week 6 Protein Targeting

Protein Targeting and Secretory Pathways

  • By the end of this lecture, you should be able to:

    • Describe the difference between prokaryotic and eukaryotic cells in terms of protein targeting.

    • Understand the role of cell compartmentalisation.

    • Describe the endomembrane system.

    • Understand what the cell uses for transport signals and pathways.

    • Know the molecular machinery involved in protein transport.

Central Dogma Refresh

  • DNA is faithfully copied to create more DNA.

  • DNA is transcribed into RNA.

  • mRNA is translated into protein.

  • The lecture focuses on what happens to proteins after translation, specifically protein targeting and secretory pathways.

Prokaryotic vs. Eukaryotic Cells

  • Major difference: presence of a nucleus in eukaryotic cells.

  • Eukaryotic cells have organelles; prokaryotic cells do not.

  • Eukaryotic Cell Compartmentalisation:

    • Endoplasmic reticulum (ER): essential for protein translation in eukaryotes.

    • Golgi body/apparatus: processes proteins.

    • Endosomes, lysosomes, and secretory vesicles: each has a specialised function.

Endomembrane system: protein trafficking

  • Protein trafficking and secretory pathways are more complex in eukaryotic cells.

  • Proteins are made in the ER, processed in the Golgi apparatus, and then trafficked to their destination.

  • Organelles have specialised functions, and proteins are recycled to different organelles = continuous trafficking of proteins around the cell, not just after they’re made = adds another level of complexity

  • Each organelle is bounded by a highly specialised membrane that is defined by its constituent parts, with specific phospholipids and specific structural/functional proteins.

Golgi Apparatus

  • ( Discovered by Camellio Golgi.

  • Has a specialised function in post-translational modification of proteins. )

    Cargo Transportation

  • Proteins made in the ER are transported to the Golgi for post-translational processing.

  • Proteins are then incorporated into vesicles for transport to their destinations.

  • Proteins can be transported into the nucleus through nuclear pores (via specific transport mechanisms) and across membranes (via vesicle transport)

Proteins have different signals - molecular clues

  • Proteins contain molecular clues encoded within their amino acid sequences that determine their destination.

  • These are a group of conserved amino acid sequences found on the N-terminal part of a protein.

  • Analogous to a molecular address or postcode. (e.g. tell the cell where to go)

  • Examples:

    • Proteins targeted for import into the ER.

    • Proteins targeted for retention in the lumen of the ER.

    • Proteins targeted for import into mitochondria.

    • Proteins targeted for import into the nucleus.

    • Proteins targeted for import into peroxisomes.

Proteins have different signals - signal peptide structure

  • signal peptide at the N-terminus of the protein.

  • Composed of three regions:

    • N-terminal region: positively charged amino acids (1-5).

    • Middle hydrophobic region (H region): 7-15 residues of hydrophobic amino acids, typically including at least 3 leucines, forming an alpha helix structure.

    • C-terminal region: 3-7 uncharged amino acids, potentially forming a beta sheet structure.

  • Cleavage site: A specific protease (cleaves between the C region and a negatively charged region of 1-6 residues)

  • Variations: Signal peptides can vary in length, which can sometimes make it difficult to find a signal peptide sequence through bioinformatics

Transport molecular machinery- secretory pathways

  • About 50% of proteins made in the cell are trafficked into specific organelles or secreted.

  • Cells are "factories" for proteins found in the extracellular space (e.g., blood, cerebrospinal fluid, lymphatic tract).

  • ( Controversy in literature regarding the proportion of secreted proteins.

    • About 34% of genes have a signal peptide.

    • Bioinformatics predictions suggest only about 9% of proteins are secreted (just under 2,000 proteins). )

  • Secretory pathways within the cell unite at least 10 cellular compartments.

  • complex process which requires the endomembrane system and ligand-receptor interactions to determine the direction that secretion occurs and the specificity of secretion.

  • ( Ligand-receptor interactions: analogous to a lock and key. )

Vesicles mediate cargo transport- vesicle formation

  • Protein transport is mediated through the formation of vesicles.

  • Vesicles transport protein cargo to specific destinations.

  • Vesicles bud off from the Golgi membrane and can become secretory vesicles (releasing proteins to the cell surface, where they merge with the plasma membrane and are released).

  • Vesicles can also go to endosomes and lysosomes.

  • Donor-acceptor vesicle interactions determine specificity, relying on receptor-ligand interactions = proteins on the surface of vesicles determine their destination.

Vesicles have coat proteins - vesicle transport proteins

  • Examples: Clathrin, COP1, and COP2 = specific vesicle transport proteins

  • Clathrin: targets vesicles to the trans-Golgi network which enables transport to the endosome and endocytic transport.

  • COP1: targets vesicles from the Golgi to the ER (retrieval pathway).

  • COP2: moves vesicles from the ER to the Golgi (forward pathway) for post-translational modifications.

  • This is the way that protein trafficking is controlled: donor-acceptor molecules that are important in the specific transport of vesicles within the cell

  • ( Analogy: Vesicles "dress appropriately" with specific coat proteins to reach their destinations. )

Bidirectional transport between ER and Golgi

  • Retrieval pathway (coming in): from outside the cell through the golgi and towards the ER; uses COP1 coat protein.

  • Forward pathway (going out): from the ER to the Golgi, into endosomes, and for secretion; mediated through COP2.

Vesicle Shapes

  • Clathrin vesicles are smaller.

  • COP1 vesicles are larger and looser.

  • COP2 vesicles are in between.

  • The shape and protein components of a vesicle play a role in its function.

Clathrin coated vesicles

  • Key protein in vesicles that transport cargo from the trans-Golgi network to the endosome.

  • Clathrin-coated vesicles:

    • Diameter: Approximately 50 nanometers.

    • Shape: Characteristic, rigid structure.

  • Size varies based on the type and amount of cargo.

Production and release of clathrin-coated vesicle

  • Cargo receptors, initially unengaged, start coat assembly and cargo selection.

  • This leads to a bulging out of the membrane.

  • The structure continues to develop into a vesicle, still attached to the plasma membrane, ER membrane, or Golgi membrane.

  • Eventually, the clathrin-coated vesicle buds off completely.

  • The coat assembly dissociates from the coated vesicle, leaving a "naked" transport vesicle, which takes the contents of the vesicle to its destination

  • Disassembled coat constituents are recycled for the next coat assembly.

COP1 coated vesicles

  • Transport from the Golgi towards the ER, and within the Golgi compartments.

  • Arranged in pentagons and hexagons, forming a cage structure around the vesicle

  • Average diameter: just under 50 nanometers.

  • The size distribution depends on the type and amount of cargo.

COP2 coated vesicles

  • Take their cargo from the ER to the Golgi.

  • Cage-like structure.

  • Average diameter: approximately 65 nanometers.

Vesicle Function Summary

  • Vesicles are delineated by the membrane they come from and are full of cargo.

  • They transport proteins for secretion, targeting to lysosomes, and to the cell surface, as well as extracellular matrix components.

  • The vesicle membrane composition dictates its fate.

  • Coat proteins are vital.

  • Cargo receptors determine the cargo composition.

  • Coat proteins determine the target compartment.

Accessory proteins - SNAREs

  • Increase the specificity of vesicle transport.

  • V-SNAREs are on the vesicles.

  • T-SNAREs (target snares) are on the target organelle and are complementary to the V-SNAREs = enhances the specificity from the donor organelle to the target organelle

  • ( This interaction is analogous to a lock and key or receptor-ligand interaction.)

Accessory proteins - Rab Family (Small GTPase Proteins)

  • Add to the specificity of vesicle transport.

  • also power the opening of the vesicle to release its contents.

  • Process:

    • Vesicle buds off the donor membrane with V-SNARE and Rab GTPase.

    • Vesicle reaches the target compartment via binding of V-SNARE to its complementary T-SNARE

    • Rab GTPase binds an effector molecule.



    • T-SNARE binding with V-SNARE pulls the vesicle in toward the target compartment membrane.

    • During this process, GTP hydrolysis (GTP to GDP) releases energy, causing membrane fusion and opening up of the vesicle which then releases the cargo

    • GDP Rab is solubilised via the addition of a lipid group and GDI (GDP dissociation inhibitor factor).

    • Diffuses across the cytoplasm (back to the donor compartment), contacts GEF (guanine nucleotide exchange factor), which regenerates GDP to GTP.

    • Rab GTP is then completely recycled to go into the next vesicle that is required for transporting its cargo

Different RABS- Rab Protein Family Specificity

  • About 70 different Rab small GTPases which are essential for vesicle transport

  • Examples:

    • RAB1: involved in the ER and Golgi complexes.

    • RAB7: important in targeting to late endosomes.

  • Different Rabs performing functions including early endosome trafficking, late endosome trafficking, and recycling etc.

Molecular Specificity of Rab

  • Shows dynamic changing of RAB during vesicle maturation

  • Early phagosome: RAB5 expressed.

  • Intermediate phagosome: RAB7 is important.

  • Late phagosome/phagolysosome: Rabs are no longer present.

Roles of RABs and SNAREs

  • Rab GTPases on the vesicle and V-SNARE.

  • V-SNARE and T-SNARE recognise each other.

  • Once Rab GTPase interacts with its effector molecule on the target membrane, V-SNARE and T-SNARE interact and pull the vesicle into the membrane

  • Hydrolysis of GTP to GDP by Rab GTPases.

  • Cargo released into the target organelle.

  • Rab proteins get recycled, and snares remain in the membrane and are eventually recycled.

True face of vesicles

  • ( Actual vesicles are much more complex including: membrane; clathrin, COP1, or COP2 coat; snares; rabs; and many other proteins. )

Exocytosis vs Endocytosis

  • Exocytosis: Vesicle fuses to release contents such as waste or secreted protein outside the cell

  • Endocytosis: Molecules are taken into the cell by a reverse process of exocytosis

  • Not the same as phagocytosis, which is usually associated with removing pathogens