Chapter 14.4

Cargo proteins transition from the cis-Golgi to the trans-Golgi via cisternal maturation.
Modifications to oligosaccharide chains of cargo proteins are performed by Golgi-resident enzymes.
Retrograde trafficking of COPI vesicles ensures that sufficient levels of carbohydrate-modifying enzymes are maintained in their respective compartments.
The trans-Golgi network (TGN) serves as the final processing site, from which these proteins are sorted into various vesicles for delivery to target destinations (plasma membrane, endosomes, lysosomes).
This sorting at the TGN establishes the unique identity of various organelles.
This section will cover:
- Different vesicle types budded from the trans-Golgi network.
- Mechanisms of cargo segregation.
- Key processing events occurring late in the secretory pathway.

COPI vesicles (purple) - Mediate retrograde transport within Golgi (step 1).
Clathrin-coated vesicles (red) - Transport proteins to the lysosome via late endosomes (step 2).
- After uncoating, these vesicles fuse with late endosomes, delivering contents to lysosomes.
Some vesicles bypass late endosomes and fuse directly with lysosomes (step 3).
- These vesicles are coated with AP3 complex (blue).
Uncharacterized coats surround constitutive (step 4) and regulated secretory (step 5) vesicles, transporting proteins to the cell surface.

Clathrin vesicles display a two-layered coat:
- Outer layer - Composed of clathrin, forming a polymer lattice.
- Inner layer - Composed of adapter protein (AP) complexes that aid in cargo selection and stabilizing the vesicle coat.
Clathrin triskelion structure:
- Comprised of three heavy chains and three light chains, exhibiting intrinsic curvature essential for vesicle shape. (Figure 14-19a).
Clathrin assembles with AP complexes on the donor membrane, filling the space between the coat and the membrane.

Multiple AP complexes: AP1, AP2, AP3, and monomeric GGA.
- Each complex has specific binding properties and selects cargo proteins for transport, indicative of their role in the late secretory pathway.
- AP complexes contain four distinct protein subunits, while GGA functions as a single polypeptide with clathrin- and cargo-binding domains.
Coat assembly activation by ARF:
- Initiator for coat assembly on donor membranes.
Targets for clathrin-coated vesicles:
- AP1 binds proteins containing Tyr-X-X-Φ sequences (tyrosine-based signals).
- GGA binds cargo with distinct sorting sequences: Asp-X-Leu-Leu and Asp-Phe-Gly-X-Φ.
Functionality of AP3: Not fully dependent on clathrin, involved in trafficking to lysosome, bypassing late endosomes, and delivering proteins to melanosomes and storage vesicles.

Dynamin is a cytosolic protein crucial for vesicle release.
Covers the neck of the vesicle bud, then hydrolyzes GTP to facilitate neck constriction and vesicle release (Figure 14-20).
Cells lacking dynamin form long-necked, unpinched vesicle buds, affirming dynamin's essential role (Figure 14-21).

After membrane fusion, clathrin coats are removed by cytosolic Hsp70 chaperones through ATP hydrolysis, releasing triskelions for reuse.
Uncoating is supported by co-chaperone auxillin and regulated by ARF state changes (GTP/GDP).

Key sorting signal for lysosomal enzyme trafficking in the trans-Golgi network (TGN) is a carbohydrate residue, specifically mannose 6-phosphate (M6P).
M6P formation involves:
- Step 1: Phosphorylation of mannose residues by N-acetylglucosamine phosphotransferase.
- Step 2: Removal of GlcNAc by a phosphodiesterase, leaving M6P attached. (Figure 14-22).
M6P receptors in late endosomes facilitate enzyme release at low pH (5.0-5.5) and prevent rebinding of enzymes to receptors post-release.
Retromer complex recycles M6P receptors to TGN after their function.

Lysosomal enzymes acquire M6P in the cis-Golgi (Figure 14-23).
Interaction with M6P receptors directs contents into clathrin/AP1-coated vesicles (step 1).
After uncoating (step 2), transport vesicles merge with late endosomes (step 3).
Phosphate removal facilitates enzyme action post-delivery to lysosomes (step 4 and 5).
Any M6P-containing enzymes released into the bloodstream can be internalized by receptor-mediated endocytosis (step 6-9).

Disorders arise from the absence of lysosomal enzymes, causing accumulation within lysosomes.
I-cell disease: A notable form caused by missing phosphotransferase, leading to secretion of lysosomal enzymes rather than delivery to lysosomes.
Research on I-cell disease revealed M6P as a critical sorting marker, showing fibroblasts can internalize M6P-tagged enzymes via receptors (indicating normal M6P receptor function).

Distinct sorting pathways for apical and basolateral membrane proteins are governed by tight junctions and specific sorting proteins.
Vesicles carry distinct apical and basolateral proteins from the trans-Golgi (Figure 14-25).
Proteins of various origins, such as hormones and neurotransmitters, are sorted into regulated secretory vesicles and released in response to signals like glucose elevation in pancreatic cells.
The sorting mechanisms engage in aggregation states, often determining vesicle type and function (e.g., hormone secretion during glucose spikes).

Many secretory proteins, termed proproteins, undergo proteolytic cleavages after passing the trans-Golgi (Figure 14-24).
Cleavage actions define functionality, impacting secretion processes of proinsulin and other precursor proteins.
Endoproteases and carboxypeptidases regulate proprotein processing within secretory vesicles.

The trans-Golgi network manages the routing of proteins for secreted products, lysosomal future, and apical/basolateral membrane arrangements.
Various vesicle types bear clathrin and AP complexes, with dynamin critical for vesicle formation.
Soluble lysosomal enzymes feature M6P markers, directing their trafficking and recycling of receptors.
Distinct sorting mechanisms cater to the polarized features of epithelial cells, managing protein distribution between apical and basolateral regions.
Proteins within this pathway often undergo vital processing to achieve maturity before cellular release