Endomembrane System, Sorting and Vesicular Transport

Presented by: Paula A. F. Waziry, PhD, MA
Key Topics:
- The Endomembrane System
- Sorting Mechanisms
- Vesicular Transport

Composed of:
- Endoplasmic Reticulum (ER)
- Golgi apparatus
- Lysosomes
- Various small vesicles carrying lipids and proteins
Each organelle is defined by a single membrane
Double Membrane Organelles:
- Nucleus
- Mitochondrion

All compartments are closed but can exchange proteins and lipids through various mechanisms.
Topologically Equivalent Compartments: Molecules can move between compartments without crossing membranes. Examples:
- ER, Golgi, Transport vesicles, Perinuclear space (nuclear envelope)
Topologically Distinct Compartments: Mitochondrion, nucleus, and peroxisomes.

Synthesis Locations: Most proteins begin synthesis in the cytosol.
N-terminus Importance: Determines the fate of a protein based on a signal sequence (15-60 amino acids). Types of destinations include:
- A) Remain in cytosol
- B) Post-translational import into organelles (mitochondria, peroxisome, nucleus, ER)
- C) Co-translational import to RER.

Translocation (Post-translational)
- Targeted to mitochondria, peroxisome, and ER through protein translocators.
- Proteins get stretched and pass through protein translocators, refolding inside the compartments.
Co-translational Import into the ER
- Signal Peptide composed of several hydrophobic amino acids binds Signal Recognition Particle (SRP).
- The SRP directs the complex to the ER membrane.
- SRP binds SRP receptor using GTP energy. As ribosomes continue synthesizing, protein is translated into the ER via a translocon channel.
- The signal peptide is cleaved, resulting in protein release into the ER lumen.
Destiny Post ER Synthesis
- Proteins enter transport vesicles which follow:
  - Anterograde transport to the cis-Golgi
  - Later to the trans-Golgi
  - Secretory vesicles towards destination.

Most mitochondrial matrix proteins are synthesized in the cytoplasm.
They possess specific N-terminal uptake sequences that are amphipathic with positively charged residues (Arg and Lys).
This import requires chaperones and ATP.
Transport Mechanism: Occurs at membrane contact points (TOM and TIM)
- After import, proteins are processed, and the signal sequence is cleaved.

Binding of the signal sequence to TOM (translocator of outer membrane).
Chaperones Removal: ATP-dependent.
Proteins needed in the inner membrane interact with TIM22 for the electron transport chain.
Proteins required in the matrix interact with TIM23.
Final Processing in the mitochondrial matrix.

The nuclear pore complex (NPC) is essential for import and export:
- Importin binds cargo in the cytoplasm and translocates into the nucleus.
- In the nucleus, GEF (G-protein Exchange Factor) exchanges GDP for GTP on Ran, leading to dissociation of importin from cargo and subsequent export via the NPC.
- Nuclear Export: Processed mRNA is exported with modifications (cap, poly-A tail) via mRNPs which interact with the nuclear pore complex.

Only processed eukaryotic mRNAs can exit the nucleus - incorrect splicing leads to failure to export.
Importance bridged to Protein Translation in the cytoplasm.

Cargo is transported via vesicles, which bud off from donor compartments and fuse with target compartments.

Exocytosis: Vesicles transport materials out of the cell.
Endocytosis: Vesicles take up materials from the extracellular environment, forming endosomes that mature into lysosomes.

Coat Proteins:
- COPI: Retrograde transport from Golgi to ER.
- COPII: Anterograde transport from ER to Golgi.
- Clathrin: Mediates transport from cell membrane to lysosome and between Golgi and lysosome.

Coated regions concentrate cargo and form bud-like structures before pinching off as vesicles.
Clathrin-coated vesicles are formed by:
- Clathrin subunits
- Adaptin
- Cargo receptors
- Dynamin (facilitates vesicle pinch-off).

Tethering: Rab GTPases recognize tethering proteins on the target membrane.
Docking: Additional recognition via v-SNARE and t-SNARE.
Fusion: SNAREs wrap together instead of interfering with water, allowing lipid bilayers to coalesce and fuse, delivering cargo.

Ensure specificity between vesicles and their target membranes by cycling between active (GTP-bound) and inactive (GDP-bound) states.
Activate vesicle targeting through binding to specific effectors on target membranes.

Two main pathways: Vesicle-mediated and Non-vesicle mediated.
Vesicle-mediated: Involves coat proteins like COPI, COPII, and Clathrin and specific signal peptides.
The general pathway for protein secretion involves transport from ribosomes through the ER, to Golgi, to vesicles, ultimately to the plasma membrane.
Viruses often disrupt normal trafficking as part of their life cycle, targeting transport mechanisms.

Lysosomes are critical for intracellular digestion with a very acidic pH necessary for function and enzyme activity.
Endosomes mature into lysosomes that digest macromolecules, distributing products throughout the cell.
Biosynthetic-Secretory Pathway: Involves synthesis and regulation of macromolecules, where signals trigger storage and release into extracellular environments.

Understanding the endomembrane system and vesicular transport is crucial for studying cell function and pathology, particularly in connection to diseases that affect these processes.