CBIO3400 - Anterograde Trafficking: ER-to-Golgi

CBIO3400 Lecture Notes: Anterograde Trafficking: ER to Golgi and Protein Modifications

Introduction

  • Date of Lecture: 03/30/2026
  • Key Focus: Anterograde transport involving the movement of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus, including details on vesicle formation and modifications that occur in the Golgi.

Learning Objectives

  • Review of vesicular traffic.
  • Understanding of anterograde transport (ER to Golgi).
  • Detailed look at N-linked glycosylation processes that occur in the Golgi.
  • Participation in three in-class activities (total of 6 points).
  • Additional questions related to the lecture can be found on eLC under "CBIO3400 Study Questions L18."

General Concept of Vesicular Transport

  • Key Components in Vesicular Transport:
    • Donor Compartment: The origin where the vesicle forms.
    • Acceptor Compartment: The destination where the vesicle fuses.
    • Cytoskeletal Elements: Provide tracks on which vesicles move.
    • Coat Proteins: Play a crucial role in vesicle budding and cargo selection.
    • Soluble Cargo: Proteins and other molecules transported within vesicles.
    • Transmembrane Cargo: Proteins embedded within the vesicle membrane.
    • SNARE Proteins: Facilitate vesicle fusion with target membranes.
    • Tethering Factors: Assist in bringing vesicles close to their target membrane.

Mechanism of Vesicular Transport

  • Budding Process:
    • Coat proteins assemble on the cytosolic side of the ER membrane, forcing the lipid bilayer to bend, creating a vesicle.
    • Cargo selection occurs at this stage, where specific cargo is recognized and packaged into budding vesicles.
  • Vesicle Characteristics:
    • COPII-Coated Vesicles: Specific to ER to Golgi transport, composed mainly of Sec23/24 and Sec13/31 proteins, along with Sar1.

Anterograde Transport (ER to Golgi)

  • Process Overview:
    • Vesicles bud off the ER at specific exit sites, potentially undergo fusion to form larger structures, and subsequently fuse with the cis-Golgi network.
    • Membrane Transport: The movement is facilitated through motor proteins along cytoskeletal tracks, such as kinesin.
  • Steps Involved:
    • Budding: Vesicle coats form and encapsulate cargo.
    • Uncoating: Upon reaching their destination, the vesicles shed their coats, exposing proteins needed for fusion.
    • Scission: The process of a vesicle detaching from its donor membrane.
    • Fusion: The vesicle interacts with the target membrane, facilitated by SNARE proteins.

COPII Coat Formation

  • Sar1 is a small GTPase that regulates COPII coat assembly:
    • Inactive Form (GDP-bound): Found in the cytosol and does not participate in vesicle formation.
    • Active Form (GTP-bound): Activated by Sar1-GEF, which is located within the ER membrane, causing a conformational change that allows it to insert into the membrane and initiate vesicle budding.
    • Sar1-GTP also recruits the inner coat proteins Sec23/24 and the outer coat proteins Sec13/31.
  • Function:
    • Cargo Selection: Sec24 binds to cargo receptors and selectively packages proteins with exit signals.
    • Cage Formation: Sec13/31 forms a cage-like structure that assists in vesicle budding.

Protein Modification in the Golgi

  • Golgi Functions:
    • Carbohydrate synthesis, including N-linked and O-linked glycosylation, lipid modification.
    • Protein sorting for transport throughout the secretory pathway.
  • Cis and Trans Faces:
    • Cis Face: Closest to the nucleus; receives vesicles from the ER and buds vesicles returning to the ER.
    • Trans Face: Points away from the nucleus; releases vesicles for transport to final destinations.

N-linked Glycosylation in the Golgi

  • Process Overview:
    • Initial glycosylation occurs in the ER, with further modifications in the Golgi lumen.
    • Enzymes like glycosyltransferases and glycosidases modify oligosaccharides attached to proteins.
  • Endo-H Analysis:
    • Endo-H is used to differentiate complex glycans from high-mannose types, recognized by their resistance to hydrolysis.

Mechanisms of Tethering and Docking

  • Rab GTPases: Mediators that help tether vesicles to target membranes and ensure specificity in vesicle targeting.
    • Activation: Inactive Rab-GDP is activated to Rab-GTP at donor membranes by Rab-GEF.
    • Interaction with Effectors: Rab-GTP interacts with effectors at target membranes, facilitating docking.

Vesicle Fusion Mechanism

  • SNARE Proteins:
    • Specific v-SNAREs from vesicles and t-SNAREs from target membranes interact and coil around each other, leading to membrane fusion.
    • The process brings membranes into close proximity, facilitating removal of water and overcoming electrostatic repulsion.
    • Energy Release: The energy from this process drives the fusion and integrates the vesicle contents into the target membrane.

Key Takeaways

  • Vesicular transport is a critical process for protein trafficking and modification within the cell.
  • The mechanisms of vesicle formation, cargo selection, and membrane fusion are complex yet highly regulated, involving various proteins and biochemical processes.

In-Class Activities

  • Engage actively with the demonstrations and the experiments illustrated, such as the photo-crosslinking experiment aimed at identifying the translocation channels used in protein translocation.
  • Review literature suggestions as guidance for deeper understanding.
    • Reference materials include the 6th and 7th editions of standard texts with specified pages for review.