module 4

• Protein Trafficking & Vesicular Transport

  • Efficiently delivers and regulates the levels of over 5,000 transmembrane proteins and 2,000 secreted proteins in humans
  • Correctly locates each protein within the cell to allow it to function
  • Dynamically moves proteins within the cell as required
• Trafficking pathways

  1. Anterograde - Secretory Pathway/Exocytosis

  2. Retrograde - Endocytosis/Uptake
• Temperature Sensitive O45 variant of the vesicular stomatitis virus G Protein is retained in the ER in a unfolded state
• Transport between different compartments occurs via transport vesicles
• Vesicle buds can be visualized during in vitro budding reactions
• Coated vesicles accumulate during in vitro budding reactions in the presence of a nonhydrolyzable analog of GTP
• Transport Vesicles

  Come in different flavors
• Vesicular Transport

  Enables the transport of membrane and proteins between intracellular compartments
• Live Cell Video Microscopy reveals many transport carriers are tubular in nature - often referred to as tubularvesicular carriers
• Laminin molecule

  70–90 nm length and <8 nm height
• COPI vesicle

  50 nm diameter
• Synaptic vesicle membrane

  • Contains 7000 phospholipid molecules and 5700 cholesterol molecules
  • Contains close to 50 different integral membrane protein molecules, which vary widely in their relative abundance and together contribute about 600 transmembrane α helices
  • The most abundant protein is a transmembrane v-snare (~70 copies per vesicle)
• Phenotypes of yeast sec mutants identified five stages in the secretory pathway
• Each vesicular transport step involves a unique combination of distinct proteins
• Proteins involved in vesicular trafficking

  • Small GTPase
  • SNAREs
  • Coat Proteins
• Protein Trafficking associated with the Endoplasmic Reticulum

  1. Delivery of newly synthesized transmembrane proteins and secreted proteins from the ER to the Cis-Golgi

  2. Retrieval of escaped ER resident proteins and membrane

  3. Anterograde transport from ER to CGN occurs via COPII Vesicles

  4. Retrograde transport from CGN to ER occurs via COPI vesicles
• Vesicular Transport Stage - Budding

  1. Budding of Vesicles is driving by the recruitment and assembly of coat proteins on the donor membrane

  2. Cargo is incorporated into the Vesicle through interactions with coat proteins

  3. Two types of cargo are incorporated: 1) Passengers to be moved (i.e. newly synthesized proteins) 2) Crew to enable transport to occur (i.e. proteins required for fusion to acceptor membrane including SNARES)

  4. The assembly of coat induces curvature of the membrane creating an attached membrane bud
• COPII Dependent Vesicular Transport - Initiation


  1. The COPII vesicle formation is initiated by GDP–GTP exchange on Sar1 catalyzed by the transmembrane guanine nucleotide exchange factor Sec12

  2. Activated Sar1-GTP binds to the ER membrane
• COPII Dependent Vesicular Transport - Coat Recruitment & Assembly


  1. Activated Sar1-GTP binds to the ER membrane and recruits the Sec23/24 subcomplex

  2. The cytoplasmically exposed signal of transmembrane cargo is captured by direct contact with Sec24, forming the "prebudding complex"

  3. These prebudding complexes are clustered by the Sec13/31 subcomplex, generating COPII-coated vesicles
• Dynamin-mediated pinching off or scission of a transport vesicles
• GTPase activity

  Regulates pinching off or scission of the vesicle
• COPII Dependent Vesicular Transport - Coat diassembly


  1. After the vesicle coat is complete, the Sec23 coat subunit promotes the hydrolysis of GTP by Sar1

  2. This promotes the release of Sar1-GDP from the vesicle which causes disassembly of the coat
• Cyclical activation/inactivation of Arf/SarI proteins during coat assembly
• Maintenance of the ER-Resident proteins

  1. Indirect Exclusion - as ER resident proteins are functioning in the ER they are less motile

  2. Retrieval of escaped ER residents from the cis-Golgi Network - Most soluble and transmembrane ER resident proteins have sorting signals within their amino-acid sequences (ie KDEL) which bind to receptors or the COPI coat directly in the CGN
• Vesicular Transport enables the transport of membrane and proteins between intracellular compartments
• Microtubules


  25 nm diameter, involved in vesicle transport, cell polarity, and division
• Intermediate filaments

  1. 12 nm diameter, provide elasticity and tensile strength
• Microfilaments

  1. 7 nm diameter, involved in contraction, transport, locomotion, and division
• Microtubule organizing centre - centriole; anchor of the microtubule "minus end"
• Polimerization of microtubules only happens at "plus end"
• Kinesin

  Motor for vesicle transport, has 2x light chains to bind cargo and 2x heavy chains with N-terminal motor domains
• Cytoplasmic dynein

  Minus-end motor for vesicle transport, has 2x heavy chains, multiple light and intermediate chains, and a dynactin complex
• Microtubules are "tracks" vesicles and organelles use for moving around the cell
• Microtubules are anchored at the MTOC
• Kinesin and dynein are microtubule-associated motors that pull vesicles/organelles in opposite direction
• Tethers are peripheral membrane proteins that are recruited from the cytoplasm to membranes
• Tethers initially detect transport vesicles and the prolonged tethering to the membrane enables time for the establishment of a productive SNARE interaction if compatible
• Different Tethers are found associated with a range of acceptor membranes throughout the cell
• Mechanism of vesicle fusion

  1. Rab protein

  2. SNARE proteins

  3. α-SNAP complex
• Vesicles are tethered to membranes which brings it in close proximity to the acceptor membrane