Lecture 11: Vesicular Trafficking and the Late Secretory Pathway

Overview of the Late Secretory Pathway

• This lecture, the final installment for Exam 2, focuses on vesicular trafficking within the late secretory pathway.

• The journey of a protein through the cell progresses from the Endoplasmic Reticulum (ER) to the Golgi apparatus, where it undergoes specific post-translational modifications like glycosylation in different Golgi cisternae to become a mature glycoprotein.

• The late secretory pathway encompasses the sorting and transport of proteins from the trans-Golgi network (TGN) to their final destinations, including:

  • The plasma membrane (for membrane-bound proteins).

  • The extracellular space (for soluble secreted proteins).

  • The lysosome (for degradative enzymes).

Coated Vesicle Fundamentals and Clathrin Structure

• Protein transport is mediated by coated vesicles that bud from donor membranes (e.g., the Golgi or plasma membrane).

• Protein Coats: These provide the physical structure to deform the membrane into a bubble. Types include:

  • COP1: Mediate retrograde transport from the Trans-Golgi back to earlier Golgi cisternae.

  • AP3 Coated Vesicles: Directly transport cargo from the TGN to the lysosome.

  • Clathrin-Coated Vesicles (CCVs): Use adapter proteins ($AP1$, $AP2$, $AP3$, or $GGA$) to transport contents from the TGN to late endosomes/lysosomes or from the plasma membrane during endocytosis.

• Triskelion Structure: The fundamental building block of clathrin is the triskelion, which consists of:

  • Three clathrin heavy chains.

  • Three clathrin light chains.

• Each heavy chain ends in a binding site essential for assembling the coat. A digitally reconstructed clathrin coat average consists of $36$ triskelia interacting to form the polyhedral cage.

Vesicle Budding, Fission, and Uncoating

• Budding Mechanism: Vesicles form a two-layered coat.

  • Inner Layer: Composed of adapter proteins (like the $AP$ complex). These recruit cargo by binding to cargo receptors or integral membrane cargo proteins and cause clathrin polymerization.

  • Outer Layer: Composed of clathrin triskelia.

• Dynamin and Fission: Dynamin is a $GTPase$ that polymerizes at the neck of the budding vesicle.

  • $GTP$ hydrolysis by dynamin drives a conformational change that pinches the vesicle off from the donor membrane.

  • Experiments using GTP̳γ S (a non-hydrolyzable derivative) result in stalled vesicles where dynamin polymers form long necks but cannot complete the pinching-off process.

• Uncoating: Before a vesicle can fuse with its target, the coat must disassemble.

  • The $GTPase$ $ARF$ initiates both assembly and disassembly by switching between $GTP$ and $GDP$ states.

  • $HSP70$, an $ATPase$, uses the energy from $ATP$ hydrolysis to drive the depolymerization of the clathrin coat back into individual triskelia.

Sorting to the Lysosome and Mannose 6-Phosphate (M6P) Signaling

• Proteins destined for the lysosome (lysosomal enzymes) are tagged with Mannose 6-Phosphate (M6P) in the cis-Golgi.

• M6P Tag Formation:

  1. The enzyme $GLCNAC$ phosphotransferase ($N$-acetylglucosamine 1-phosphate transferase) recognizes recognition sequences on the lysosomal enzyme and transfers a $UDP$-$GLCNAC$ group to a mannose residue.

  2. A second enzyme, phosphodiesterase, removes the $N$-acetylglucosamine sugar, leaving the phosphate group behind on the mannose.

• The M6P Receptor Pathway:

  • The $M6P$ receptor in the TGN binds to the M6P-tagged protein.

  • A clathrin-coated vesicle pinches off and uncoats.

  • The transport vesicle fuses with a late endosome.

  • In the late endosome, the $pH$ drops significantly. This acidic environment causes the $M6P$ receptor to release the lysosomal protein.

  • The receptor is recycled back to the TGN or the plasma membrane. The protein is delivered to the lysosome.

Secretory Pathways: Constitutive vs. Regulated

• Constitutive Secretion: A continuous process where vesicles move proteins and lipids to the cell surface without the need for external signals.

• Regulated Secretion: Proteins are stored in concentrated secretory vesicles that only fuse with the plasma membrane in response to specific regulatory signals.

• Aggregation in TGN: Proteins destined for regulated secretion often aggregate in the acidic conditions of the TGN, along with three specific proteins found in almost all regulated secretory vesicles.

Proteolytic Processing of Pro-proteins

• Many proteins are synthesized in an inactive pro-protein form and require proteolytic cleavage (processing) to become active.

• Constitutive Secretion (Albumin): Proalbumin is cleaved by the endoprotease Furin at a specific diarginine site to produce mature, active albumin.

• Regulated Secretion (Insulin):

  • Proinsulin contains $A$, $B$, and $C$ chains with disulfide bridges.

  • Endoproteases $PC2$ and $PC3$ recognize di-amino acid sequences and cleave the $C$ peptide.

  • Carboxypeptidase then removes the remaining diarginine residues at the junction to produce mature insulin.

  • Experimental evidence using antibodies shows proinsulin is found in immature vesicles near the TGN, while mature insulin is found in older vesicles further from the Golgi.

Protein Sorting in Polarized Epithelial Cells

• Polarized Cells: Epithelial cells (like $MDCK$ cells) have two distinct membrane domains: the apical domain (top) and the basolateral domain (bottom/sides), separated by tight junctions.

• Asymmetric Sorting: The TGN sorts proteins into different transport vesicles targeted to specific domains.

  • Example: In cells infected with both VSV and influenza, the $VSV\text{ }G$ glycoprotein is directed to the basolateral membrane, while the influenza hemagglutinin (HA) glycoprotein is directed to the apical membrane.

Receptor-Mediated Endocytosis: The LDL Pathway

• Low-Density Lipoprotein (LDL): A particle containing a phospholipid monolayer, an apolar core of cholesterol (esterified and unesterified), and a surrounding protein called Apolipoprotein B.

• Internalization Process:

  1. The LDL receptor ($LDLR$) binds to Apolipoprotein B on the LDL particle.

  2. The $LDLR$ cytosolic tail contains an $NPXY$ sorting signal that associates with the $AP2$ adapter complex.

  3. A clathrin-coated pit forms, pinches off via dynamin-mediated fission, and uncoats into an early endosome.

  4. The early endosome fuses with a late endosome. At approximately $pH\text{ }5$, the $LDLR$ beta-propeller domain becomes positively charged, causing the ligand-binding arms to release the LDL and bind to the propeller itself.

  5. The receptor recycles to the surface (at neutral $pH\text{ }7$, the arms relax and are free to bind LDL again).

  6. The LDL particle is degraded in the lysosome into fatty acids, cholesterol, and amino acids.

• Hypercholesterolemia: High blood-cholesterol levels common in patients with mutations in the $LDLR$ gene. Mutations can block protein synthesis, cause misfolding, reduce LDL binding, or inhibit internalization.

Metal Ion Internalization: The Transferrin/Iron Pathway

• Ferrotransferrin: The protein form that carries iron ($Fe^{3+}$) in the bloodstream.

• Mechanism:

  1. Ferrotransferrin binds to naming the Transferrin Receptor at the cell surface ($pH\text{ }7$).

  2. The entire complex is internalized via clathrin-coated vesicles.

  3. In the late endosome ($pH$ drop), iron is released from the protein and reduced from $Fe^{3+}$ to $Fe^{2+}$ before moving to the cytosol via a transporter.

  4. The iron-free protein (apotransferrin) remains bound to the receptor at low $pH$.

  5. The complex recycles back to the plasma membrane. Upon exposure to neutral $pH$, the apotransferrin dissociates from the receptor and is released back into the blood.

Multivesicular Bodies (MVB) and Selective Protein Degradation

• Multivesicular Bodies: Specialized late endosomes that allow the cell to distinguish between resident lysosomal membrane proteins and proteins marked for degradation.

• Mechanism:

  1. Proteins targeted for degradation (like certain cell-surface receptors) are tagged with ubiquitin.

  2. The $HRS$ protein on the endosome membrane is also ubiquitinated and recruits the $ESCRT$ complex (Endosomal Sorting Complex Required for Transport).

  3. $ESCRT$ mediates the inward budding of the endosome membrane to form internal vesicles containing the ubiquitinated cargo.

  4. $VSP4$ uses $ATP$ hydrolysis to disassemble the $ESCRT$ complex for recycling.

  5. When the $MVB$ fuses with the lysosome, the internal vesicles (and their blue transmembrane proteins) are degraded, while the membrane proteins on the endosome's outer surface (green lysosomal proteins) are spared.

Viral Mimicry and Autophagy Mechanisms

• HIV Budding: The HIV virus hijacks the $ESCRT$ pathway. The viral $Gag$ protein mimics the $HRS$ protein; it is ubiquitinated and recruits $ESCRT$ to the plasma membrane to facilitate the pinching off of new virus particles into the extracellular space.

• Autophagy: A recycling process for whole organelles like mitochondria.

  • A cup-shaped membrane structure forms around the organelle, closing to create a double-membrane autophagosome.

  • This structure fuses with a lysosome to break down the organelle.

  • Regulatory proteins involved include $ATG12$, $ATG5$, $ATG6$, and $ATG8$ (which is found on the autophagosome membrane).