16.2 Vesicular transport and protein secretion

Cells rely on a complex system of vesicle formation and transport to move proteins and other molecules between compartments. From coat protein assembly to membrane fusion, each step is carefully orchestrated to ensure cargo reaches the right destination.

Protein secretion builds on this transport machinery. Cells can release proteins continuously (constitutive secretion) or store them for release in response to a specific signal (regulated secretion). Both pathways are essential for processes like maintaining the extracellular matrix, signaling between neurons, and regulating blood glucose.

Vesicle Formation and Transport

Steps of transport vesicle formation

Vesicle transport follows a consistent sequence regardless of which coat protein or pathway is involved. Here are the seven key steps:

1. Initiation: Coat proteins assemble on the donor membrane, recognizing specific cargo molecules and membrane receptors (e.g., the mannose-6-phosphate receptor for lysosomal enzymes).

2. Budding: Coat proteins deform the membrane by inducing curvature, shaping the emerging vesicle. Clathrin and COPI coats are well-studied examples of this membrane-bending activity.

3. Scission: The vesicle pinches off from the donor membrane. Accessory proteins drive this step. For clathrin-coated vesicles, the GTPase dynamin wraps around the neck of the bud and uses GTP hydrolysis to sever it.

4. Movement: Vesicles travel along cytoskeletal elements powered by motor proteins.

   • Microtubules serve as tracks for long-distance transport (e.g., axonal transport in neurons).
   • Kinesins move vesicles toward the plus end of microtubules (anterograde transport, away from the cell body), while dyneins move vesicles toward the minus end (retrograde transport, toward the cell body).

5. Tethering: Tethering factors loosely capture vesicles near the target membrane, providing the first layer of specificity. The exocyst complex, for example, tethers vesicles destined for exocytosis at the plasma membrane.

6. Docking: SNARE proteins on the vesicle (v-SNAREs, such as VAMP/synaptobrevin) and on the target membrane (t-SNAREs, such as syntaxin) interact, pulling the two membranes into close contact.

7. Fusion: The SNARE complex zips together fully, generating enough force to merge the lipid bilayers. Vesicle contents are released into the target compartment or the extracellular space. Synaptic vesicle fusion at nerve terminals is a classic example.

Role of coat proteins

Three major coat protein systems handle different transport routes. Each one selects specific cargo and buds vesicles from a particular donor membrane.

• COPII (Coat Protein Complex II)
  • Assembles on the ER membrane
  • Mediates anterograde (forward) transport from the ER to the Golgi apparatus
  • Built from two subcomplexes: Sec23/24 (inner coat) and Sec13/31 (outer cage)
  • Sec24 is the cargo adaptor. It directly binds ER export signals on cargo proteins, selecting which proteins get packaged into the vesicle.
• COPI (Coat Protein Complex I)
  • Assembles on Golgi membranes
  • Mediates retrograde transport from the Golgi back to the ER, as well as intra-Golgi transport between cisternae
  • Composed of seven subunits (α, β, β', γ, δ, ε, ζ)
  • Recognizes retrieval signals on ER-resident proteins: KKXX motifs (on transmembrane proteins) and KDEL sequences (on soluble ER luminal proteins, recognized indirectly via the KDEL receptor). This retrieval system returns escaped ER proteins to where they belong.
• Clathrin coats
  • Assemble on the plasma membrane and the trans-Golgi network (TGN)
  • Mediate endocytosis (plasma membrane) and transport from the TGN to endosomes/lysosomes
  • Clathrin heavy and light chains assemble into a three-legged structure called a triskelion, which polymerizes into a lattice cage around the vesicle
  • Clathrin doesn't bind cargo directly. Instead, adaptor protein (AP) complexes bridge clathrin to cargo receptors: AP-2 operates at the plasma membrane, while AP-1 operates at the TGN.

Function of SNARE proteins

SNAREs (Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptors) are the core machinery for membrane fusion. They ensure that vesicles fuse only with the correct target.

The two classes of SNAREs:

• v-SNAREs sit on the vesicle membrane. The best-known example is VAMP/synaptobrevin.
• t-SNAREs sit on the target membrane. Examples include syntaxin and SNAP-25.

How the SNARE complex drives fusion:

• A v-SNARE on the vesicle and t-SNAREs on the target membrane wind together into a highly stable four-helix bundle.
• This "zippering" proceeds from the membrane-distal ends toward the membrane-proximal ends, progressively pulling the two bilayers together.
• The energy released by forming this tight coiled-coil is what overcomes the repulsive forces between membranes and drives fusion.

How SNAREs ensure specificity:

• Different transport steps use distinct SNARE combinations. Syntaxin-1 and SNAP-25 pair with VAMP2 for synaptic vesicle fusion, while syntaxin-5 participates in ER-to-Golgi transport. These specific pairings help guarantee that vesicles fuse with the right compartment.

Recycling and regulation:

• After fusion, the SNARE complex is extremely stable and must be actively disassembled. NSF (an ATPase) and its cofactor α-SNAP use ATP hydrolysis to pry apart the spent SNARE complex, freeing individual SNAREs for reuse.
• SM (Sec1/Munc18) proteins regulate SNARE complex assembly. Munc18-1, for instance, binds syntaxin-1 and controls when and how the SNARE complex forms during synaptic vesicle fusion.

Protein Secretion

Regulated vs constitutive secretion

All cells use constitutive secretion, and many specialized cells also use regulated secretion. The key difference is whether cargo is stored before release.

Constitutive secretion:

• Proteins move continuously from the ER through the Golgi to the plasma membrane without any storage step.
• This is the "default" pathway. If a protein has no special sorting signal directing it elsewhere, it gets secreted constitutively.
• Examples: extracellular matrix proteins like collagen and fibronectin, as well as newly synthesized membrane proteins (receptors, channels) being delivered to the cell surface.

Regulated secretion:

• Proteins are concentrated and stored in secretory granules until a specific extracellular signal triggers their release.
• The trigger typically involves a rise in intracellular $\text{Ca}^{2+}$ or another second messenger, which promotes fusion of the granules with the plasma membrane.
• Examples:
  • Insulin secretion by pancreatic β-cells when blood glucose rises
  • Neurotransmitter release (glutamate, GABA) at synapses in response to action potentials

How proteins enter and navigate the secretory pathway:

1. A signal peptide (a short N-terminal hydrophobic sequence) directs the ribosome-mRNA complex to the ER membrane, where the protein is co-translationally translocated into the ER lumen.

2. Inside the ER and Golgi, proteins undergo post-translational modifications that affect folding, stability, and sorting:

   • Glycosylation adds sugar chains. N-linked glycosylation begins in the ER; O-linked glycosylation occurs in the Golgi.
   • Disulfide bond formation in the oxidizing ER lumen stabilizes protein tertiary structure.

3. Sorting receptors then direct proteins to the correct destination:

   • The mannose-6-phosphate receptor recognizes the mannose-6-phosphate tag added to lysosomal hydrolases in the Golgi and routes them to lysosomes via clathrin-coated vesicles.
   • Transmembrane domains anchor proteins in the lipid bilayer for delivery to the plasma membrane, while GPI (glycosylphosphatidylinositol) anchors attach proteins to the outer leaflet of the membrane.