Study Notes for BIOL 4210 - Lecture 17

BIOL 4210 – Cell and Molecular Biology Lecture 17: Intracellular Membrane Traffic: Part 1

Instructor Details

  • Name: Dr. David E. Nelson

  • Contact Email: david.e.nelson@mtsu.edu

  • Course: Lectures on Cell and Molecular Biology

Learning Objectives

  1. Understand the molecular mechanisms of membrane transport.

  2. Know how materials are transported from the ER through the Golgi apparatus.

Objective 1: The Molecular Mechanisms of Membrane Transport

Exocytosis and Endocytosis

  • Exocytosis: A process involving the biosynthetic-secretory pathway.

  • Endocytosis: Involves internalization of membranes and associated proteins.

  • Vesicular Transport:

    • A vesicle buds from a donor compartment carrying ‘cargo’ (substances transported within the vesicle) from the lumen and transmembrane proteins from its surface.

    • The vesicle fuses with the target compartment, delivering cargo to it.

Key Characteristics of Membrane Transport

  • The flow of membrane between compartments is balanced.

  • This process is highly selective, ensuring that the proper cargo goes to the proper destination.

  • Main pathways involved:

    • Secretory Pathway

    • Endocytic Pathway

Coated Vesicles in Trafficking

  • Types of Coated Vesicles:

    • Mediate trafficking between different compartments.

    • Coats concentrate specific membrane proteins in a specialized patch, directly regulating membrane curvature and formation of vesicles.

  • Clathrin:

    • Major protein involved, composed of three large and three small polypeptide chains forming a structure known as a triskelion.

    • This structure forms a basket-like convex configuration on the cytosolic surface of the plasma membrane.

Assembly and Disassembly of Clathrin Coats

  • Adaptor Proteins:

    • Link transmembrane proteins to clathrin, enabling packaging of selected proteins into clathrin-coated transport vesicles.

  • Binding to four molecules of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the cytosolic face of the plasma membrane exposes binding sites for cargo receptors in the AP2 adaptor protein complex.

Phosphoinositides in Vesicle Transport

  • Phosphoinositides:

    • The 3’, 4’, and 5’ positions of the inositol sugar head group can be phosphorylated/dephosphorylated.

    • Different proteins recognize different phosphoinositide head groups, providing a mechanism to recruit various proteins to membranes.

  • The distribution of PI and PIP kinases and PIP phosphatases influences the steady-state distribution of various PIPs.

    • For instance, the membrane of secretory vesicles contains PI(4)P, which is transformed into PI(4,5)P2 by a PI 5-kinase upon vesicle fusion with the plasma membrane.

    • PI(4,5)P2 aids in recruiting adaptors for endocytosis and forming clathrin-coated pits.

    • Post-budding, PI(4,5)P2 is hydrolyzed by a PI(5)P phosphatase, which stimulates the uncoating of vesicles.

The Role of Dynamin

  • Dynamin:

    • A soluble cytosolic protein that binds phosphatidylinositol and destabilizes noncovalent interactions between lipid bilayers to promote vesicle budding.

Regulation by Monomeric GTPases

  • GTPases:

    • Control clathrin coat assembly and most COPI/COPII coats.

    • COPII-Coated Vesicles:

    • Important for budding from the ER.

Guidelines for Vesicle Targeting

  • Rab GTPases:

    • Guide vesicle targeting; over 60 family members.

    • Regulate vesicular trafficking between membranes.

  • Rab Proteins and Their Locations:

    • Rab1, Rab2: ER and Golgi complex.

    • Rab3A, Rab4/Rab11: Synaptic vesicles, secretory vesicles.

    • Rab5: Early endosomes, clathrin-coated vesicles.

    • Rab6: Medial and trans Golgi.

    • Rab7: Late endosomes.

    • Rab8: Cilia.

    • Rab9: Late endosomes, trans Golgi.

Function of Rab Proteins

  • Rab Effectors:

    • Can be tethering or motor proteins.

    • Rab proteins maintain residence in the cytosol until activated by binding membranes.

    • GTP-bound Rab Proteins:

    • Engage with membranes, facilitating transport.

Activation of Rab Proteins

  • Active Rab5:

    • Activates phosphatidylinositol 3-kinase to produce PI(3)P, which binds Rab5 effector proteins with PI(3)P binding sites including tethering proteins.

  • Rab domains determine singularity of endosomal organelles and transition when swapping with subsequent Rab domains, e.g., Rab5 transitioning into Rab7 for late endosome development.

Objective 2: Transport from the ER Through the Golgi Apparatus

  • Golgi Apparatus/Complex:

    • Major site for carbohydrate synthesis; nearly all proteins leaving the ER traffic through the Golgi.

    • Proteins can be directed to their final destinations or recycled back to the ER via retrieval pathways.

Mechanism of COPII-Coated Vesicles

  • COPII-Coated Vesicles:

    • Bud from ER exit sites (regions of ER membrane devoid of ribosomes).

  • Cargo Proteins:

    • Transmembrane and soluble cargo proteins possess ER exit signals that either bind COPII coat components or cargo receptor membrane proteins.

    • Unfolded or misfolded proteins are retained in the ER, although the system can exhibit leakage.

    • Coats are disassembled post-budding.

Homotypic Membrane Fusion

  • Involves v- and t-SNARE proteins on both vesicles, enabling the fusion process.

    • If required, ATPase NSF separates v- and t-SNAREs before they function.

Retrieval of ER Resident Proteins

  • COPI Vesicles:

    • Bud from vesicular tubular clusters to move ER resident proteins and cargo receptors back to the ER where they perform their functions.

  • Retrieval Signals:

    • ER membrane proteins bind directly to COPI coat proteins using the KKXX motif.

    • Soluble ER resident proteins utilize KDEL sequences for retrieval.

KDEL Receptor Functionality

  • The KDEL receptor binds both the KDEL sequence of soluble resident ER proteins and COPI coat proteins, facilitating retrieval.

Structure of the Golgi Apparatus

  • Cis and Trans Faces:

    • The cis face is the entry face (nearest the ER).

    • The trans face serves as the exit face.

  • Consists of flattened membrane-enclosed compartments (cisternae).

Golgi Processing Mechanism

  • Processing of N-linked Oligosaccharides:

    • Occurs in distinct regions of the Golgi apparatus.

    • All resident Golgi proteins are membrane-bound and thus enzymatically functional.

    • Reactions occur on the membrane surface, evident by staining with osmium.

Biochemical Gradient in the Golgi

  • Earlier steps in processing occur nearer to the cis-face, while later steps take place towards the trans-face.

  • Enzyme distribution forms a gradient, with all Golgi resident proteins being membrane-bound—a difference from the ER.

Glycobiology Insights

  • There is a diverse array of glycosyl transferases and glycosidases which can be expressed in a cell-type-specific manner, yielding complex patterns of protein glycosylation.

  • Generation of complex oligosaccharide chains follows a well-ordered pathway.

Activities

  1. Attempt the study guide questions.

  2. Complete the following from the Problems Book:

    • Definitions: 13-1 to 13-12 and 13-29 to 13-35

    • True/False: 13-13 to 13-14 and 13-36 to 13-38

    • Thought problems: 13-15, 13-17, and 13-39

Reading Material

  • Today: Chapter 13, pages 695-718.

  • Next Lecture Prep: Chapter 13, pages 719-742.

  • Watch:

    • Lecture 17 – Part 1: The molecular mechanisms of membrane transport

    • Lecture 17 – Part 2: Transport from the ER through the Golgi apparatus

Homework

  • Review the material presented in today's lecture and complete all assigned questions and readings.