CBIO3400 Lecture L19 - Retrograde Transport and Exocytosis

CBIO3400 Lecture Notes: Retrograde Transport from Golgi to ER & Exocytosis

General Information

  • Course Code: CBIO3400

  • Lecture Date: 03/26/2025

  • Lecture Topic: Retrograde Transport: from Golgi to ER & Beyond: Exocytosis

  • Key Terms: COPI, COPII, Golgi, TGN (Trans-Golgi Network), cis, medial, trans

Learning Objectives

  • Relevant questions are posted on eLC “CBIO3400 Study Questions L19”

  1. Review of last In-Class Activity: “The EndoH Experiment”

  2. Retrograde Traffic – Golgi to ER Traffic

  3. Transport Post Golgi: Exocytosis

    • (a) Understand the process of exocytosis.

    • (b) Learn about exocytosis related diseases.

    • (c) Understand the different pathways of exocytosis.

    • (d) Explore how exocytosis releases secretory vesicles into the synaptic cleft.

  4. Exam #3 Insights

EndoH Experiment

  • EndoH:

    • Definition: Endoglycosidase, an enzyme that cleaves N-linked glycans between the two GlcNAc sugars.

    • Significance:

    • Complex glycans are resistant to hydrolysis.

    • Treatment with this enzyme is used to distinguish complex from high-mannose oligosaccharides.

    • Relevant in various experiments, including tracking glycosylation processes.

  • Hypothesis related to Sec61:

    • Sec61 is the channel through which nascent proteins are threaded into the ER (translocator).

Retrograde Transport: Golgi to ER

Mechanism of Transport

  • Transport Overview:

    • Some ER-resident proteins mistakenly transported to the Golgi to facilitate ER to Golgi traffic for further N-glycosylation before functional.

    • Sorting Signals:

    • Transmembrane Proteins: Lys-Lys-X-X (KKXX) at C-terminus, interacts with COPI coats.

    • Soluble Proteins: KDEL (Lys-Asp-Glu-Leu) at the extreme C-terminal end.

      • Requires a receptor for retrieval.

KDEL Sequence and Its Function

  1. KDEL Sequence Details:

    • Always on C-terminus of cytoplasmic face of soluble ER resident proteins, such as BiP.

    • Needs a receptor to function correctly.

  2. KDEL Receptor:

    • Cycles between ER and Golgi.

    • Binds proteins with KDEL sequence:

      • Binding Affinity:

      • Binds tightly in Golgi (pH 6.0 - 6.7) and loosely in ER (pH 7.2).

    • Proteins returned to the ER in COPI-coated vesicles.

COPI and Retrograde Transport Mechanics

  • COPI Vesicles:

    • Mediate retrograde and retrieval pathways, composed of seven coatamer proteins.

    • Mechanism Elements:

    • Similar to ER to Golgi transport.

    • Arf1 regulates coat assembly.

    • Docking requires Rab and Rab effectors.

    • Fusion involves v-SNAREs and t-SNAREs.

Clinical Relevance

  • Human Diseases Associated with Golgi Traffic Disruptions:

    • Neurodegenerative diseases, autoimmune diseases, cancer, and other disorders related to Golgi fragmentation or dysfunction (e.g., ALS, CDG).

Exocytosis Overview

Definition and Mechanism

  • Definition of Exocytosis:

    • Process by which cells release molecules or particles to the extracellular environment through fusion of secretory vesicles from the Trans-Golgi Network (TGN) with the plasma membrane (PM).

    • Types of Secretion:

    1. Constitutive Secretion:

      • Present in all cells as a default pathway, delivers lipids and proteins continuously.

      • Important for extracellular matrix production.

    2. Regulated Secretion:

      • Used by specialized cells to release secretory products rapidly in response to external signals (e.g., insulin secretion).

Exocytosis-Related Diseases

  • Diseases Caused by Defective Exocytosis:

    1. Cystic Fibrosis: Affects lung and digestive systems due to defective CFTR protein functioning (impairs secretory pathways).

    2. Type 2 Diabetes Mellitus (T2DM): Leads to insulin resistance and impaired secretion.

    3. Hemophilia: Resulting from deficiencies in blood clotting factors due to exocytotic dysfunction.

    4. Lysosomal Storage Diseases: Enzymatic delivery failures often in the nervous system.

    5. Cancer Metastasis: Cancer cells hijack exocytosis for invasive behavior.

Cystic Fibrosis Insights

  • CFTR Mutations:

    • Includes Class I (no synthesis), Class II (reduced trafficking), Class III (reduced gating), Class IV (decreased conductance), and Class V (reduced synthesis).

    • Most common mutations F508del and G551D lead to mis-processed CFTR and its absence on the plasma membrane.

  • Symptoms and Treatments of Cystic Fibrosis:

    • Symptoms include cough, repeated lung infections, salty skin, and inability to gain weight.

    • Treatment focuses on symptom management and complication reduction.

Stepwise Process of Exocytosis for Synaptic Vesicles

  1. Docking:

    • Synaptic vesicle docks to the presynaptic membrane.

  2. Priming (Two Steps):

    • Prepares vesicles for fusion ensuring SNARE partially pairs.

  3. Opening of Fusion Pore:

    • Triggered by Ca2+ influx upon action potentials.

  4. Fusion and Release:

    • Neurotransmitter release and functional reuse of the fusion machinery.

Additional Resources

  • Textbook Readings:

    • 6th edition: pp. 713-714; 716-718; 720-724, 741-743

    • 7th edition: pp. 768-771; 775-780, 798-800

Exam Preparation

  • Exam #3 Announcements & Insights to review for better understanding of the discussed topics.