A-ai-review.yaml

RAB7A gene review (human)

Early-to-late endosome maturation and lysosome transport (UniProtKB:P51149, GO:0003924, GO:0045022, GO:0008333) matches the completed human RAB7A review.

file:mouse/Rab7/Rab7-ai-review.yaml

Rab7 gene review (mouse)

Conserved mouse Rab7 late endosome maturation and LNP escape regulation (UniProtKB:P51150, PMID:41814093) matches the completed mouse Rab7 review.

file:human/CTSD/CTSD-ai-review.yaml

CTSD gene review (human)

Lysosomal lumen aspartic endopeptidase proteolysis (UniProtKB:P07339, GO:0004190, GO:0006508) matches the human CTSD review.

8Nodes

7Parts

0Variant Sets

0Variants

8Annotons

7Connections

Executable models all models

Agent-based derived from this module

Agent-based cargo routing model of endosomal recycling, lysosomal degradation, and cytosolic escape

✓ Wild-type baseline trafficking (recycling + lysosomal degradation dominate; ~10% cytosolic escape)
✓ RAB7A / late-endosomal maturation loss (diverts flux from lysosomes and increases cytosolic escape)
✓ Clathrin / AP-2 endocytosis block (cargo trapped at the cell surface)
✓ V-ATPase acidification block (bafilomycin A1 / ATP6V1A loss; arrest in nascent endocytic vesicles)
✓ EHD1 recycling block (diverts sorting-endosome cargo toward lysosomal degradation and escape)
✓ CTSD lysosomal proteolysis block (undegraded cargo accumulates in late endosomes/lysosomes)

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Derived QC

Recommended-field compliance

100.0% recommended fields populated

All recommended fields populated.

Module deep research

✗ none found

No MODULE:endosomal_trafficking_routing deep-research report alongside the module YAML.

Leaf nodes lacking representative members

1 leaf node(s) with no concrete protein grounding:

Endosomal escape to cytosol (productive LNP/mRNA or payload delivery)

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Function consistency and core coverage

9 gene-function check(s) reflected in core functions · 0 conflict(s) · 7 evidence or coverage gap(s) (17 comparison(s))

Retained noncore functions are reported separately; they do not require promotion to core functions. Support from representative genes does not establish conservation across the whole family. Missing reviews or identifiers leave a claim unverified.

Annoton	Participant	Function	Assessment
tfrc_receptor_binding	TFRC (human) UniProtKB:P02786 Family Representative	transferrin receptor activity GO:0004998	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ap2m1_cargo_selection	AP2M1 (human) UniProtKB:Q96CW1 Family Representative	signal sequence receptor activity GO:0005048	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
cltc_coat_lattice	CLTC (human) UniProtKB:Q00610 Family Representative	structural molecule activity GO:0005198	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
atp6v1a_proton_pump	ATP6V1A (human) UniProtKB:P38606 Family Representative	proton-transporting ATPase activity, rotational mechanism GO:0046961	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ehd1_membrane_fission	EHD1 (human) UniProtKB:Q9H4M9 Family Representative	ATP hydrolysis activity GO:0016887	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
tsg101_escrt_sorting	TSG101 (human) UniProtKB:Q99816 Family Representative	ubiquitin binding GO:0043130	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
rab7a_late_maturation	RAB7A (human) UniProtKB:P51149 Family Representative	GTPase activity GO:0003924	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
rab7a_late_maturation	Rab7 (mouse) UniProtKB:P51150 Family Representative	GTPase activity GO:0003924	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ctsd_proteolysis	CTSD (human) UniProtKB:P07339 Family Representative	aspartic-type endopeptidase activity GO:0004190	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
tfrc_receptor_binding	Transferrin receptor protein 1 PANTHER:PTHR10404:SF26 Family	transferrin receptor activity GO:0004998	Family Function Unreviewed The family review has no applicable adjudication supporting or excluding GO:0004998.
ap2m1_cargo_selection	AP-2 complex subunit mu PANTHER:PTHR10529 Family	signal sequence receptor activity GO:0005048	Family Supported Family review supports GO:0005048 FAMILY_WIDE. Mu subunits across AP-1, AP-2, AP-3, and AP-4 directly bind peptide sorting signals (such as YxxPhi motifs) in the cytosolic tails of transmembrane cargos via a conserved hydrophobic pocket on the C-terminal MHD beta-sandwich.
cltc_coat_lattice	Clathrin heavy chain 1 PANTHER:PTHR10292:SF7 Family	structural molecule activity GO:0005198	Family Review Missing No supported structured family review is available for PANTHER:PTHR10292:SF7.
atp6v1a_proton_pump	V-type proton ATPase catalytic subunit A PANTHER:PTHR43607:SF9 Family	proton-transporting ATPase activity, rotational mechanism GO:0046961	Family Review Missing No supported structured family review is available for PANTHER:PTHR43607:SF9.
ehd1_membrane_fission	EH domain-containing protein 1 PANTHER:PTHR11216:SF127 Family	ATP hydrolysis activity GO:0016887	Family Review Missing No supported structured family review is available for PANTHER:PTHR11216:SF127.
tsg101_escrt_sorting	Tumor susceptibility gene 101 protein PANTHER:PTHR23306:SF17 Family	ubiquitin binding GO:0043130	Family Review Missing No supported structured family review is available for PANTHER:PTHR23306:SF17.
rab7a_late_maturation	Ras-related protein Rab-7a PANTHER:PTHR47981:SF13 Family	GTPase activity GO:0003924	Family Review Missing No supported structured family review is available for PANTHER:PTHR47981:SF13.
ctsd_proteolysis	Cathepsin D PANTHER:PTHR47966:SF42 Family	aspartic-type endopeptidase activity GO:0004190	Family Review Missing No supported structured family review is available for PANTHER:PTHR47966:SF42.

Feedback loops (advisory, prose-driven: a regulator counts as "feedback" when its own description says so)

✓ every negative regulator described as feedback has an upstream edge closing the loop.

escrt_rab7_late_endosome ⊣ cytosolic_cargo_escape [closed]
escrt_rab7_late_endosome is regulated by early_endosome_acidification: feedback loop closed

Gene-review completeness (9/9 grounded genes reviewed)

7 complete review(s) · 6 with deep research · 0 missing review · 3 reviewed but lacking deep research

Gene	Review	Complete	Deep research
AP2M1 Q96CW1	✓	129/184	✗
ATP6V1A P38606	✓	✓	✓
CLTC Q00610	✓	✓	✓
CTSD P07339	✓	84/91	✗
EHD1 Q9H4M9	✓	✓	✓
Rab7 P51150	✓	✓	✓
RAB7A P51149	✓	✓	✓
TFRC P02786	✓	✓	✓
TSG101 Q99816	✓	✓	✗

Tree

Intracellular endosomal trafficking, sorting, and cytosolic escapeTransport Stependosomal_trafficking_routing

Parts
1. 1. cell-surface cargo receptor binding (TfR1 / transferrin and targeted payload capture)
2. Cell-surface cargo receptor binding (TfR1 / transferrin & targeted payload capture)Molecular Functiontfrc_cargo_binding
  
  Annotons
  - TFRC (TfR1/CD71): cell-surface cargo receptor Family: Transferrin receptor protein 1
3. 2. clathrin/AP-2 coated pit assembly and cargo internalization
4. Clathrin/AP-2 coated pit assembly and cargo internalizationTransport Stepclathrin_ap2_internalization
  
  Annotons
  - AP2M1 (AP-2 mu2): tyrosine-motif cargo adaptor Family: AP-2 complex subunit mu
  - CLTC (CHC17): clathrin heavy chain coat lattice Family: Clathrin heavy chain 1
5. 3. V-ATPase-driven early endosome acidification and cargo/lipid protonation
6. V-ATPase early endosome acidification (pH drop, ligand uncoupling & lipid protonation)Transport Stepearly_endosome_acidification
  
  Annotons
  - ATP6V1A: V-ATPase catalytic subunit A Family: V-type proton ATPase catalytic subunit A
7. 4. EHD1-mediated tubular endocytic recycling to the plasma membrane
8. EHD1 recycling endosome tubulation and plasma-membrane returnTransport Stepehd1_recycling_branch
  
  Annotons
  - EHD1: recycling endosome tubulation/fission ATPase Family: EH domain-containing protein 1
9. 5. ESCRT-I MVB cargo sorting and RAB7A-mediated early-to-late endosome maturation
10. TSG101/ESCRT-I MVB cargo sorting and RAB7A late endosome maturationTransport Stepescrt_rab7_late_endosome
  
  Annotons
  - TSG101: ESCRT-I ubiquitin-binding MVB sorting subunit Family: Tumor susceptibility gene 101 protein
  - RAB7A / Rab7: late endosome maturation and HOPS-tethering GTPase Family: Ras-related protein Rab-7a
11. 6. endosomal membrane destabilization and productive cytosolic payload escape
12. Endosomal escape to cytosol (productive LNP/mRNA or payload delivery)Transport Stepcytosolic_cargo_escape
13. 7. lysosomal fusion and CTSD-mediated luminal cargo proteolysis
14. Lysosomal fusion and CTSD luminal cargo proteolysisMolecular Functionctsd_lysosomal_degradation
  
  Annotons
  - CTSD (cathepsin D): lysosomal aspartic endopeptidase Family: Cathepsin D

Details

Intracellular endosomal trafficking, sorting, and cytosolic escapeTransport Stependosomal_trafficking_routing

receptor-mediated endocytosisGO:0006898 endosomal lumen acidificationGO:0048388 endocytic recyclingGO:0032456 endosome to lysosome transportGO:0008333

Constructed for the DELIVEROME and ABM_TRAFFICKING projects to represent intracellular endosomal routing and cytosolic cargo escape as an executable agent-based model (ABM) derived directly from the module graph. All protein participants are grounded to completed AIGR gene reviews (human TFRC, AP2M1, CLTC, ATP6V1A, EHD1, TSG101, RAB7A, CTSD, and mouse Rab7) and PANTHER family/subfamily identifiers verified against interpro/panther/panther.obo and PANTHER sequence classifications.

Connections

tfrc_cargo_binding -> clathrin_ap2_internalization Provides Input For

Receptor-bound cargo complexes at the cell surface are captured into clathrin/AP-2 coated pits.

clathrin_ap2_internalization -> early_endosome_acidification Provides Input For

Internalized clathrin-coated vesicles uncoat and fuse with V-ATPase-acidified early sorting endosomes.

early_endosome_acidification -> ehd1_recycling_branch Provides Input For

Acid-uncoupled receptors and recycling cargo enter EHD1-dependent tubular recycling endosomes for plasma-membrane return.

early_endosome_acidification -> escrt_rab7_late_endosome Provides Input For

Cargo retained in sorting endosomes is sorted by ESCRT-I (TSG101) and matures via RAB7A into late endosomes/MVBs.

early_endosome_acidification -> cytosolic_cargo_escape Provides Input For

Protonated LNP/macromolecular cargo in acidified endosomes destabilizes the limiting membrane to escape into the cytosol.

escrt_rab7_late_endosome -> cytosolic_cargo_escape Negatively Regulates

Active RAB7A/ESCRT late endosomal maturation sequesters cargo into the degradative lysosomal route, restricting productive cytosolic escape (PMID:41814093).

escrt_rab7_late_endosome -> ctsd_lysosomal_degradation Provides Input For

Mature RAB7A-positive late endosomes fuse with lysosomes where CTSD degrades luminal cargo.

Part 1: cell-surface cargo receptor binding (TfR1 / transferrin and targeted payload capture)

Cell-surface cargo receptor binding (TfR1 / transferrin & targeted payload capture)Molecular Functiontfrc_cargo_binding

Annotons

TFRC (TfR1/CD71): cell-surface cargo receptor

tfrc_receptor_binding

Participant: Family: Transferrin receptor protein 1

Family:

Transferrin receptor protein 1PANTHER:PTHR10404:SF26

Representative Members: TFRC (human)UniProtKB:P02786

Function

transferrin receptor activityGO:0004998

Substrates: diferric transferrin / receptor-targeted macromolecular cargo

Processes

receptor-mediated endocytosisGO:0006898 transferrin transportGO:0033572

Locations

external side of plasma membraneGO:0009897

Captures extracellular diferric transferrin (and TfR1-targeted payloads) at the plasma membrane and presents its cytoplasmic YTRF sorting motif to the AP-2 adaptor complex.

Part 2: clathrin/AP-2 coated pit assembly and cargo internalization

Clathrin/AP-2 coated pit assembly and cargo internalizationTransport Stepclathrin_ap2_internalization

Annotons

AP2M1 (AP-2 mu2): tyrosine-motif cargo adaptor

ap2m1_cargo_selection

Participant: Family: AP-2 complex subunit mu

Family:

AP-2 complex subunit muPANTHER:PTHR10529

Representative Members: AP2M1 (human)UniProtKB:Q96CW1

Function

signal sequence receptor activityGO:0005048

Processes

clathrin-dependent endocytosisGO:0072583 receptor internalizationGO:0031623

Locations

clathrin-coated pitGO:0005905

Recognizes tyrosine-based cytoplasmic sorting signals (such as the TfR1 YTRF motif) at PtdIns(4,5)P2-rich plasma-membrane patches to concentrate cargo into clathrin-coated pits.

CLTC (CHC17): clathrin heavy chain coat lattice

cltc_coat_lattice

Participant: Family: Clathrin heavy chain 1

Family:

Clathrin heavy chain 1PANTHER:PTHR10292:SF7

Representative Members: CLTC (human)UniProtKB:Q00610

Function

structural molecule activityGO:0005198

Processes

receptor-mediated endocytosisGO:0006898 clathrin coat assemblyGO:0048268

Locations

clathrin-coated endocytic vesicleGO:0045334

Assembles polyhedral clathrin cages on AP-2-selected membrane patches to invaginate and internalize cargo-bearing endocytic vesicles.

Part 3: V-ATPase-driven early endosome acidification and cargo/lipid protonation

V-ATPase early endosome acidification (pH drop, ligand uncoupling & lipid protonation)Transport Stepearly_endosome_acidification

Annotons

ATP6V1A: V-ATPase catalytic subunit A

atp6v1a_proton_pump

Participant: Family: V-type proton ATPase catalytic subunit A

Family:

V-type proton ATPase catalytic subunit APANTHER:PTHR43607:SF9

Representative Members: ATP6V1A (human)UniProtKB:P38606

Function

proton-transporting ATPase activity, rotational mechanismGO:0046961

Processes

endosomal lumen acidificationGO:0048388 proton transmembrane transportGO:1902600

Locations

endosome membraneGO:0010008 lysosomal membraneGO:0005765

Acidifies sorting endosomes so that receptor-bound ligands dissociate (or release iron) and ionizable lipid nanoparticles become protonated prior to downstream routing into recycling, late-endosomal maturation, or cytosolic escape.

Part 4: EHD1-mediated tubular endocytic recycling to the plasma membrane

EHD1 recycling endosome tubulation and plasma-membrane returnTransport Stepehd1_recycling_branch

Annotons

EHD1: recycling endosome tubulation/fission ATPase

ehd1_membrane_fission

Participant: Family: EH domain-containing protein 1

Family:

EH domain-containing protein 1PANTHER:PTHR11216:SF127

Representative Members: EHD1 (human)UniProtKB:Q9H4M9

Function

ATP hydrolysis activityGO:0016887

Processes

endocytic recyclingGO:0032456

Locations

recycling endosome membraneGO:0055038

Tubulates and vesiculates recycling endosome membranes to return internalized receptors (such as apo-TfR1) and non-degraded cargo back to the cell surface.

Part 5: ESCRT-I MVB cargo sorting and RAB7A-mediated early-to-late endosome maturation

TSG101/ESCRT-I MVB cargo sorting and RAB7A late endosome maturationTransport Stepescrt_rab7_late_endosome

Context

late endosome membraneGO:0031902

Annotons

TSG101: ESCRT-I ubiquitin-binding MVB sorting subunit

tsg101_escrt_sorting

Participant: Family: Tumor susceptibility gene 101 protein

Family:

Tumor susceptibility gene 101 proteinPANTHER:PTHR23306:SF17

Representative Members: TSG101 (human)UniProtKB:Q99816

Function

ubiquitin bindingGO:0043130

Processes

multivesicular body assemblyGO:0036258 ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathwayGO:0043162

Locations

late endosome membraneGO:0031902 early endosome membraneGO:0031901

Recognizes ubiquitinated cargo and recruits ESCRT machinery to bud intraluminal vesicles into maturing multivesicular bodies.

RAB7A / Rab7: late endosome maturation and HOPS-tethering GTPase

rab7a_late_maturation

Participant: Family: Ras-related protein Rab-7a

Family:

Ras-related protein Rab-7aPANTHER:PTHR47981:SF13

Representative Members: RAB7A (human)UniProtKB:P51149 Rab7 (mouse)UniProtKB:P51150

Function

GTPase activityGO:0003924

Processes

early endosome to late endosome transportGO:0045022 endosome to lysosome transportGO:0008333

Locations

late endosomeGO:0005770 late endosome membraneGO:0031902

Executes the Rab5-to-Rab7 conversion on maturing endosomes and recruits HOPS/RILP effectors to commit cargo to late endosomes and lysosomal fusion, competing with productive cytosolic escape.

Part 6: endosomal membrane destabilization and productive cytosolic payload escape

Endosomal escape to cytosol (productive LNP/mRNA or payload delivery)Transport Stepcytosolic_cargo_escape

Context

cytosolGO:0005829 endosome membraneGO:0010008

PMID:41814093

In vivo endosomal escape assay identifies mechanisms for efficient hepatic LNP delivery.

Approximately 8% of branched ionizable phospholipid LNPs escape acidified endosomes into the hepatic cytosol within 30 min in vivo, and blocking Rab7-mediated late endosomal maturation increases cytosolic escape.

Loss of Rab7, a mediator of late endosomal maturation, increased LNP escape.

Part 7: lysosomal fusion and CTSD-mediated luminal cargo proteolysis

Lysosomal fusion and CTSD luminal cargo proteolysisMolecular Functionctsd_lysosomal_degradation

Annotons

CTSD (cathepsin D): lysosomal aspartic endopeptidase

ctsd_proteolysis

Participant: Family: Cathepsin D

Family:

Cathepsin DPANTHER:PTHR47966:SF42

Representative Members: CTSD (human)UniProtKB:P07339

Function

aspartic-type endopeptidase activityGO:0004190

Processes

proteolysisGO:0006508

Locations

lysosomal lumenGO:0043202 endosome lumenGO:0031904

Hydrolyzes internalized protein cargo delivered to the acidic lysosomal lumen, acting as the terminal degradative sink of the endolysosomal pathway.