8.1 Processing and sorting in the Golgi #
The study of Processing and sorting in the Golgi provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.
-en.svg/1280px-Golgi_apparatus_(borderless_version)-en.svg.png)
Golgi Apparatus (Borderless Version)
8.1.1 What arrives and what leaves #
Cargo arrives at the cis-Golgi in vesicles or in the larger vesicular-tubular clusters that leave ER exit sites. ER residents that accidentally came along are returned. Through the stack, N-linked oligosaccharides are trimmed and rebuilt. The high-mannose tree received from the ER can remain high-mannose, become hybrid, or become a complex oligosaccharide with added GlcNAc, galactose, and sialic acid. Different cells stop at different points, which is why the same protein can carry different sugars in different tissues. O-linked chains are started in the Golgi, and the long GAG chains of proteoglycans are polymerised there.
8.1.2 The trans-Golgi network as a decision point #
Three outbound routes matter in a first course.
.svg/1280px-Golgi_apparatus_(editors_version).svg.png)
Golgi Apparatus (Editors Version)
- Lysosomes. Hydrolases receive a mannose 6-phosphate tag. In the cis-Golgi, GlcNAc-1-phosphate is added to selected mannoses; an uncovering enzyme then removes the GlcNAc, leaving mannose 6-phosphate. The tag binds the mannose 6-phosphate receptor in the trans-Golgi network. Receptor plus hydrolase leave in clathrin-coated vesicles for late endosomes. The low pH there releases the hydrolase, and the receptor recycles. I-cell disease is the loss of the tagging enzyme: hydrolases follow the default route and are secreted.
- Regulated secretion. Proteins such as insulin, digestive enzymes, and peptide neurotransmitters are packed into dense secretory granules and held until a signal, usually a rise in Ca2+, triggers fusion with the plasma membrane. Packing involves aggregation of the cargo and often a proteolytic maturation inside the granule.
- Constitutive secretion. The default. Proteins that are folded and soluble, and membrane proteins with no special retention or lysosomal signal, leave in a steady stream of vesicles and keep the plasma membrane and the extracellular space supplied. Collagen, serum albumin, and ordinary plasma-membrane carriers travel this way.
Polarised cells add a further choice, apical versus basolateral, using signals in the cytosolic tails of membrane proteins and, in some epithelia, lipid domains. The glucose transporters of Chapter 3 reach opposite faces of the enterocyte by this logic.
8.1.3 Retrieval signals #
ER soluble residents carry a C-terminal KDEL sequence (or a close variant). If they reach the Golgi, the KDEL receptor binds them and returns them in COPI vesicles. Membrane proteins of the ER often carry a cytosolic KKXX motif that binds COPI directly. Golgi residents are held by the length of their transmembrane segments and by aggregation into enzyme complexes that vesicle machinery does not package efficiently. Location is the result of forward movement plus continuous retrieval, not of a protein being glued in place.
Signal Transduction Insulin
8.2 Vesicle coats and fusion #
8.2.1 Three coats #
| Coat | GTPase that recruits it | Where it forms | Main direction |
|---|---|---|---|
| COPII | Sar1 | ER exit sites | Forward, ER toward the Golgi |
| COPI | Arf1 | Golgi cisternae | Backward, Golgi toward the ER, and between cisternae |
| Clathrin, with adaptors | Arf1 at the TGN; no Arf at the plasma membrane (AP2 binds PI(4,5)P₂ and cargo) | Trans-Golgi network and plasma membrane | TGN to endosomes; plasma membrane into the cell |
Coat assembly does two things at once. It concentrates cargo, because the coat binds cargo receptors, and it curves the membrane into a bud. After the vesicle pinches off, the coat comes off. A coated vesicle cannot fuse; an uncoated vesicle can, because the fusion proteins are then exposed.
At the plasma membrane, the GTP-binding protein dynamin polymerises around the neck of a clathrin pit and, with membrane bending, pinches the vesicle free. Mutations in dynamin block endocytosis at the stage of deep pits that will not let go.
8.2.2 Addressing and fusion #
A vesicle needs an address as well as a coat. Rab GTPases provide it. Each organelle and vesicle population has its own Rab, active when GTP-bound, and Rabs recruit tethering proteins that hold the vesicle near the correct membrane. Tethering is reversible and long-range compared with fusion.
Fusion itself is driven by SNARE proteins. A v-SNARE on the vesicle and t-SNAREs on the target membrane zipper into a four-helix bundle. Zippering pulls the two bilayers together and overcomes the water barrier to fusion. After fusion, the ATPase NSF, with SNAP proteins, spends ATP to pull the bundle apart so that SNAREs can be reused. Botulinum toxin and tetanus toxin are proteases that cut specific neuronal SNAREs. Synaptic vesicles then cannot fuse, which is why these toxins paralyse. They are evidence that SNAREs are the fusion machine, and they are also the reason the diseases are so dangerous.
8.3 Endocytosis #
8.3.1 Three ways in #
- Phagocytosis engulfs large particles: bacteria, dead cells, debris. Actin drives the membrane around the particle. It is efficient in professional phagocytes (macrophages, neutrophils) and is how apoptotic bodies are cleared without inflammation.
- Pinocytosis takes in fluid in small vesicles, continuously.
- Receptor-mediated endocytosis concentrates a specific cargo at clathrin-coated pits. It is how cells take up LDL, transferrin, and many signalling receptors.
8.3.2 The LDL receptor as the worked example #
Low-density lipoprotein carries cholesterol in the blood. The LDL receptor in coated pits binds it. The vesicle uncoats and fuses with the early endosome, whose pH of about 6, set by V-ATPase, releases LDL from the receptor. The receptor recycles to the plasma membrane within minutes. LDL travels on to the late endosome and the lysosome, where the cholesterol ester is hydrolysed and the cholesterol is released for membrane synthesis or stored.
Familial hypercholesterolaemia, studied by Brown and Goldstein, is what happens when this receptor is absent or fails to enter coated pits. LDL stays in the blood, and cholesterol deposits in artery walls. One class of mutation removes the cytosolic internalisation signal of an otherwise normal receptor, so the receptor binds LDL and never enters a pit. That mutation separated, experimentally, the binding function from the endocytic function.
8.3.3 The endosomal system #
Early endosomes are the sorting lobby. Receptors and some membrane return to the cell surface, either directly or via the recycling endosome. Cargo destined for destruction is passed to late endosomes. Membranes and receptors that must be destroyed are sorted into intraluminal vesicles by the ESCRT machinery, producing a multivesicular body. Fusion with a lysosome delivers everything to the hydrolases. Signalling receptors are often silenced by this route: endocytosis does not only feed the cell, it turns signals off.
Endomembrane System
Transcytosis is the variant in which cargo crosses a polarised cell, entering on one face and leaving on the other. In many mammals, antibodies from milk cross the intestinal epithelium of the newborn by this route. In humans, most maternal IgG reaches the fetus across the placenta, again by receptor-mediated transcytosis.
8.4 Key terms, summary, and self-check #
Key terms. Mannose 6-phosphate; constitutive secretion; regulated secretion; KDEL; COPI; COPII; clathrin; Sar1; Arf1; dynamin; Rab; SNARE; NSF; early endosome; LDL receptor.
Summary. The Golgi finishes sugars and the trans-Golgi network sorts lysosomal enzymes, regulated cargo, and default secretory cargo. COPII moves material forward from the ER, COPI returns residents, and clathrin serves the TGN and endocytosis. Rabs tether and SNAREs fuse. Endosomes recycle receptors and send LDL and other cargo to lysosomes.
Self-check.
1. A lysosomal hydrolase is found outside the cell, and all other lysosomal enzymes are in the same condition. Which tagging step is the likely failure?
2. Why must the clathrin coat come off before fusion?
3. What do botulinum toxins cut, and why does the synapse fall silent?
4. LDL dissociates from its receptor in the early endosome. What physical condition causes the release, and why is that useful?
5. A soluble ER protein appears in the medium. Which four-letter signal is probably missing?
