Secretory Pathways, Vesicular Transport, and Golgi Function

Endoplasmic Reticulum Stress and the Unfolded Protein Response (UPR)

• Healthy Cell ER Function: In normal cellular conditions, proteins are synthesized and brought into the Endoplasmic Reticulum (ER) lumen where they fold and interact with chaperones through the Calnexin cycle. If proteins cannot fold properly after sufficient time, they are ejected back into the cytosol and degraded.

• ER Stress Conditions: High levels of stress or specific exposures can lead to an accumulation of misfolded or unfolded proteins that the ER cannot manage, leading to cell death.

• The Unfolded Protein Response (UPR): This process kicks in to mitigate damage from misfolded protein accumulation.

  • Sensors and Chaperones: In a healthy ER, UPR sensors are typically bound to chaperones and remain inactive. When misfolded proteins accumulate, these chaperones dissociate from the sensors to deal with the misfolded proteins.

  • Dimerization and Signaling: Once sensors are free of chaperones, they allow for dimerization and mutual phosphorylation, triggering signaling cascades.

  • Gene Expression Changes: The UPR focuses gene expression specifically on:

    • Increasing the production of ER chaperones.

    • Promoting the synthesis of proteasomal components for protein degradation.

    • Turning down the synthesis of non-chaperone proteins to reduce the loading stress on the ER.

  • Outcomes: If the problem is cleared, the cell returns to normal function. If the UPR fails to resolve the stress, the cell will eventually die.

Clinical Implications: Cystic Fibrosis and Protein Misfolding

• Genetic Basis: Mutations in the primary sequence of a protein can prevent correct folding. These proteins are recognized as problematic by the Calnexin cycle and are degraded in the cytosol instead of reaching their destination.

• Cystic Fibrosis Mechanism:

  • CFTR Channel: This channel is responsible for moving chloride ($Cl^-$) ions from the inside of the cell to the outside.

  • Ion Concentration and Mucus: Proper ion movement maintains the salt concentration in the extracellular environment of the lungs, keeping mucus at a thin, manageable consistency.

  • Pathology: In Cystic Fibrosis, both gene copies of the CFTR channel typically carry mutations. The resulting proteins are misfolded and degraded, never reaching the plasma membrane. Without the channel, the salt balance is lost, mucus becomes extremely thick and hard to clear, causing severe breathing problems.

  • Current Treatments: Treatments focus on thinning and removing mucus. Investigations into CRISPR-mediated gene editing are ongoing to resolve the underlying mutations.

Vesicular Transport Mechanisms

• General Process: Transport begins when a donor compartment membrane buds to form a vesicle. This bud pinches off, travels to a target compartment, and fuses with it to release contents.

• Coat Proteins and Pathways:

  • COPII: Mediates forward transport from the ER to the Golgi.

  • COPI: Mediates transport within the Golgi stacks or retrograde transport from the Golgi back to the ER.

  • Clathrin: Mediates transport from the Golgi to lysosomes or the plasma membrane, as well as endocytosis from the plasma membrane.

Clathrin-Coated Vesicle Formation

• Structure of Clathrin: Clathrin possesses a unique "triskelion" structure consisting of three heavy chains (visualized in red) and three light chains (visualized in yellow). These triskelions nest within each other to form a basket-like cage around the budding vesicle.

• Adaptor Proteins: Adaptors like AP1 and AP2 act as bridges between the clathrin coat and the vesicle membrane. They simultaneously bind to:

  1. The lipid composition of the donor compartment.

  2. Cargo receptors that hold the specific cargo proteins.

• Lipid Identity (Phosphoinositides): Organelles are identified by different combinations of phosphoinositides (modified membrane phospholipids). Different compartments have different phosphorylation patterns on the carbons of the inositol sugar (e.g., PI 4-phosphate vs. PI 4,5-bisphosphate).

  • Example: At the plasma membrane, the specific lipid PI(4,5)P2 and cargo receptors must both be present for the AP2 adaptor protein to bind efficiently and initiate endocytosis.

• The Fission Process (Dynamin):

  • Dynamin Function: A GTP-binding protein that forms a spiral or "collar" around the neck of the budding vesicle.

  • Mechanism: Upon GTP hydrolysis, the dynamin subunits slide over one another, causing the spiral to tighten and constrict the membrane until the vesicle is pinched off.

  • Mutant Observation: If a mutant GTP-binding protein is used that cannot hydrolyze GTP, the dynamin collar continues to grow into a long spiral without ever severing the vesicle.

COPII Coat Assembly: ER to Golgi

• Step-by-Step Assembly:

  1. Sar1 Activation: Sar1 protein interacts with a Guanine Exchange Factor (GEF) on the ER membrane, swapping GDP for GTP.

  2. Membrane Embedding: Active Sar1 undergoes a conformational change where an amphiphilic arm swings down and embeds into the ER membrane.

  3. Adaptor Recruitment: Active Sar1 recruits the Sec23 and Sec24 protein complex. Sec24 specifically interacts with cargo receptors.

  4. Outer Coat Recruitment: Additional proteins, Sec13 and Sec31, associate with the complex to provide the structural curvature needed to bend the membrane.

• Fission and Uncoating: Unlike Clathrin, COPII coats can pinch themselves off without needing separate fission proteins like dynamin. Once the vesicle is released, GTP hydrolysis occurs, causing the coat proteins to shed (uncoat) before the vesicle fuses with the target.

Vesicle Tethering, Docking, and Fusion

• Rab Proteins: These are Ras-associated binding proteins (GTPases) that provide identity to vesicles and target membranes.

  • Tethering: A vesicle travels through the cytosol until its specific Rab protein interacts with a compatible Rab effector (tethering protein) on the target membrane. This stops the vesicle's movement.

  • Rab Domains: Certain regions of an organelle (like an endosome) can become enriched with specific Rab proteins (e.g., Rab5) through a positive feedback loop involving lipid modifications and GEFs.

• SNARE Proteins: Mediate the docking and fusion of the vesicle.

  • v-SNAREs: Located on the vesicle membrane.

  • t-SNAREs: Located on the target membrane.

  • Docking: The v-SNARE and t-SNARE interact and twist around each other to form a stable four-protein coiled-coil. This interaction squeezes out water molecules between the two membranes.

  • Fusion: The outer lipid layers fuse first (hemifusion), followed by the inner lipid layers, resulting in a single continuous bilayer.

• SNARE Recycling: After fusion, the SNARE proteins are tightly bound. NSF, an ATPase, uses ATP hydrolysis to untwist and separate the v-SNARE and t-SNARE so they can be recycled for future transport events.

ER Retrieval and the KDEL Signal

• Accidental Escape: Sometimes ER resident proteins (proteins meant to stay in the ER) are incorrectly packaged into COPII vesicles and sent to the Golgi.

• KDEL Sequence: Soluble ER resident proteins contain a specific four-amino acid signal sequence at their C-terminus: Lysine-Aspartic Acid-Glutamic Acid-Leucine ($K-D-E-L$).

• The Retrieval Pathway: KDEL receptors located throughout the Golgi detect this sequence and package the "straggler" proteins into COPI vesicles to be returned to the ER.

The Golgi Apparatus Structure and Maturation

• Structure: The Golgi consists of multiple flattened, membrane-enclosed stacks called cisternae. It is typically positioned near the nucleus and the Rough ER.

• Compartments:

  • Cis Golgi Network (CGN): Closest to the ER; involved in protein sorting and receiving vesicles from the ERGIC (ER-Golgi Intermediate Compartment).

  • Medial Golgi: The middle stacks where progressive glycan modification occurs.

  • Trans Golgi Network (TGN): The final exit face where proteins are sorted for transport to lysosomes, the plasma membrane, or secretory vesicles.

• Transport Models:

  1. Stationary Cisterna Model: Proposes that Golgi stacks are static, and proteins move between them exclusively via vesicles.

  2. Cisternal Maturation Model: Proposes that the cisternae themselves are dynamic. The ERGIC matures into the Cis Golgi, which then matures into the Medial, and eventually the Trans Golgi. This is the predominant hypothesis. Evidence includes the fact that if the formation of the ERGIC is blocked, the entire Golgi disappears.

• Structural Maintenance: Golgi Reassembly and Sorting Proteins (GRASPs) hold the stacks together. Golgin proteins act as long, flexible tethers to catch vesicles. During mitosis, these proteins are phosphorylated, causing the Golgi to fragment so it can be divided equally between daughter cells.

Glycosylation in the Golgi

• N-linked Glycosylation Modifications: The core oligosaccharide added in the ER is further processed in the Golgi.

  • High Mannose Type: Formed when enzymes remove several mannose sugars but do not add new ones. This occurs if the glycan is not easily accessible by Golgi enzymes.

  • Complex Type: Formed when further sugars such as N-acetylglucosamine (GlcNAc), Galactose, and Sialic Acid are added.

• EndoH Tool: The enzyme EndoH can cleave glycans between the two N-acetylglucosamines at the base, but only if the glycan has not yet been modified by GlcNAc in the medial Golgi. This allows researchers to track how far a protein has traveled through the secretory pathway.

• O-linked Glycosylation: Sugars are added to the hydroxyl ($-OH$) group of Serine or Threonine amino acids. This is critical for the production of mucus and the fluidity of the extracellular environment.

Lysosomes and Acid Hydrolase Sorting

• Lysosomal Properties: Highly acidic compartments ($pH$ approx. 5.0) used for the breakdown of macromolecules. They contain acid hydrolases that only function at low $pH$.

• Maintenance of Acidity: Proton ($H^+$) pumps on the lysosomal membrane use ATP to pump protons into the lumen against their concentration gradient.

• Protection: The membrane proteins of the lysosome are heavily glycosylated; these carbohydrate decorations act as a protective shield against the acid hydrolases.

• The Mannose-6-Phosphate (M6P) Sorting Pathway:

  1. Signal Patch: Lysosomal enzymes have a signal patch of positively charged amino acids.

  2. Phosphorylation: In the Cis Golgi, a phosphotransferase enzyme adds a phosphate and a GlcNAc molecule to the 6th carbon of a mannose sugar on the protein's N-linked glycan.

  3. Signal Exposure: The GlcNAc is later removed, leaving a Mannose-6-Phosphate tag.

  4. Receptor Binding: M6P receptors in the TGN recognize and bind the tagged proteins.

  5. Transport: Clathrin-coated vesicles transport the receptor-cargo complex to the endosome/lysosome.

  6. Release: The low $pH$ of the lysosome/endosome causes the enzyme to dissociate from the receptor. The phosphate is removed, and the receptor is recycled back to the Golgi.

• Plant Equivalent: Plant cells use a "vacuolar targeting sequence" to direct proteins to the vacuole, performing a function similar to the M6P signal in animal cells.