Module 3 · Cargo Delivery
Delivery Pathways
Cargo reaches the lysosome via three principal pathways — endocytosis, phagocytosis, and autophagy — each with distinct upstream machinery but converging at lysosomal fusion for degradation. Yoshinori Ohsumi shared the 2016 Nobel Prize for elucidating autophagy genetics in yeast. The three pathways together constitute the cell’s principal catabolic input.
1. Endocytosis
Internalisation of surface material through vesicles budding inward. Multiple sub-pathways:
- Clathrin-mediated endocytosis (CME): the most heavily studied. Triskelia-forming clathrin coats with AP-2 adaptor select cargo from the plasma membrane; dynamin pinches vesicles (80–120 nm). Delivers receptors (LDLR, transferrin receptor, EGFR) to early endosomes.
- Caveolae-mediated: caveolin-based invaginations; slower and more specialised.
- Clathrin- and caveolin-independent(CLIC/GEEC, flotillin, Arf6-dependent): handles GPI-anchored proteins and bulk fluid.
- Macropinocytosis: actin-driven bulk fluid uptake via large (> 1 μm) membrane ruffles that close into macropinosomes. Dominant in macrophages and dendritic cells; hijacked by tumour cells for protein scavenging under nutrient limitation (Ras-driven pancreatic and lung cancers).
Sorted cargo progresses: early endosome → late endosome → lysosome. The progression involves Rab5-to-Rab7 conversion, ESCRT-mediated intraluminal vesicle formation, and a gradient of progressive acidification. Recycling cargo (transferrin receptor, integrins) diverts back to the PM from early endosomes.
2. Phagocytosis
Engulfment of particles > 0.5 μm via actin-driven membrane extension. Used constitutively by professional phagocytes (macrophages, neutrophils, dendritic cells) and sparingly by most cell types. Triggers:
- FcγR-mediated: IgG-coated targets bound by Fcγ receptors trigger actin polymerisation via Rac1/Cdc42 and mDia.
- Complement-mediated (CR3): C3b-coated targets.
- Apoptotic-cell recognition: phosphatidyl-serine exposure on dying cells recognised by TIM4, stabilin, or BAI1, engulfed through ELMO/Dock180/Rac1.
- Pathogen recognition: TLRs and NLRs drive anti-microbial phagocytosis.
Phagosomes mature through Rab5/Rab7 exchanges, fuse with lysosomes to form phagolysosomes, and digest contents. Some pathogens (Mycobacterium tuberculosis, Legionella, Salmonella) block phagosome maturation to survive inside macrophages — the central immunological battle of lysosome biology.
3. Autophagy: Ohsumi’s 2016 Nobel
Self-degradation of the cell’s own cytoplasm. Yoshinori Ohsumi’s 1993 yeast mutant screens identified the ATG (autophagy-related) gene series (ATG1–ATG41+), establishing the mechanism. Three flavours:
- Macroautophagy: the dominant form. A double-membrane phagophore nucleates from the ER at an omegasome site (PI3P, ATG14L-VPS34-Beclin1 complex), expands, engulfs cytoplasm (bulk or selective), closes into an autophagosome, and fuses with the lysosome to form an autolysosome where contents are degraded. Core machinery: ULK1 initiator kinase, VPS34 PI3K, ATG7/ATG3 E1/E2 for LC3 lipidation, ATG5-12-16 E3, LC3-PE conjugation.
- Microautophagy: direct invagination of the lysosomal membrane engulfing small cytoplasmic regions.
- Chaperone-mediated autophagy (CMA): Cuervo & Dice. Substrates bearing a KFERQ motif are recognised by Hsc70, delivered to the lysosomal membrane, bound by LAMP2A receptor, and translocated one by one across the limiting membrane. Declines with age.
Selective autophagy variants: mitophagy (Parkin/PINK1 — mitochondria), ER-phagy (FAM134B), pexophagy (peroxisomes), nucleophagy, lipophagy (lipid droplets), aggrephagy (protein aggregates), xenophagy (intracellular pathogens). Each uses specific receptors that bind ubiquitinated cargo and LC3/GABARAPs on phagophore.
4. Autophagy Regulation
Macroautophagy is induced by nutrient stress. The master regulator is mTORC1 (next module): under nutrients, mTORC1 phosphorylates and inhibits ULK1; under starvation, mTORC1 releases ULK1 and autophagy initiates within minutes. AMPK phosphorylates ULK1 positively when ATP is low, reinforcing starvation-induced autophagy. Transcriptional regulation via TFEB (the master biogenic switch, translocates to the nucleus under stress) expands lysosomal/autophagic capacity over hours.
5. SNARE-Mediated Fusion with the Lysosome
Autophagosomes, late endosomes, and phagosomes all converge at the lysosome via SNARE-mediated fusion. Key SNAREs: STX17 (autophagosome), VAMP8 (lysosome), SNAP29. Tethering involves HOPS complex, Rab7, and RILP. Pathogenic bacteria evolved dedicated effector proteins to disrupt this step and evade lysosomal killing.