Membrane Trafficking Overview

Intracellular Compartments

  • Key Compartments in Animal Cells

    • Cytosol

    • Nucleus

    • Endoplasmic Reticulum (ER)

    • Golgi Apparatus

    • Lysosome

    • Peroxisome

    • Mitochondrion

    • Plasma Membrane

    • Endosome

    • Polyribosomes

    • Diagram illustrating these compartments is critical for visualizing internal structures (15 μm scale).

Secretory Pathway Details

  • General Plan (Inside to Out):

    • Endoplasmic Reticulum

      • Newly synthesized proteins inserted

      • Folding

      • N-linked glycosylation

      • Quality control

    • Pre-Golgi Intermediates (e.g. VTCs)

    • Golgi Apparatus: Intra-Golgi transport

      • N-linked oligosaccharides are modified

      • Sorting to various destinations occurs

    • Post-Golgi carriers, secretory vesicles

    • Destinations

      • Cell surface

      • Secretory granules

      • Endosomes

Endocytic Pathway Details

  • General Plan (Outside to In):

    • Beginning at the cell surface and moving to external environments.

    • Transition through early endosome ➜ late endosome ➜ lysosome.

    • Often targeted to the lysosomes for degredation

Historical Overview

  • Notable Discoveries:

    • Keith Porter (1945): Discovered the structure of the endoplasmic reticulum; significant figure in cell biology.

    • Camillo Golgi (1898): First to identify the Golgi apparatus, pivotal in understanding secretory pathways.

    • George Palade (1950s-1960s): Utilized electron microscopy, defined the secretory pathway involving the ER, Golgi, and cell surface.

Techniques in Studying Membrane Trafficking

  • Autoradiography:

    • In Palade’s work, he used radioactive amino acids (often tritium-labeled leucine) to track protein synthesis.

    • Newly synthesized proteins incorporated the radioactive label inside the rough endoplasmic reticulum (RER).

    • After different time intervals (pulse-chase method), samples were fixed and sectioned to trace the movement of labeled proteins through the cell.

      • The radioactive amino acids are incorporated for a certain amount of time, then washed away, and non-radioactive amino acids are incorporated

    • Palade’s experiments followed proteins from the RER → Golgi → secretory vesicles → extracellular space.

    • Method for visualizing proteins’ locations via isotopes in living cells.

    • Detailed steps: Sample preparation, photo-sensitive emulsion exposure, development to show silver grains indicating protein location.

    • The sample was coated with a photosensitive silver emulsion that captures radiation from tritium decay.

    • Over time, the radioactivity exposes the emulsion, forming metallic silver grains where radioactive proteins were located.

    • The grains of silver were visualized under an electron microscope

    • Palade used pancreatic cells because they are synthesizing digestive enzymes at great quantities, making it easier to ignore the “background” synthesis

    • ER → Golgi → Condensing vacuole → Zymogenic granule

Protein Transport Mechanisms

  • Categories of Protein Transport:

    • Transmembrane Transport: Movement to organelles like ER, mitochondria, and peroxisomes.

    • Gated Transport: Occurs through nuclear pores.

    • Vesicular Transport: Inside secretory and endocytic pathways; involves vesicle fusion and transport mechanisms.

    • Direct Connections: Less common pathways including "kiss-and-run" methods.

Steps in Vesicular Trafficking

  • Key Steps:

    • Sorting of cargo: Identification of protein/lipid that needs transport.

      • Involves coproteins

      • Coproteins bind to the cytoplasmic domain concentrating them in the vesicles

    • Budding: Formation of vesicles from donor membranes.

      • Involves coproteins

      • Tubular transport intermediates do not use coats

    • Separation: Completing vesicle closure before transferring.

    • Transfer: Moving to another compartment.

      • Involves motor proteins

      • ER to golgi (towards minus end - dynein)

      • Golgi to somewhere else (toward plus end - kinesin)

    • Recognition and Fusion: Ensuring the vesicle accurately merges with target membranes

      • Tethering proteins for docking

      • SNAREs drive the fusion

  • Motor Proteins:

    • Kinesins and dyneins direct vesicle movement along microtubules, crucial for effective trafficking.

Molecular Mechanisms and Proteins Involved

  • Coat Proteins:

    • Clathrin: Involved in endocytosis and Golgi processes.

      • Found in glgo, endosomes, and on cell surface

    • COPI & COPII: Categorize vesicular transport between ER and Golgi.

      • COPI found in the golgi, not on endosomes

      • COPII on endoplasmic reticulum

    • Retromer: Functions in recycling cargo receptors.

      • Found on cell surface

    • Caveolin:

      • Found on endosomes

  • Tethering Proteins and SNAREs:

    • Involved in initial docking and fusion; specific recognition processes to enhance transport accuracy.