Protein Trafficking through the Endomembrane System

Chapter 12: Protein Trafficking through the Endomembrane System

Learning Objectives

  • Understand the structure and formation of vesicles in endomembrane trafficking.
  • Why cargo proteins move in one direction during endomembrane trafficking but fluid and lipid components move both anterograde (forward) and retrograde (backward).
  • The process of vesicle formation and budding from the rough ER up through fusion with the target cis-Golgi apparatus membrane.

Vesicle Budding and Fusion

Key Aspects of Transport Pathways

  • Vesicle formation, docking, and fusion are crucial for the movement of proteins and materials through the endomembrane system.

Endomembrane Transport

From ER to Golgi

  • Proteins translated on the rough ER include:
    • Resident rough ER proteins: Stay within the ER.
    • Most proteins: Transported to the Golgi for modification and sorting.
  • Golgi function: Some resident proteins stay in the Golgi while others are trafficked to various destinations.
  • Transport is facilitated by transport vesicles, which are vehicles for protein movement.

Transport Vesicles

  • Definition: Small spherical membrane-bound sacs that carry proteins between organelles and to outside of the cell.
    • Inner contents: Soluble proteins reside in the lumen, while transmembrane proteins are embedded in the vesicle membrane.

Membrane Orientation

  • Important points regarding membrane orientation:
    • The lumen of the ER and Golgi is equivalent to the extracellular environment.
    • Provides a separate and distinct environment for proteins compared to the cytoplasm.

Transport: Two-Way Street

Secretory Pathway (Outward)

  • Proteins are moved from the rough ER towards the cell membrane:
    • Some proteins are released from the cell (secretion).
    • Some proteins are integrated into the cell membrane.
    • Proteins may also be trafficked to organelles.
    • A subset of proteins remains within the ER.

Endocytic Pathway (Inward)

  • Materials from the extracellular solution or cell membrane are delivered via endocytosis to endosomes:
    • Some endosomes mature into lysosomes.

Overview of Vesicle Formation

  • Budding and Fusion Process:
    1. Vesicle formation from the donor compartment containing cargo proteins in its lumen.
    2. Vesicle transports cargo proteins to the target compartment.
    3. Membrane fusion occurs, releasing proteins to new destinations.

Vesicle Anatomy

  • Characteristics:
    • Size: Typically 40-100 nm in diameter; some can be larger.
    • Outer membrane: Often associated with coat proteins:
    • Types of coat proteins include Clathrin, COPI, and COPII.
    • Coat proteins are crucial for cargo selection and bending the vesicle membrane to form a sphere.

Lipid and Fluid Supply Maintenance

  • Donor compartments lose membrane, proteins involved in trafficking, and fluids to vesicles.
  • Target compartments gain vesicle membranes, thus maintaining supply:
    • Targeted proteins move in one direction, while lipids should be recycled via retrograde transport.

Overview of Vesicle Budding

Key Steps in Budding, Uncoating, Docking, and Fusion

  1. Vesicles bud off from donor compartments, mediated by coat proteins.
  2. Coated vesicles contain transmembrane and soluble cargo proteins bound to transmembrane receptors.
  3. Coat proteins detach as vesicle uncoats.
  4. Vesicle docks with its target compartment via tether proteins.
  5. Docking is mediated by SNARE proteins between the vesicle and target membranes.
  6. SNAREs facilitate the fusion of the two membranes, allowing cargo delivery.

Vesicle Formation Initiation

Role of GEF (Guanine Nucleotide Exchange Factor)

  • Promotes the exchange of GDP for GTP, activating the Sar1 G-protein which then:
    • Exposes a hydrophobic residue, inserting into the membrane.
  • Sar1 proteins in the membrane recruit coat proteins (notably COPII for ER to Golgi transport) which:
    • Fold to bend the membrane into a spherical shape, concentrating cargo proteins in vesicle formation.

Concentration of Cargo Proteins

  • COPII Mechanism:
    • Binds to both Sar1 and cargo proteins or soluble protein receptors.
    • Promotes cargo protein specificity through interaction with receptors or recognition signals.

Vesicle Budding Process

  • Aggregation of coat proteins causes:
    • Membrane to form a bud (coated pit).
    • Resulting in the full separation of the vesicle from the membrane.

Vesicle Uncoating

  • Once fully budded off from the rough ER:
    • GTP on Sar1 hydrolyzes to GDP, causing Sar1 to return to the cytosol, leading to coat proteins falling off for reuse.
  • Uncoated vesicle proceeds to the target compartment.

Vesicle Docking and Fusion

  • Docking Steps:
    • Vesicle recognizes the target membrane.
  • Fusion Steps:
    • Vesicle membrane fuses with the target membrane, releasing soluble cargo proteins.

Role of Rab G-proteins in Docking

  • Rab-GTP incorporates during budding:
    • Binds to tethering proteins in the target membrane—allows specificity for membrane fusion.

Vesicle Fusion Process

  • Fusion initiates when v-SNAREs bind t-SNAREs from target membranes:
    • SNARE proteins coil, strongly interacting to pull membranes close together, initiating fusion through hydrophobic forces.

Post-Fusion Process

  • Rab-GTP is hydrolyzed to Rab-GDP, which:
    • Conceals the hydrophobic domain and leads to its release from the membrane.
  • v-SNARE Recycling:
    • Energy for membrane fusion is sourced from resetting SNARE proteins, facilitated by ATPase NSF and SNAP complex, recycling v-SNARE back to their origin.

Trafficking through the Golgi Apparatus

Overview of Golgi Structure and Function

  • Golgi body construction: Stacked membrane-bound flattened sacks (cisternae) arranged in networks:
    • Cis-Golgi network (near ER), Medial-Golgi network (middle), and Trans-Golgi network (farthest from ER).
    • Golgi size and number of cisternae vary across cell types.
    • Cisternae are maintained in a curved structure supported by protein interactions.

Function of Golgi

  • Cisternal Maturation Model: Cargo remains within the same cisterna while moving to the trans network; cisternae mature and change
    • COPI-coated vesicles facilitate retrograde transport of Golgi-resident proteins back to earlier cisternae.

Chemical Modification and Sorting

  • Detailed process of glycosylation:
    • Begins with a 10-sugar oligosaccharide in the ER, continued in the Golgi via glycosyltransferases (adding) and glycosidases (removing) sugars systematically.
  • Modifications occur in a specific sequence as cargo progresses through the Golgi, where resident enzymes discriminate and catalyze necessary changes.

Cargo Protein Sorting

  • As cargo proteins travel, they are sorted effectively as they reach the trans-Golgi:
    • Final preparations for vesicle packaging before leaving for designated destinations (secretory pathway or recycling).

Clathrin-Coated Vesicles

  • Clathrin plays a pivotal role in transporting proteins to endosomes and lysosomes:
    • Forms cage-like structures (
      -triskelions) around vesicles, recruited via Arf G-proteins.

Comparison of Vesicle Coat Proteins

  • Key distinctions between Clathrin and COPII:
    • Clathrin: Involves adaptor proteins and dynamin for vesicle pinching off;
    • COPII: Direct binding without adaptors and relies on Sar1 for recruitment but lacks a complex pinching mechanism.

Cellular Export and Import

Exocytosis and Endocytosis

  • Processes for moving cargo in and out of the cell:
    • Exocytosis: Trafficking vesicles to the cell membrane for protein release.
    • Endocytosis: Bringing substances into the cell via endocytic vesicles that mature into early then late endosomes and finally lysosomes.

Types of Endocytosis

  • Pinocytosis (i.e., "cell drinking") and Phagocytosis (i.e., "cell eating"):
    • Pinocytosis involves intake of small materials via endocytic vesicles.
    • Phagocytosis involves larger substances.

Endosome Functions

  • Endosomes are organelles functioning in sorting processes, either degrading or recycling materials, evolving into acidic environments through proton pumps.
    • These structures manage cargo contents, affecting downstream fates based on protein processing needs.

Receptor-Mediated Endocytosis

  • LDL complex uptake is a prominent example:
    • Incorporates binding to specific receptors, followed by disassembly and recapturing of receptors for repeated use.

Phagocytosis and Lysosomes

  • Phagosomes facilitate the engulfing process, subsequently fusing with lysosomes for digestion:
    • Lysosomes: Maintain highly acidic environments to support macromolecule degradation, housing multiple hydrolytic enzymes.

Targeting Enzymes to Lysosomes

  • Enzymatic designation process begins with a unique Mannose-6-phosphate conjugate on oligosaccharides, forming clathrin-coated vesicles that transport them into lysosomes post-Golgi.
    • The Lysosomal process ensures recycled proteins return for further enzymatic cycles.