Intrinsic (Mitochondrial) Apoptosis Pathway

Core Concept

The intrinsic (mitochondrial) pathway of apoptosis is activated by intracellular stress — DNA damage, growth-factor withdrawal, ER stress, or developmental cues — rather than by an external death ligand. Its central event is mitochondrial outer-membrane permeabilization (MOMP), which releases cytochrome c (and other intermembrane-space proteins such as SMAC/DIABLO) into the cytosol. Cytochrome c binds the adaptor Apaf-1, which oligomerizes into a wheel-shaped "apoptosome" that recruits and activates the initiator caspase-9. Caspase-9 then activates executioner caspases that dismantle the cell. The pathway is governed by the Bcl-2 protein family — pro-apoptotic and anti-apoptotic members that regulate MOMP — and by p53.

Key Components

• Bcl-2 family: Anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1) vs. pro-apoptotic. Pro-apoptotic members split into multi-domain effectors (Bax, Bak) and BH3-only sensors (Bid, Bim, Puma, Noxa, Bad).
• Bax/Bak: Effector proteins that oligomerize in the outer mitochondrial membrane to form pores (MOMP).
• BH3-only proteins: Stress sensors that either activate Bax/Bak or neutralize anti-apoptotic Bcl-2 proteins.
• Cytochrome c: Heme protein released from the mitochondrial intermembrane space.
• Apaf-1: Cytosolic adaptor; binds cytochrome c and dATP.
• Apoptosome: Heptameric Apaf-1/cytochrome c complex that activates caspase-9.
• SMAC/DIABLO: Releases caspase inhibition by neutralizing IAPs (inhibitor of apoptosis proteins).
• p53: Transcriptionally upregulates Puma/Noxa/Bax and can act directly at mitochondria.

Mechanism / How It Works

1. Stress sensing: DNA damage activates p53, which induces BH3-only proteins (Puma, Noxa) and Bax. Growth-factor withdrawal causes Bim (via FOXO) and Bad (dephosphorylated) activation. These signals converge on the mitochondria.
2. Bcl-2 family control of MOMP: BH3-only proteins bind and neutralize anti-apoptotic Bcl-2/Bcl-xL/Mcl-1 and/or directly activate Bax and Bak. Activated Bax/Bak undergo conformational change, insert into the outer mitochondrial membrane, and oligomerize into pores — MOMP.
3. Cytochrome c release: Pores release cytochrome c (and SMAC/DIABLO, Omi/HtrA2) from the intermembrane space.
4. Apoptosome assembly: In the cytosol, cytochrome c binds Apaf-1, inducing a conformational change (aided by dATP) that exposes its oligomerization domain. Seven Apaf-1 molecules assemble with cytochrome c into the heptameric apoptosome, which presents caspase-recruitment domains (CARDs).
5. Caspase-9 activation: Procaspase-9 is recruited to the apoptosome via CARD–CARD interactions and is activated by proximity-induced dimerization (the induced-proximity model).
6. Execution: Caspase-9 cleaves and activates executioner caspases-3 and -7, which dismantle the cell. SMAC neutralizes IAPs (XIAP) that would otherwise inhibit caspases.

Energy and Directionality

The intrinsic pathway is an energy-dependent, stepwise amplification cascade. MOMP is the point of no return: once the outer membrane is permeabilized, cytochrome c is released and the cell is committed to death, even if the initial stimulus is removed — because the downstream caspase cascade is self-amplifying and feeds forward (caspase-9 → caspase-3 → more caspase-9). Apoptosome assembly requires dATP/ATP, and caspase proteolysis is irreversible (proteolysis, not reversible phosphorylation). The Bcl-2 family acts as a rheostat: the balance of pro- vs. anti-apoptotic members, not any single protein, sets the threshold for MOMP.

Experimental Evidence / Technique

• Cytochrome c release assay: Immunofluorescence or subcellular fractionation shows cytochrome c translocating from mitochondria to cytosol upon apoptosis induction.
• Cell-free systems: Adding cytochrome c and dATP to Xenopus or mammalian cytosol activates caspases — Liu et al. (1996) identified cytochrome c and Apaf-1 as the cytosolic factors.
• Bax/Bak double knockout: Cells lacking both Bax and Bak resist MOMP and apoptosis, proving they are the essential pore-forming effectors.
• Apoptosome structure: Cryo-EM revealed the heptameric Apaf-1–cytochrome c wheel.
• BH3 profiling and BH3 mimetics: Drugs like ABT-737/venetoclax (a Bcl-2 inhibitor) trigger apoptosis in Bcl-2-dependent cancer cells, validating the pathway clinically.

How it works

1. Stress sensing: DNA damage activates p53, which induces BH3-only proteins (Puma, Noxa) and Bax. Growth-factor withdrawal causes Bim (via FOXO) and Bad (dephosphorylated) activation. These signals converge on the mitochondria.
2. Bcl-2 family control of MOMP: BH3-only proteins bind and neutralize anti-apoptotic Bcl-2/Bcl-xL/Mcl-1 and/or directly activate Bax and Bak. Activated Bax/Bak undergo conformational change, insert into the outer mitochondrial membrane, and oligomerize into pores — MOMP.
3. Cytochrome c release: Pores release cytochrome c (and SMAC/DIABLO, Omi/HtrA2) from the intermembrane space.
4. Apoptosome assembly: In the cytosol, cytochrome c binds Apaf-1, inducing a conformational change (aided by dATP) that exposes its oligomerization domain. Seven Apaf-1 molecules assemble with cytochrome c into the heptameric apoptosome, which presents caspase-recruitment domains (CARDs).
5. Caspase-9 activation: Procaspase-9 is recruited to the apoptosome via CARD–CARD interactions and is activated by proximity-induced dimerization (the induced-proximity model).
6. Execution: Caspase-9 cleaves and activates executioner caspases-3 and -7, which dismantle the cell. SMAC neutralizes IAPs (XIAP) that would otherwise inhibit caspases.

Common confusions

• Intrinsic vs. extrinsic: Intrinsic is triggered by internal stress and runs through mitochondria/cytochrome c; extrinsic is triggered by external death ligands and runs through death receptors (caspase-8). They converge on the executioner caspases (and can cross-talk via Bid).
• Bax/Bak vs. Bcl-2: Bax/Bak are pro-apoptotic pore formers; Bcl-2/Bcl-xL are anti-apoptotic. All are structurally related (Bcl-2 family), but functionally opposite.
• Cytochrome c's dual role: In healthy cells it shuttles electrons in respiration; only when released to the cytosol does it trigger apoptosis.
• Caspase-9 is an initiator, not an executioner: It activates caspases-3/7; the executioners do the demolition.

Quick review

• Stress → p53/BH3-only → neutralize Bcl-2, activate Bax/Bak → MOMP → cytochrome c (+SMAC) release.
• Cytochrome c + dATP + Apaf-1 → apoptosome → procaspase-9 → caspase-9 (initiator) → caspase-3/7 (executioners) → cell death.
• Point of no return = MOMP; regulated by Bcl-2 family balance.