Vesicular Transport and the Secretory Pathway

The Unfolded Protein Response (UPR)

• Proteins are synthesized and folded within the Endoplasmic Reticulum (ER) lumen, often interacting with chaperones through the Calnexin cycle. Under normal, healthy conditions, proteins that fail to fold correctly are retrotranslocated to the cytosol and degraded.

• In scenarios of excessive stress or exposure to certain stimuli, misfolded or unfolded proteins accumulate in the ER beyond the capacity of standard degradation pathways. This accumulation leads to ER stress and potential cell death.

• The Unfolded Protein Response (UPR) is a cellular mechanism designed to mitigate ER stress. In a stressed ER, chaperones dissociate from specific ER membrane sensors to assist with the high volume of misfolded proteins.

• Once sensors are no longer bound to chaperones, they undergo activation. One mechanism of activation involves sensors dimerizing and phosphorylating each other. This triggers signaling cascades that result in:

  • Increased expression of gene-encoding chaperones to enhance folding capacity.
  • Turning down the synthesis of non-chaperone proteins to reduce the load on the ER.
  • Increased production of proteasomal components to aid in clearing misfolded proteins.

• If the UPR fails to resolve the protein misfolding conflict after a certain duration, the cell will eventually undergo apoptosis (cell death).

Protein Misfolding and Human Disease: Cystic Fibrosis

• Many genetic diseases are caused by mutations that alter the primary sequence of a protein, leading to improper folding. These misfolded proteins are recognized by the Calnexin cycle and degraded in the cytosol, preventing them from reaching their destination.

• Cystic Fibrosis serves as a primary example of this phenomenon. It involves the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) channel, which is responsible for moving chloride ions ($Cl^-$) from the interior of cells to the extracellular environment.

• Proper ion movement maintains a specific salt concentration in the extracellular space of the lungs, keeping mucus at a thin consistency. This allows for normal breathing while protecting the lungs.

• In Cystic Fibrosis, mutations in both copies of the CFTR gene result in proteins that do not fold correctly. Consequently, they are degraded and never reach the plasma membrane.

• The absence of functional CFTR channels leads to an imbalance of salts, causing the mucus to become extremely thick and difficult to clear. This thick mucus blocks the ability to breathe and leads to severe health consequences.

• Current treatments involve thinning the mucus manually, though research into CRISPR-mediated gene therapy is currently under investigation to resolve the underlying mutations.

Principles of Vesicular Transport

• Vesicular transport is the primary mechanism for moving proteins between organelles in the secretory pathway. The process follows a specific sequence:

  • 1. A donor compartment membrane forms a bud.
  • 2. This bud pinches off to create a vesicle.
  • 3. The vesicle travels to the target compartment.
  • 4. The vesicle docks and fuses with the target membrane, releasing its contents.

• The direction of transport dictates the type of coat proteins used:

  • ER to Golgi: COP II (Coat Protein II).
  • Within the Golgi or Golgi back to the ER: COP I (Coat Protein I).
  • Golgi to lysosomes, secretory vesicles, or plasma membrane: Clathrin.

Clathrin-Coated Vesicles

• Clathrin possesses a distinct structure known as a triskelion, consisting of three heavy chains ($red$) and three light chains ($yellow$). These triskelions assemble into a basket-like cage around the budding vesicle.

• Adaptor proteins, such as AP2, mediate the connection between the clathrin coat and the vesicle membrane. These adaptors bind simultaneously to cargo receptors (carrying the cargo protein) and specific membrane phospholipids.

• Organelle identity is defined by the lipid composition of their membranes, specifically phosphoinositides (modified membrane phospholipids). Different compartments are enriched with different phosphoinositides:

  • The plasma membrane is characterized by the presence of $PI(4,5)P_2$.
  • Endosomes and Goljis contain different concentrations of phosphoinositides with varying phosphorylation states (e.g., phosphorylation on the 4th and 5th carbons for blue-coded lipids versus only the 4th carbon for red-coded lipids).

• The process of Clathrin-coated budding involves:

  • 1. Cargo receptors binding to cargo.
  • 2. Adaptor proteins recognizing the cargo-receptor complex.
  • 3. Clathrin triskelions associating with adaptor proteins, inducing membrane curvature.
  • 4. A fission protein called Dynamin severs the vesicle.

• Dynamin is a GTP-binding protein that forms a spiral collar around the neck of the budding vesicle. Upon GTP hydrolysis, the collar tightens and slides, pinching the lipid connection and releasing the vesicle. If a mutant GTP-binding protein is used that cannot hydrolyze GTP, the Dynamin collar continues to grow without releasing the vesicle.

• Once in the cytoplasm, clathrin coats fall off, allowing the naked vesicle to fuse with its target.

COP II-Coated Vesicles: ER to Golgi

• The assembly of COP II coats involves a multi-step GTPase-regulated process:

  • 1. Sar1-GDP (inactive) interacts with Sar1-GEF (Guanine Exchange Factor) on the ER membrane.
  • 2. GDP is swapped for GTP, activating Sar1. A conformational change causes an amphiphilic arm to swing out and embed into the ER membrane.
  • 3. Active Sar1 recruits adaptor proteins Sec23 and Sec24, which bind to cargo receptors.
  • 4. Additional proteins, Sec13 and Sec31, join the complex to induce membrane bending.

• Unlike clathrin, COP II coats do not require Dynamin for fission; the coat proteins themselves facilitate the pinching-off process. After budding, GTP hydrolysis causes the COP II coat to shed before docking.

Vesicle Tethering, Docking, and Fusion

• Transport and fusion are mediated by Rab proteins and SNARE proteins.

• Rab proteins (Ras-associated binding proteins) are GTPases that function in vesicle tethering. Every organelle has specific Rab subtypes (e.g., Rab5 is associated with endosomes).

• Rab-GDP in the cytosol is activated by a GEF on the target membrane to become Rab-GTP, which then embeds in the membrane. This creates a domain that recruits Rab effectors or tethering proteins. The vesicle possesses its own Rab proteins that interact with these long, thread-like tethering proteins to hold the vesicle in place.

• Docking occurs via SNARE proteins. There are two types:

  • v-SNAREs: Located on the vesicle membrane.
  • t-SNAREs: Located on the target membrane.

• When v-SNAREs and t-SNAREs meet, they wrap around each other to form a four-protein coiled interaction. This twisting force squeezes out water molecules between the membranes, bringing them into extremely close proximity.

• Fusion involves two stages:

  • 1. Hemifusion: The outer lipid layers of the two membranes fuse first.
  • 2. Full Fusion: The inner lipid layers fuse, creating a continuous bilayer and releasing cargo.

• After fusion, the SNARE complex must be disassembled for recycling. NSF (an ATPase) and a SNAP accessory protein use the energy from ATP hydrolysis to untwist and separate the v-SNARE and t-SNARE.

The Golgi Apparatus Structure and Maintenance

• The Golgi is organized into stacks of membrane-enclosed sacs called cisternae. It is typically located near the nucleus, sitting atop the Rough ER.

• The Golgi is divided into three functional regions:

  • Cis Golgi Network (CGN): Closest to the ER; receives incoming vesicles.
  • Medial Golgi: The middle cisternae stacks.
  • Trans Golgi Network (TGN): Furthest from the ER; sorts and buds vesicles toward final destinations.

• The ER-Golgi Intermediate Compartment (ERGIC) or vesicular tubular clusters form when vesicles leaving the ER fuse with each other via SNAREs before reaching the Golgi.

• Two models explain Golgi transport:

  • 1. Stationary Cisterna Model (Vesicular Transport): Each cisterna is static, and cargo moves between them via vesicles.
  • 2. Cisternal Maturation Model: Cisternae are dynamic structures that physically move forward, changing their identity (e.g., a Cis cisterna matures into a Medial cisterna). In this model, only proteins meant to be returned to previous stacks are moved via retrograde vesicular transport.

• Golgi maintenance involves GRASP proteins (Golgi Reassembly and Stacking Proteins) and Golgins (long tethering proteins). During mitosis, these proteins are phosphorylated, causing the Golgi to fragment so it can be divided between daughter cells.

Retrieval Signals

• Proteins that accidentally escape the ER or are ER-resident proteins are returned via COP I-coated vesicles.

• Soluble ER resident proteins contain a KDEL signal sequence ($Lys-Asp-Glu-Leu$) at their C-terminus. KDEL receptors in the Golgi recognize this sequence and package the protein into COP I vesicles for retrieval to the ER.

Glycosylation and Protein Processing

• N-linked glycosylation (started in the ER) is modified in the Golgi:

  • The core structure consists of two N-acetylglucosamines ($GlcNAc$) and three mannoses.
  • In the Cis Golgi, three mannoses are removed to form a high-mannose oligosaccharide.
  • In the Medial Golgi, further trimming and the addition of $GlcNAc$ occur.
  • In the Trans Golgi, galactose and sialic acid are added to form complex oligosaccharides.

• Endo H is an enzyme used to study these modifications; it can cleave glycans between the two $GlcNAc$ sugars, but only before they are modified by certain Golgi enzymes.

• O-linked glycosylation occurs in the Golgi, where glycans are added to the hydroxyl groups of Serine ($Ser$) or Threonine ($Thr$). This is important for mucus formation and extracellular fluidity.

Lysosomes and the Mannose-6-Phosphate Pathway

• Lysosomes are highly acidic ($low pH$) organelles responsible for degrading macromolecules into building blocks using acid hydrolases. These enzymes only function optimally at low pH, which protects the rest of the cell in case of leakage.

• The low pH is maintained by V-type ATPases (proton pumps) that use ATP to pump $H^+$ ions into the lysosome against their concentration gradient.

• Lysosomal proteins are protected from self-degradation by a heavy layer of glycosylation that blocks enzyme access to the membrane proteins.

• Targeting to the lysosome uses the Mannose-6-Phosphate ($M6P$) signal:

  • 1. In the Cis Golgi, a phosphotransferase recognizes a signal patch on lysosomal enzymes and adds a $GlcNAc-phosphate$ to a mannose on the N-linked glycan.
  • 2. The $GlcNAc$ is removed, exposing the $M6P$ signal.
  • 3. In the TGN, $M6P$ receptors bind the signal and package the enzyme into Clathrin-coated vesicles.
  • 4. The vesicle fuses with an endosome/lysosome. The low pH causes the enzyme to dissociate from the receptor.
  • 5. The phosphate is removed from the enzyme, and the empty $M6P$ receptor is recycled back to the Golgi.