lecture 2 recording

Introduction

  • The session began with an instructor addressing students after a slight delay due to exam grading.

  • Key focus: Discuss the intricate processes of protein translation and transport, fundamental to cell function and homeostasis, as covered in a specific set of slides.

Protein Translation & Transport

Translation Locations

  • Proteins can be translated in two primary locations:

    • Cytoplasm: Occurs on free ribosomes, synthesizing proteins destined for the cytoplasm, nucleus, mitochondria, or peroxisomes. These proteins are typically synthesized entirely in the cytoplasm and then imported post-translationally.

    • Co-translationally imported into the ER: Occurs on ribosomes bound to the endoplasmic reticulum (ER) membrane. This pathway is for proteins destined for the ER lumen, Golgi apparatus, lysosomes, plasma membrane, or secretion outside the cell.

  • Proteins entering the ER, either fully integrated into the membrane or residing within the lumen, subsequently become part of the secretory/endomembrane system, which includes a network of organelles involved in protein modification, sorting, and transport.

Pathways and Destinations

  • Once inside the ER, proteins can be further transported to various destinations:

    • Golgi apparatus: For further processing, glycosylation, and sorting.

    • Plasma membrane: For integration as membrane proteins (e.g., receptors, transporters) or for secretion outside the cell (e.g., hormones, digestive enzymes).

    • Endosomes or lysosomes: For degradation of old organelles or macromolecules, or for specific sorting pathways.

Coated Vesicles & Address Labels

  • Coating proteins (coat proteins) are essential for the formation and targeted transport of vesicles.

    • Function: They induce membrane curvature to create vesicle buds, select specific cargo molecules for transport, and provide a unique identity that acts as an "address label" for guiding vesicles to their correct target compartments.

  • Examples of coat proteins discussed include:

    • COPII (Coat Protein Complex II): Mediates anterograde transport by forming vesicles that bud from the ER and carry cargo forward to the Golgi apparatus.

    • COPI (Coat Protein Complex I): Mediates retrograde transport, primarily by forming vesicles that bud from the Golgi and return to the ER, playing a crucial role in recycling ER resident proteins and ensuring membrane lipid balance.

Anterograde & Retrograde Transport

  • Anterograde transport: Represents the forward movement of proteins along the secretory pathway:

    • ER → Golgi → Plasma Membrane (or secretion). This movement is facilitated by COPII-coated vesicles from the ER to the Golgi.

  • Retrograde transport: Refers to the backward movement of vesicles:

    • Golgi → ER. This pathway, mediated by COPI-coated vesicles, is vital for retrieving ER resident proteins that have escaped into the Golgi and for recycling SNARE proteins.

Resident Proteins & Retention Tags

  • Retention tags are specific amino acid sequences that ensure resident proteins remain in their correct organelles, preventing their accidental secretion or mislocalization.

  • An important retention tag is:

    • KDEL: This four-amino acid sequence (Lysine-Aspartic acid-Glutamic acid-Leucine) acts as an ER retrieval signal. It is found on the C-terminus of many soluble ER resident proteins.

    • When an ER resident protein with a KDEL sequence accidentally reaches the Golgi apparatus, it binds to a specific KDEL receptor.

Mechanism of KDEL Function

  1. A KDEL-containing protein, having escaped the ER, binds to the KDEL receptor in the cis-Golgi network. This binding affinity is higher in the slightly acidic environment of the Golgi than in the ER.

  2. This binding event triggers the recruitment of COPI coat proteins, initiating the formation of a retrograde transport vesicle.

  3. The COPI-coated vesicle transports the protein-receptor complex back to the ER.

  4. Upon arrival in the ER, the more neutral pH causes the KDEL protein to dissociate from its receptor, releasing the ER resident protein back to its functional location. The KDEL receptor is then recycled back to the Golgi.

SNARE Proteins & Vesicle Fusion

SNARE Functionality

  • SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical components in mediating the fusion between vesicles and their target compartments.

    • V-SNAREs (vesicle-SNAREs): Located on the vesicle membrane.

    • T-SNAREs (target-SNAREs): Located on the target compartment membrane.

  • They form stable, coiled-coil complexes called trans-SNARE complexes, which pull the vesicle and target membranes into close apposition, overcoming the energetic barrier to membrane fusion. This tight docking interaction is essential for the specificity and efficiency of vesicle fusion.

Metaphor for SNARE Action

  • Think of a boat docking at a harbor:

    • As the boat approaches, it secures ropes to the dock to pull itself close enough to berth. Similarly, SNARE proteins act like these ropes, specifically recognizing each other and drawing the vesicle and target membranes together until they fuse, allowing the vesicle's contents to be released.

Examples of SNARE Proteins

  • Synaptobrevin (a V-SNARE, also known as VAMP) is found on synaptic vesicles.

  • SNAP-25 and Syntaxin (T-SNAREs) are found on the presynaptic plasma membrane.

  • These specific SNARE proteins interact to enable synaptic vesicles to fuse with the presynaptic membrane, releasing neurotransmitters into the synaptic cleft. This process is crucial for neuronal communication.

  • The critical importance of SNAREs is highlighted by the action of botulinum and tetanus toxins: Both are proteases that specifically cleave and inactivate SNARE proteins in neurons. This blocks neurotransmitter release (e.g., acetylcholine in botulism, GABA/glycine in tetanus), leading to paralysis (flaccid in botulism, spastic in tetanus).

Proteins in the ER & Golgi Apparatus

Post-Translational Modifications

  • Proteins entering the ER undergo several crucial modifications, most notably glycosylation.

    • Oligosaccharide addition (N-linked glycosylation): A pre-formed core oligosaccharide (consisting of specific N-acetylglucosamine, mannose, and glucose residues) is transferred en bloc from a lipid carrier molecule (dolichol phosphate) to an asparagine residue (N) within a specific consensus sequence (Asn-X-Ser/Thr) of the nascent polypeptide chain. This reaction is catalyzed by the enzyme oligosaccharyl transferase.

    • Glycoproteins are produced as proteins mature within the ER and continue their journey through the secretory pathway. This initial glycosylation is important for protein folding, stability, and subsequent sorting.

Golgi Apparatus Function

  • The Golgi apparatus is comprised of a series of flattened membrane-bound sacs called cisternae, typically organized into cis, medial, and trans compartments. It is considered the primary site for extensive modification and sorting of glycoproteins and lipids.

    • Enzymatic addition and removal of various sugar moieties occur as proteins transit through the Golgi's distinct cisternae. Each cisterna (e.g., cis-Golgi network (CGN), cis, medial, trans, trans-Golgi network (TGN)) contains a unique set of enzymes that carry out sequential processing steps.

    • Multiple cycles of glycosylation, as well as proteolysis and lipid modifications, lead to diverse protein functions and modifications, tailoring proteins for their specific roles.

Glycosylation Process Overview

  1. Core oligosaccharide is synthesized on the dolichol phosphate in the ER and then transferred to the nascent protein (N-linked glycosylation).

  2. As the protein moves from the cis-Golgi, through the medial-Golgi, and into the trans-Golgi network, enzyme modifications occur. These involve specific glycosyltransferases and glycosidases that add, remove, or modify sugar residues, creating a wide variety of oligosaccharide chains.

  3. The end result is diverse protein glycosylation, which significantly influences protein folding, stability, intracellular transport, cell-cell recognition, and overall functionality post-Golgi secretion or targeting.

Default Pathway Overview

  • Proteins that do not possess specific targeting signals (like KDEL for ER retention or M6P for lysosomal delivery) generally move from the Golgi to the Plasma Membrane through the default secretory pathway. This pathway assumes that without a specific